PHARMACEUTICAL PRODUCTION IN TRANSGENIC ANIMALS

PHARMACEUTICAL PRODUCTION IN TRANSGENIC ANIMALS

PHARMACEUTICAL PRODUCTION IN TRANSGENIC ANIMALS

Biotechnology Information Series (Bio-10) North Central Regional Extension Publication Iowa State University - University Extension

A New Kind of Farming
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A new brand of farming is emerging from the research and development labs of several universities and small biotechnology companies - so new they're even changing the spelling to "pharming."

Pharming is the production of human pharmaceuticals in farm animals that is presently in the development stage with possible commercialization by the year 2000. It has been gaining application among biotechnologists since the development of transgenic "super mice" in 1982 and the development of the first mice to produce a human drug, tPA (tissue plasminogen activator to treat blood clots), in 1987. Transgenic organisms have been modified by genetic engineering to contain DNA from an external source. The first drugs produced by this approach are about to enter clinical trials as part of the FDA review process. These transgenic animals will likely be raised by the pharmaceutical companies and will certainly be kept separate from the food supply.

Genetic Engineering
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During the 1970s, advances in DNA manipulation techniques provided a significant, economical alternative source for many drugs made of protein. Previously, these protein drugs were available in extremely limited supplies; for example, human cadavers were the source for human growth hormone, and insulin to treat diabetes was collected from slaughtered pigs.

By genetic engineering, the DNA gene for a protein drug of interest can be transferred into another organism that will produce large amounts of the drug. This technique (illustrated in Figure 1), can be used to impart new production characteristics to an organism, as well as to trigger the production of a protein drug:

1. The gene of interest is isolated on a strand of DNA.
2. DNA is cut at specific points by restriction enzymes. The enzymes recognize certain sequences of bases on the DNA strand and cut where those sequences appear.
3. The cut DNA joins with a vector, which may be a virus or part of a bacterial cell called a plasmid. The vector carries the gene of interest into the organism that will produce the protein.
4. Transformation occurs when the gene carried by the vector is incorporated into the DNA of another organism where it initiates the action desired (production of a drug, etc.).

[Figure 1]

The first successful products of the genetic engineering process were protein drugs like insulin and growth hormone. These drugs do not have to be produced by mammals to be active in mammals. An inexpensive, easy-to-grow culture of genetically engineered bacteria like the common E. coli can manufacture these protein drugs.

[Figure 2]

Other human drugs, such as tPA for blood clots, erythropoietin for anemia, and blood clotting factors VIII and IX for hemophilia, require modifications that only cells of higher organisms like mammals can provide. The higher costs of maintaining mammalian cell cultures that produce only small amounts of the drugs have been an enormous barrier to the commercial development of this type of cell culture production method.

Animal Pharming
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By genetic engineering, the DNA gene for a protein drug of interest can be transferred into another organism for production. Which organism to use for production is a technical and economic decision. For certain protein drugs that require complex modifications or are needed in large supply, production in transgenic animals seems most efficient. The farm animal becomes a production facility with many advantages - it is reproducible, has a flexible production capacity through the number of animals bred, and maintains its own fuel supply. Best of all, in most animal drug production, the drug is delivered from the animal in a very convenient form - in the milk!

Procedure

A transgenic animal for pharmaceutical production should (1) produce the desired drug at high levels without endangering its own health and (2) pass its ability to produce the drug at high levels to its offspring.

The current strategy to achieve these objectives is to couple the DNA gene for the protein drug with a DNA signal directing production in the mammary gland. The new gene, while present in every cell of the animal, functions only in the mammary gland so the protein drug is made only in the milk. Since the mammary gland and milk are essentially "outside" the main life support systems of the animal, there is virtually no danger of disease or harm to the animal in making the "foreign" protein drug.

After the DNA gene for the protein drug has been coupled with the mammary directing signal, this DNA is injected into fertilized cow, sheep, goat, or mouse embryos with the aid of a very fine needle, a tool called a micromanipulator, and a microscope (Figure 2). The injected embryos are then implanted into recipient surrogate mothers where, hopefully, they survive and are born normally.

Commercialization Issues
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Success in creating a transgenic animal that can produce the drug is far from guaranteed. About 10 to 30 percent of mouse embryos produce transgenics, but less than 5 percent of goats, sheep, or cows do. Production of the drug is measured during lactation after the animal is raised to maturity and bred. Because of the long time periods involved and low success rates, developing transgenic animals is currently very expensive, as the dollar amounts in Table 1 indicate.

Although most protein drugs are made in milk, a notable exception is human hemoglobin that is being made in swine blood to provide a blood substitute for human transfusions. Because hemoglobin is naturally a blood protein, it is likely to be one of few exceptions to the usual method of production in milk. Furthermore, the economics of blood production are less favorable, because to recover human hemoglobin, the animal producing it must be slaughtered.

Drugs currently made by or being developed in transgenic animals are listed in Table 1. Notice that pharming is expected to increase the value of animals dramatically. In general, animal pharming is considered to be 5 to 10 times more economical on a continuing basis and 2 to 3 times cheaper in start-up costs than cell culture production methods.
[Table 1]

Drug Animal Value/Animal/Yr*
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AAT sheep $15,000
tPA goat 75,000
Factor VIII sheep 37,000
Factor IX sheep 20,000
Hemoglobin pig 3,000
Lactoferrin cow 20,000
CFTR sheep, mouse 75,000
Human Protein C pig 1,000,000

*Current market price of the drug and supply produced by one animal.

Drug descriptions:

AAT alpha-1-antitrypsin, inherited deficiency leads to
emphysema
tPA tissue plasminogen activator, treatment for
blood clots
Factors VIII, blood clotting factors, treatment for hemophilia
XI
Hemoglobin blood substitute for human transfusion
Lactoferrin infant formula additive
CFTR cystic fibrosis transmembrane conductance
regulator, treatment of CF
Human Protein C anticoagulant, treatment for blood clots

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Regulatory and Ethical Issues
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Production of human pharmaceuticals in farm animals has many technical barriers to overcome, although most technologists agree that these technical difficulties will be easily resolved in the 1990s. As a production method, animal pharming is entirely unprecedented and is likely to undergo significant evaluation by the Food and Drug Administration (FDA). Human drugs purified from animal milk or blood are likely to require exceptional levels of safety testing before animal and human health concerns are addressed to the satisfaction of consumers.

At a more fundamental level, many people are genuinely concerned about animal welfare and biotechnology's redefinition of the relationship between humans and animals. Genetic engineering and transgenic animal research are essentially human endeavors to improve the availability, quality, and safety of drugs; to enhance human health; and to improve animal health. Animal breeding has gone on for centuries, but the ability to change the DNA of the animal brings breeding to a revolutionary new level.

DNA profiling can be used for identifying individuals and determining relationships

DNA profiling can be used for identifying individuals and determining relationships

17.4. DNA profiling can be used for identifying individuals and determining relationships

We use the term DNA profiling to refer to the general use of DNA tests to establish identity or relationships. DNA fingerprinting is reserved for the technique invented by Jeffreys et al. (1985) using multilocus probes. For more detail on this whole area, the reader should consult the book by Evett and Weir (Further reading).
17.4.1. A variety of different DNA polymorphisms have been used for profiling

DNA fingerprinting using minisatellite probes

These probes contain the common core sequence of a hypervariable dispersed repetitive sequence GGGCAGGAXG, first discovered by Jeffreys et al. (1985) in the myoglobin gene (see Section 7.4.2). When hybridized to Southern blots they give an individual-specific fingerprint of bands (Figure 17.19). Their chief disadvantage is that it is not possible to tell which pairs of bands in a fingerprint represent alleles. Thus, when matching DNA fingerprints, one matches each band individually by position and intensity. Other hypervariable repeated sequences have been used in the same way, for example those detected by the synthetic oligonucleotide (CAC)5 (Krawczak and Schmidtke, 1998). top link
DNA profiling using single-locus minisatellite markers

Minisatellite probes recognize single-locus variable tandem repeats on Southern blots. Each probe should reveal two bands in any person's DNA, representing the two alleles. Profiling is based on four to ten different polymorphisms. These probes allow exact calculations of probabilities (of paternity, of the suspect not being the rapist, etc.), if the gene frequency of each allele in the population is known. For matching alleles between different gel tracks, the continuously variable distance along the gel has to be divided into a number of ‘bins'. Bands falling within the same bin are deemed to match. It is imperative that the criteria used for judging matches in each profiling test should be the same binning criteria that were used to calculate the population frequencies of each allele. The binning criteria can be arbitrary within certain limits, but they must be consistent. Minor variations within repeated units of some minisatellites potentially allow an almost infinite variety of alleles to be discriminated, so that the genotype at a single locus might suffice to identify an individual (Jeffreys et al., 1991).top link
DNA profiling using microsatellite markers

Microsatellite polymorphisms (Section 7.4.3) are based on short tandem repeats, usually di-, tri- or tetranucleotides. They have the advantages over minisatellites that they can be typed by PCR and that discrete alleles can be defined unambiguously by the precise repeat number. This avoids the binning problem and makes it easier to relate the results to population gene frequencies.top link
The use of Y-chromosome and mitochondrial polymorphisms

For tracing relationships to dead persons, Y-chromosome and mitochondrial DNA polymorphisms are especially useful because of their sex-specific pattern of transmission. An interesting example was the identification of the remains of the Russian Tsar and his family, killed by the Bolsheviks in 1917, by comparing DNA profiles of excavated remains with living distant relatives (Gill et al., 1994).top link
17.4.2. DNA profiling can be used to determine the zygosity of twins

In studying nonmendelian characters (Chapter 19), and sometimes in genetic counseling, it is important to know whether a pair of twins are monozygotic (MZ, identical) or dizygotic (DZ, fraternal). Traditional methods depended on an assessment of phenotypic resemblance or on the condition of the membranes at birth (twins contained within a single chorion are always MZ, though the converse is not true). Errors in zygosity determination systematically inflate heritability estimates for nonmendelian characters, because very similar DZ twins are wrongly counted as MZ, while very different MZ twins are wrongly scored as DZ.

Genetic markers provide a much more reliable test of zygosity. The extensive literature on using blood groups for this purpose is summarized by Race and Sanger (1975). DNA profiling is nowadays the method of choice. The Jeffreys fingerprinting probe allows a very simple test - samples from MZ twins look like the same sample loaded twice, and samples from DZ twins show some differences. An error rate could be calculated from empirical data on band sharing by unrelated people, using some defined binning strategy (see above).

When single-locus markers are used, if twins give the same types, then for each locus, the probability that DZ twins would type alike is calculated. If the parents have been typed, this follows from mendelian principles; otherwise the probability of DZ twins typing the same must be calculated for each possible parental mating and weighted by the probability of that mating calculated from population gene frequencies. The resultant probabilities for each (unlinked) locus are multiplied, to give an overall likelihood PI that DZ twins would give the same results with all the markers used. The probability that the twins are MZ is then:

where m is the proportion of twins in the population who are MZ (about 0.4 for like-sex pairs). Sample calculations are given in Appendix 4 of Vogel and Motulsky (Further reading).top link
17.4.3. DNA profiling can be used to disprove or establish paternity

Excluding paternity is fairly simple - if the child has a marker allele not present in either the mother or alleged father then, barring new mutations, the alleged father is not the biological father. Proving paternity is, in principle, impossible - one can never prove that there is not another man in the world who could have given the child that particular set of marker alleles. All one can do is establish a probability of nonpaternity that is low enough to satisfy the courts and, if possible, the putative father.

DNA fingerprinting probes have been widely used for this purpose (Figure 17.19). Bands must be binned according to an arbitrary but consistent scheme, as explained above, to decide whether or not each nonmaternal band in the child fits a band in the alleged father. Then if, say, 10/10 bands fit, the odds that the suspect, rather than a random man from the population, is the father are 1:p10, where p is the chance that a random man from that population would have a band matching a given band in the child. Even for p = 0.2, p10 is only 10-7. Single-locus probes allow a more explicit calculation of the odds (Figure 17.20). A series of four to ten unlinked single-locus markers can give overwhelming odds favoring paternity if all the bands fit.top link
17.4.4. DNA profiling is a powerful tool for forensic investigations

DNA profiling for forensic purposes follows the same principles as paternity testing. Scene-of-crime material (bloodstains, hairs or a vaginal swab from a rape victim) are typed and matched to a DNA sample from the suspect. If the bands don't match, the suspect is excluded. One of the most powerful applications of DNA profiling is for preventing miscarriages of justice. If the bands do all match, the odds that the criminal is the suspect rather than a random member of the population can be calculated, based on the allele frequencies in the population. Of course, if the alternative were the suspect's brother, the odds would look very different. The fate of DNA evidence in courts provides a fascinating insight into the difference between scientific and legal cultures. There are at least three stumbling blocks for DNA data.

* The jury may simply not believe, or perhaps choose to ignore, the DNA data, as evidently happened in the OJ Simpson trial. A fascinating account of the DNA evidence is given by Weir (1995).
* The jury may be led into a false probability argument, the so-called Prosecutor's Fallacy. Suppose a suspect's DNA profile matches the scene-of-crime sample. The Prosecutor's Fallacy confuses two different probabilities:
1. the probability the suspect is innocent, given the match;
2. the probability of a match, given that the suspect is innocent.

The jury should consider the first probability, not the second.

Using Bayesian notation (Box 17.2), with M = match, G = suspect is guilty, I = suspect is innocent, we want to calculate PI | M, and not PM | I. If the suspect were guilty, the samples would necessarily match: PM | G = 1. Population genetic arguments might say there is a 1 in 106 chance that a randomly selected person would have the same profile as the crime sample: PM | I = 10-6. Suppose the guilty person could have been any one of 107 men in the local population. If there is no other evidence to implicate him, he is simply a random member of the population and the prior probability that he is guilty (before considering the DNA evidence) is PG = 10-7. The prior probability that he is innocent is PI = 1–10-7, ~ 1. Baye's theorem tells us that

The prosecutor would no doubt be happy to see the jury use 106 instead of 0.9 for the probability that the suspect is innocent! Given the Bayesian argument, it is clear that a forensic test needs PM | I to be 10-10 or less if it is to be able to convict a suspect on DNA evidence alone.
* Objections may be raised to some of the principles by which DNA-based probabilities are calculated.
1. The multiplicative principle, that the overall probabilities can be obtained by multiplying the individual probability for each band or locus, depends on the assumption that bands are independent. If the population were actually stratified into reproductively isolated groups, each of whom tended to have a particular subset of bands or alleles, the calculation would be misleading. This is serious because it is the multiplicative principle that allows such exceedingly definite likelihoods to be given.
2. For single-locus markers, the probability depends on the gene frequencies. DNA profiling laboratories maintain databases of gene frequencies - but were these determined in an appropriate ethnic group for the case being considered? Taken to extremes, this argument implies that the DNA evidence might identify the criminal as belonging to a particular ethnic group, but would not show which member of the group it was who committed the crime.

These issues have been debated at great length, especially in the American courts. Both objections are valid in principle, but the question is whether they make enough difference to matter. General opinion is that they do not. It would be ironic if courts, seeing opposing expert witnesses giving odds of correct identification differing a million-fold (105:1 versus 1011:1), were to decide that DNA evidence is hopelessly unreliable, and turn instead to eye-witness identification (odds of correct identification < 50:50).top link

PV92 Alu-Polymorphism

PV92 Alu-Polymorphism

The human genome consists to about 50% of transposable DNA elements, so-called "jumping genes". These DNA stretches constitute a major portion of our non-coding or "Junk"-DNA. Some of these elements jump, leaving one location to insert themselves into another. Others are being copied into new locations, resulting in ever growing numbers of insertions across the human genome. Whatever their mechanism, all transposable elements have the potential to affect change through mutations, duplications, or deletions.
One major transposon found in all primates is called Alu. Alu does not jump by itself but gets copied by way of reverse trancriptase encoded by another transposon, L1. Alu has emerged so early in time that it exists in all primates inserted into approximately 1,000,000 different locations across each species' genome. Thus, with the exception of roughly 2,000 human-specific insertions, most human Alu insertions can be found in their homologous positions in the genomes of other primates, too.
PV92 Alu Insertion Polymorphism detects the presence or absence of a "jumping gene" on chromosome 16. This simple genetic system has only three alleles and nine genotypes. Despite this simplicity, allele frequencies vary greatly in different world populations. Alternative explanations about the causes of this variation are consistent with opposing theories of the origins of modern humans "Out of Africa" vs. "Multiregional".

Re: Human Alu PCR Lab Report (200Pt. Due 4/15)

Re: Human Alu PCR Lab Report (200Pt. Due 4/15)

Introduction The Alu element is actually a family of DNA sequences found only in primate genomes. (Dolan, 2001) There 14 known families of the Alu element and the family relationship are based on the gene sequence similarity. (Russel 1998) All 14 families are present in the human genome. The more recently evolved the organism, the more different variations of the Alu element will be present. Alu insertion polymorphism is autosomal markers that reflect both the maternal and paternal history of a population (Nasidze, 2001). They are stable markers that reflect unique evolutionary events. Alu element is a type of gene known as a polymorphic transposon. In humans there are over 1.5 million copies of the 14 subfamilies of the Alu gene with the average similarity being between 85% and 98 % (Makalowski, 1995). The transposon is flanked by inverted short terminal repeats and encode for the protein transposase which allow the gene to be inserted into the DNA (Makalowski, 1995). How an Alu element transposes: (Dolan, 2001)-First, the inserted Alu is transcribed into messenger RNA by the cellular RNA polymerase. -Then, the mRNA is converted to a double-stranded DNA molecule by reverse transcriptase. -Finally, the DNA copy of Alu is integrated into a new chromosomal locus at the site of a single- or double-stranded break. The Alu element is considered to be a class of mobile elements known as Short Interspersed Elements (Nasidze, 2001). The elements are usually about 300 base-pairs in length and are interspersed throughout the genome. For a long period it was though that the Alu element had no function at all and the true function is still unknown. Alu rarely transposes itself into regions critical for DNA function where it would possibly corrupt the code. This leads to the conclusion that the Alu gene family has the ability to modify gene for a genetic advantage. The Alu family of genes may be involved in regulating mutations to deal with stress. (Chu et al, 1998). Most Alu have both of the paired chromosomes with an insertion at the same position. However, many Alu insertion can be dimorphic, meaning that an insertion may be present or absent on each paired chromosome (Dr. Lin’s notes, 2003).

Colin Pitchfork first to be convicted w dna fingerprinting

Colin Pitchfork

Colin Pitchfork - first murder conviction on DNA evidence also clears the prime suspect

Two schoolgirls who were murdered in the small town of Narborough in Leicestershire in 1983 and 1986 sparked a murder hunt that was only to be resolved by a intelligence-led screen, eventually leading to the conviction of a local man - Colin Pitchfork.

In 1983, a 15-year-old schoolgirl was found raped and murdered. A semen sample taken from Lynda Mann’s body was found to belong to a person with type A blood group and an enzyme profile, which matched 10 per cent of the adult male population. At that time, with no other leads or forensic evidence, the murder hunt was eventually wound down.

Three years later, Dawn Ashworth, also 15, was found strangled and sexually assaulted in the same town. Police were convinced the same assailant had committed both murders. Semen samples recovered from Dawn’s body revealed her attacker had the same blood type as Lynda’s murderer.

The prime suspect was a local boy, who after questioning revealed previously unreleased details about Dawn Ashworth’s body. Further questioning led to his confession but he denied any involvement in the first murder – that of Lynda Mann.

Convinced that he had committed both crimes, officers from Leicestershire Constabulary contacted Professor Sir Alec Jeffreys at Leicester University who had developed a technique for creating DNA profiles. Dr Jeffreys - along with Dr Peter Gill and Dr Dave Werrett of the Forensic Science Service (FSS) - had jointly published the first paper on applying DNA profiling to forensic science. Significantly, in 1985, they were the first to demonstrate that DNA could be obtained from crime stains, which proved vital in this case.

Dr Gill said: "I was responsible for developing all of the DNA extraction techniques and demonstrating that it was possible after all to obtain DNA profiles from old stains. The biggest achievement was developing the preferential extraction method to separate sperm from vaginal cells – without this method it would have been difficult to use DNA in rape cases."

Using this technique Dr Jeffreys compared semen samples from both murders against a blood sample from the suspect, which conclusively proved that both girls were killed by the same man, but not the suspect. The police then contacted the FSS to verify Dr Jeffrey’s results and decide which direction to take the investigation.

Peter Gill said: "Since the technique had not been used in criminal casework before, the FSS were asked by the police to confirm Dr Jeffrey’s conclusions. Accordingly, we carried out further tests and indeed demonstrated that the prime suspect could be excluded."

This suspect became the first person in the world to be exonerated of murder through the use of DNA profiling. Professor Alec Jeffreys said " I have no doubt whatsoever that he would have been found guilty had it not been for DNA evidence. That was a remarkable occurrence."

The police then decided to undertake the world’s first DNA intelligence-led screen. All adult males in three villages – a total of 5,000 men - were asked to volunteer and provide blood or saliva samples. Blood grouping was performed and DNA profiling carried out by the FSS on the 10 per cent of men who had the same blood type as the killer.

The murderer almost escaped again by getting a friend to give blood in his name. However, this friend was later overheard talking about the switch and that he’d given his sample masquerading as Colin Pitchfork.

A local baker, Colin Pitchfork was arrested and his DNA profile matched with the semen from both murders. In 1988 he was sentenced to life for the two murders.

An Interview with Sir Alec Jeffreys on DNA fingerprinting

An Interview with Sir Alec Jeffreys

Sir Alec Jeffreys on DNA Profiling and Minisatellites
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Sir Alec Jefferys
"The terminology that we developed for DNA typing using multi-locus probes has been hijacked and in a very misleading way," says sir Alec Jefferys of the University of Leicester.

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GO TO: The InterviewsSince the discovery of the structure of DNA in 1953, knowledge of the composition and organization of the genetic material has accumulated at an astonishing pace. By the early 1980s it had become clear that most human DNA shows very little variation from one person to another. The small percentage that does vary presents enormous potential for fruitful study.

Sir Alec Jeffreys's involvement with mammalian molecular genetics began in 1975, when, as a postdoc, he moved from Oxford University to the University of Amsterdam to work with Dick Flavell. There, the two and their colleagues tried to clone a mammalian single-copy gene. They failed, but in the process managed to develop the Southern blot hybridization technique to the point where they could directly detect single-copy genes–and, in so doing, discovered one of the first examples of introns.

When Jeffreys moved to the University of Leicester in 1977, he chose to change direction completely and study DNA variation and the evolution of gene families. As a result of this work, his laboratory produced one of the first descriptions of RFLPs–restriction fragment length polymorphisms–a common form of variation in human DNA. The aim of the work was to develop a new breed of markers using DNA to track the position of genes. To develop good markers, the researchers needed to find highly variable regions of DNA.

In 1980, another team made one of the major breakthroughs in the study of DNA polymorphism, with their fortuitous discovery of the first "hypervariable" region of human DNA. These regions were found to consist of short tandem sequences repeated over and over again.

In 1983, Jeffreys found that these repeat sequences, dubbed "minisatellites," contain certain "core" sequences. This opened the way for the development of probes, containing the core sequences, for detecting many other such regions of variable DNA. One Monday morning in September 1984, Jeffreys and colleague Vicky Wilson successfully tested the effectiveness of such a probe. "The implications for individual identification and kinship analysis were obvious.... It was clear that these hypervariable DNA patterns offered the promise of a truly individual-specific identification system," Jeffreys wrote later (see A.J. Jeffreys, Am. J. Hum. Genet., 53[1]:1-5, 1993). They had stumbled on DNA fingerprinting, and Jeffreys's life was changed.

Jeffreys, 45, gained a first-class degree in biochemistry from Oxford University in 1972, and his Ph.D., also from Oxford, in 1975. After working in Amsterdam with Flavell between 1975 and 1977, Jeffreys moved to the University of Leicester as a lecturer in genetics and became a full professor in 1987. He was elected a Fellow of the Royal Society (FRS) in 1986.

Science Watch's European correspondent Amir Amirani
spoke with Jeffreys at his laboratory in Leicester.

SW:Your most-cited paper, "Hypervariable minisatellite regions in human DNA," appeared in Nature in 1985. Is the paper highly cited because it's subsequently been used in fingerprinting, or because of the light that the paper shed on the structure of variable DNA?

Jeffreys: The citations, I think, reflect the fact that at the time this was a novel, very powerful generalized technology that could be applied to a wide range of problems in human and nonhuman genetics. The paper described for the first time a general method for getting at large numbers of highly variable regions of human DNA. Also, almost as an accidental by-product, it suggested approaches for not only developing genetic markers for medical genetic research, but for opening up the whole field of forensic DNA typing. And, from the work in that first paper, we could see immediately the potential applications in individual identification and in establishing family relationships–for example in paternity and immigration disputes.
Although it wasn't mentioned in the paper for patenting reasons, we also saw the potential for exactly the same technology being applied to nonhuman species as well. This opened up all sorts of interesting possibilities in animal breeding, conservation biology, ecological genetics, and the like.

SW:Has the potential for the animal work been fulfilled?

Jeffreys: Very much so. That original DNA fingerprinting system, for example, has been used in a fair number of zoos to try and establish family relationships within captive colonies of endangered species of animals and birds, in particular to identify cases of close relationship–those individuals that you do not want to interbreed. The aim, in other words, is to minimize inbreeding and maintain genetic diversity.

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Sir Alec Jeffreys's Most-Cited Papers
Published Since 1985
(Citations updated through 1996)

Rank


Paper
Citations
through 6/94* Citations
through 12/96

Avg. cites per year
through 1996
1 A.J. Jeffreys, V. Wilson, S.L. Thein, "Hypervariable minisatellite regions in human DNA," Nature, 314(6006):67-73, 1985. 1,407 1,778 148
2 A.J. Jeffreys, V. Wilson, S.L. Thein, "Individual-specific fingerprints of human DNA," Nature, 316(6023):76-9, 1985. 669 857 71
3 E. Solomon, R. Voss, V. Hall, W.F. Bodmer, J.R. Jass, A.J. Jeffreys, F.C. Lucibello, I. Patel, S.H. Rider, "Chromosome 5 allele loss in human colorectal carcinomas," Nature, 328(6131):616-9, 1987. 444 485 49
4 A.J. Jeffreys, N.J. Royle, V. Wilson, Z. Wong, "Spontaneous mutation rates to new length alleles at tandem repetitive hypervariable loci in human DNA," Nature, 332(6161):278-81, 1988. 309 434 48
5 Z. Wong, V. Wilson, I. Patel, S. Povey, A.J. Jeffreys, "Characterization of a panel of highly variable minisatellites cloned from human DNA," Ann. Hum. Gen., 51:269-88, 1987. 308 375 38
SOURCE: ISI's Personal Citation Report, 1981-96
*citations reported with original interview

SW:Is there a biological function for mini- and microsatellites?

Jeffreys: That is a very, very tough question. If we look at minisatellites, by and large, there seems to be no obvious biological function. There are a few cases in the human genome, and a fair number of cases outside the human genome, of minisatellites that actually form part of genes. So there are tandem repeated DNA sequences that code for tandem repeated protein sequences. But those are the exception, not the rule. The majority of the minisatellite loci we look at have no obvious function. However, one area that we are very actively examining at the moment is the whole question of how variation arises at these tandem repeat DNA sequences. And that means exploring the mutation processes that go on in sperm and eggs, creating new versions.
What's come out of that is actually a very surprising result in which the mutation process, rather than just reflecting the instability of tandem repeat DNA, seems to be actively controlled by elements external to the tandem repeats. So it looks as though the tandem repeats themselves are not so unstable, but rather the instability is being directed from a locally acting regulator. We also know that the mutation process is astonishingly complex and operates by a process that is wholly unexpected for minisatellites. We call this process "gene conversion," and it involves chunks of DNA being shifted from one allele to another during the mutation process.
We also suspect that, in males, the majority of sperm mutations are specific to the male germline and may be meiotic in origin. This suggests a type of recombinational process, controlled by some elements near the minisatellite, and it looks as if it's meiotic as well. And that really does start raising questions–such as, maybe this mutation process isn't just some sort of accidental artifact of having tandem repeat DNA, but rather reflects some basic biological process going on in the DNA. One of our main jobs now is to explore this in a lot more detail.

SW:And is that of purely theoretical interest, or are there going to be practical implications as well?

Jeffreys: This is basic biology. As to whether there will be practical implications, I don't know. However, in the course of our investigations, we've developed various new strategies for detecting new mutations in human DNA, and this does, in principle, offer practical applications. By mutations, I don't mean, for example, a cystic fibrosis mutation, which is actually not a mutation at all but a variant that's been around for thousands of years. I'm talking about new mutations–actually catching DNA at the point where it has altered its structure. If we can develop methods for measuring mutation rate in an individual undergoing this process–and this is one of my main interests–then we can start asking basic questions about environmental agents, such as ionizing radiation, which might impact upon the mutation rate.

SW:Fingerprinting has been subject to a lot of controversy, something you have alluded to in some of your papers. Do you personally have any reservations about its reliability?

Jeffreys: Before I answer that, we must clear up a point on semantics, and this is not trivial. The original DNA fingerprinting system we developed, which for technical reasons is not that useful in forensic identification, produces patterns that are idiotypes–they are, for all intents and purposes, completely unique to an individual, except for identical twins. There's no serious dispute about that in my view. Unfortunately, the second generation of DNA typing systems–which is DNA profiling using single-locus probes–do not produce individual-specific patterns test by test. Even with a typical battery of five different tests, they produce patterns where unrelated people are most unlikely, in fact extremely unlikely, to share the same pattern. However, when you come to close relatives, brothers and sisters, there is a real chance, in fact about 1 in 4 to the power of 5 chance, of a brother and sister match, which is 1 in 1,000.
So, for every 1,000 sibling pairs, over five probes, you find a complete match. So they are not DNA fingerprints, not unique to an individual. However, their variability among unrelated people is pretty spectacular over five tests. Unfortunately–and particularly in the United States–the term "DNA fingerprinting," which we specifically apply to the original multi-locus system in which we look at scores of markers, has been corrupted to be used in almost any DNA typing system. That has created a problem in court, because DNA profiling does not produce DNA fingerprints, but if you call them DNA fingerprints, then the defense lawyer can stand up in court and say, "This is misleading," and that's quite right.
So this is a semantic problem, but a serious one. Basically, the terminology that we developed for DNA typing using multi-locus probes has been hijacked, and in a misleading way. Now, if we get rid of that semantic part, we can ask how valid is the huge amount of debate that's gone on about the reliability of DNA profiling? In the early days, in particular, there was real cause for concern. Some of the laboratories doing this work were carrying out real forensic analysis with technology that had been very poorly validated and hadn't been standardized.
I think that this issue has been largely addressed now, through quality controls, the adoption of standard operating conditions, blind proficiency trials, and so on. For DNA profiling, the real source of debate now relates to how one estimates the rarity of a set of DNA profiles out in the population, and how one presents that evidence in court. If you say that a DNA profile of a forensic sample matches a given suspect and is very rare in the population, then that, depending on the context, can be pretty damning evidence.

SW:Let's turn to your current research interests.

Jeffreys: My current interests are in exploring the basics of mutation of human minisatellites. We now know they are mutating by processes that are totally unexpected. These processes are probably of biological significance and may shed light on another fascinating area of human genetics: the whole area of triplet repeat instability diseases. These are microsatellites that go horribly unstable and cause neurological disease, such as Huntington's chorea, myotonic dystrophy, fragile x syndrome, and so on. These are basically microsatellites, which suddenly become highly unstable, increase their repeat number, become very long, and wreck nearby genes.
And again, for technical reasons, it's not easy to explore the details of the mutation process going on there, but we can explore in great detail the mutation process going on in minisatellites. We can use a whole battery of techniques that we've developed, which explore these bizarre mutation processes. It's not impossible, though far from guaranteed, that what we discover in minisatellites may actually be applicable to these inherited diseases.
One sort of science-fiction scenario would be this: let's suppose that what happens in minisatellites also applies to these unstable microsatellites. In other words, instability is conferred upon the array by flanking DNA, which, we suspect, is activating an allele for mutation. It's basically switching an allele on, perhaps by introducing some kind of DNA damage, such as a double strand break into the DNA.
Now, if that is true for these neurological diseases, and these diseases manifest because of this instability, one could conceivably think of some therapy aimed at blocking that mutation initiation. That's wild fantasy, but who knows? After all, gene therapy was fairly wild fantasy 20 years ago.
Another area in which we're very much involved is developing new approaches to DNA typing. We've been heavily involved over the last couple of years in an approach called digital DNA typing, where you get a digital readout from the DNA rather than the usual sort of band length measurements in DNA profiling. And that has first of all revealed minisatellites as by far the most variable loci in the human genome. The typical minisatellite has, for example, 100 million different alleles worldwide, and that is astonishingly variable. And that in turn may give us some rather interesting markers for studying recent events in human evolution–by looking at these allele structure and how they've changed over time, how they differ between recently split populations, and so on.

SW:The field of DNA fingerprinting is relatively new. How do you expect this technique to develop, and how do you expect DNA structure studies overall to progress?

Jeffreys: The field of DNA fingerprinting has diversified to the point of incoherence. It's no longer a single unified field. For example, back in 1987-88, when we had our first congress on DNA fingerprinting, the thing that welded it together was that everybody was playing around with minisatellites, DNA fingerprinting, and DNA profiling.
What's happened since then, of course, is the advent of DNA amplification by polymerase chain reaction, or PCR. This means, first of all, that there is little doubt that in forensic DNA typing within the next few years all the classic systems of DNA fingerprinting and DNA profiling will be totally replaced by PCR-driven systems. Such systems have their powers and their weaknesses as well–contamination and the like. But the advantage of PCR is that it offers great sensitivity, potential for automation, lower costs, and information that is much less ambiguous in terms of a DNA profiling result.
Now, what the ultimate DNA forensic typing system will be, I don't know. But to suppose that we've actually arrived there now is naive in the extreme, bearing in mind that information about PCR, or user-friendly PCR, was published only seven years ago. To pretend that we've gone from that to the ultimate DNA typing system is nonsense. There'll be other ones coming along, and that actually creates a major problem for the forensic scientist who is interested in databasing, because once you go in for very large-scale databasing of many thousands of people–you are trapped in that technology. You cannot change that technology because you've got to retype everybody in the database if you do. So the drive towards databasing, I think, is in fundamental conflict with the still rapidly evolving field of forensic DNA typing–the technology itself.
So I see all kinds of developments on the forensic front. People may actually come up with what everyone is talking about: DNA chips, oligonucleotide chips that will be used to interrogate PCR reactions. At the moment, these are not chips at all in the electronic sense. If one could, however, create a chip in which an oligonucleotide could detect and transduce the detection of a product (such as a PCR product) into an electronic signal, that would open up not just forensic typing, but DNA typing, medical diagnostics, and just about everything else one can think of.

Rare blood donor registry; need of the hour - Cover Story - Express Healthcare Management

Rare blood donor registry; need of the hour - Cover Story - Express Healthcare Management

Rare blood donor registry; need of the hour

Shardul Nautiyal - Mumbai

Reena Mathews lost blood heavily during delivery and urgently required blood. A sample of her blood was sent to the blood bank for matching. The red cells grouped like O group, while her serum reacted with all O group cells available in the blood bank during cross-matching or compatibility test, making the blood bank official realise that the lady may be carrying the rare Bombay Blood group.

Experts inform that a rare genotype (blood group) of people was detected in Mumbai, a few decades back, who neither had A, AB, B or O group. This rare genotype was labelled as the Bombay Blood Group. If a Bombay Blood Group recipient is not transfused the blood of a Bombay Blood Group person, it can lead to a haemolytic transfusion reaction, which can be fatal and lead to death.

According to Dr Anand Deshpande, consultant, transfusion medicine and haematology, Hinduja Hospital, “Transfusion of ‘O’ group blood to these persons would result in immediate red cell lysis because of the presence of anti H antibodies in the serum of Bombay Blood Group patients. Therefore blood from only a Bombay Blood Group individual should be transfused to a Bombay Blood Group recipient.”

Studies reveal that this is due to the absence of the H substance (antigen) in the red cells. The absence of the H substance is attributed to the deficiency of the enzyme fucosyl transferase. The Bombay Blood Group phenotypes lack H antigen in the red cells and have anti-H in the serum.

Says Dr Maya Parihar Malhotra, blood bank in-charge, Bombay Hospital, “Family studies have shown that the Bombay phenotype, called as Oh, is due to the presence in homozygous state of a rare recessive gene.”

The precursor protein from which the blood group proteins are formed is termed as the H substance. This is bio-chemically produced by the binding of Fucose to the surface glycoproteins, the process being catalysed by Fucosyl transferase. If N-acetyl galactosamine binds to the H substance, it forms the blood group A, whereas if galactose binds to it, it forms the group B. Absence of any binding substance produces the O blood group.

Studies reveal that all human red blood cells with exceedingly rare exceptions carry the red cell H antigen. It is present in greatest amount on type O red cells and least on type A1B cells. The H antigen is an intermediate stage in the production of the A and B antigens. The individuals with the so-called Bombay phenotype are recognised with the presence of anti-H in the serum, in addition to anti-A and anti-B, as in type O persons.

Experts say that if proper blood grouping or testing practices is not followed, it can lead to people with Bombay blood group not being detected. According to Dr Mukesh Desai, haematologist, H N Hospital, “During cell grouping or routine grouping, Bombay Blood Group would be categorised as O group because they wouldn’t show any reaction to anti-A and anti-B antibodies just like a normal O group. When a cross matching with different blood bags of O group is done, then it would show cross-reactivity or incompatibility. Therefore Reverse grouping or Serum grouping has to be performed to detect the Bombay Blood group.”

“Other issues related to Bombay Blood Group is that blood is incompatible with all A, B and O donors. In routine forward grouping, this blood group would give reaction as an ’O’ blood group where as in serum grouping it would show reaction with ’O’ cells due to the presence of anti H in their serum,” says Dr Deshpande.

Most of the cases once detected are registered at Institute of Immuno-haematology (IIH)) for further studies as well as for availability of information regarding the donors of this group.

According to Dr Kanjaksha Ghosh, deputy director, IIH, “Since Bombay Blood Group is the rarest of the rare group, it is desirable to develop cryopreservation facilities for rare donor units. Every blood bank can easily maintain a rare blood type donor file from amongst their regular voluntary donors.”

“If these blood banks can borrow or exchange rare blood units in times of need, lot of problems related to rare blood groups like Bombay Blood Group can be solved. This is only possible if each blood bank has a large number of committed regular voluntary donors,” added Dr Ghosh.

“The wrong notion among people need to be dispelled that of the possibility of getting infections like HIV1 through blood donation. The public need to be informed that there is no way a donor can get such infection through blood donation,” opined Dr Ghosh.


Cell Grouping

Serum Grouping

Interpretation
Anti A

Anti B

Anti AB

A cells

B cells

O cells

+

-

+

-

+

-

A
-

+

+

+

-

-

B
+

+

+

-

-

-

AB
-

-

-

+

+

-

O
-

-

-

+

+

+

Bombay Blood Group

Diagram As shown in above diagram, cell grouping is carried out using anti A, anti B and anti AB commercially available sera. Serum grouping is carried out using A cells, B cells and O cells.

blood and vampires

Satantra's Cathedral

Vampires in Literature by Sarah Abrahams
Vampire: Historical Origins

The image of the vampire has a long and complicated history. Many different cultures have a notion of a vampire-like character within their respective mythologies. In order to understand a modern critique of the vampire as a figure representing an array of subverted meanings, it might first be appropriate to provide a summary of vampire figures in a variety of cultural perspectives, noting both their differences and similarities. The vampire image, in a more basic sense, represents our human preoccupation with death, blood and darkness. With the advent of modern science, many of the mystical beliefs surrounding blood were abandoned. "Blood was the sight of death" (Leatherdale 16). "The concept of the vampire is founded upon two precepts: the belief in life after death, and the magical power of blood" (Leatherdale 15). The connection between the mysticism of blood and death is one founded upon observation. "Blood was the sight of death" (Leatherdale 16).
Blood

Blood has throughout history served many purposes. It was believed that both "human strength and health resided within blood" (Leatherdale 16). Blood had the power to sustain life as well as "[consuming] [it] could restore, rejuvenate, bring back life" (Leatherdale 17). The connection between blood and life is a complicated one. Blood is present in female menstruation, a symbol of the transition into adulthood, as well as in the breaking of the hymen. Blood is also present in the birth of a child. Clearly this has significance within the vampire legend.
The Living Dead

Along with notions of the magical properties of blood lied a fear in the 'living dead.' As humans have known that death is inevitable, the land of the dead seemed to be a place apart, governed by its own customs and laws of nature, which the living could not penetrate but to which they were inexorably drawn in the course of time. In certain periods of history, especially periods of disease (the plague, and more recently AIDS) humans became even more aware of the presence of death within their own communities. Because in some respects, death is the inexplicable other, humans seem to have to formed a sort of dualism where the living and the world in which they operate appears on one side and the dead the world in which they operate appears on the other.
The near universally-held belief in these two supposed laws of nature-the rejuvenating power of blood and the presumption of life after death-meant that the product of their combination (the vampire) was equally universal. The presence of the vampire can be located within a wide array of cultures.
The Transcultural Presence of the Vampire

* ·Chinese tales spoke of blood-sucking creatures that were green, covered with mold, and which had a propensity to glow in the dark.
* The Melanesian talamaur-known as the soul of the dead which preyed on the ebbing vitality of the dying.
* In India, Kali is revered as a blood-sucking mother goddess of disease, war, and death. Siva is identified with ghoulish (flesh-eating) propensities.
* In Africa, the Ashanti's asambosam, likes to suck blood through the thumbs of the sleeping. Rather than feet, asabosam stands on a pair of books.
* West Indies lore contains the loogaroo (from the French expression for werewolf- loup garou) who, disguised as an old woman, in a pact with the devil, sheds her skin and reforms as a blob of light in order to draw blood .
* A jaracara, in Brazil, resembles a snake and seeks either blood or breast milk.
* In Europe, the vampire concept seems to have developed from idea of the succubus-a female entity who would seduce young men in their sleep and "withdraw their vital fluids at the moment of peak distraction during climax." The incubus, the male counterpart associated with the devil, "would impregnate suitable female victims, such as witches" (Leatherdale 20). It is interesting to note here that only the female vampire acts out the withdrawal of essential liquid. The male vampire only serves to assist in the creation of new demonic creatures.

Though I will focus on the European history and literature of the vampire, there seems to be vampiric figures (in some form) in the folklore or religion of almost every culture.
The Vampire in Victorian Literature

In the Victorian period, the image of the vampire became a popular one. Bram Stoker's Dracula, published in 1897, most centrally explored the idea of vampirism as an interesting and complex image within literature. From Stoker's work came the figure of Dracula, who's continued portrayal in literature and film is proof of transcendence from mere literary figure to cultural icon. The Bronte's Jane Eyre and Wuthering Heights both make direct allusion to the vampire. In Jane Eyre particularly, the character of Bertha Mason is described with reference to vampirism.
Vlad Tepes- Vlad The Impaler

There has been much speculation over the creation of the vampire Dracula. Historicists have discovered the life of Vlad Tepes (Vlad the Impaler) as a potential inspiration for Bram Stoker's work and other European vampire lore. Bram Stoker occupies a large portion of Dracula to a detailed description of Romania and the conflicts surrounding the Turks. The story presented by Stoker is Vlad Tepes', other similarities can be located elsewhere in Tepes' military flair for violence and the absolutely gory nature of his destruction of his country's enemies. One scene depicted by artists, show Vlad the Impaler seated and dining among the skewered corpses of his enemies. Tepes' connections to the character of Dracula, though, are otherwise difficult to discern, though his political exile, like Dracula's societal exile, may be another area of potential similarity.
Representations of Vampirism and the Critics

There has been a long history of critical exploration in attempts to undercover the symbolic significance of the vampire. In terms of the girlhood narrative and issues of women in literature, three main themes appear most prominent and most often, all of which are based in a form of psychoanalytic theory.

Vampirism has long had associations with female sexuality. In Bram Stoker's Dracula, for example, Dracula's vampiric women epitomize the threat/fear of unleashed female sexuality. In the Victorian period, specifically with the appearance of what was called the "New Woman" (Victorian women who were more financially and emotionally independent from their families and men), the dangers of female sexuality became an issue of urgency. Traditionalists of the period worried that with women's newfound independence, these women would be more likely to explore their sexuality in ways that were considered inappropriate for women.

When one examines the specifics of dangerous female sexuality, one notices a fear of excess. The possibility of women having the potential to experience an eroticized way of life and a sexuality not limited to genital contact becomes extremely dangerous to patriarchal power. The ability for women to enact a polymorphous type of sexuality enables them, and empowers them. In order for patriarchal systems to remain in control, they insist on labeling and attempting to teach society that this form of sexuality is unhealthy and abnormal. Indeed, much of the image of the vampire deals with issues of taboo nature.

The second theme most often discussed in recent literature criticism of works containing vampire (or vampire-like) figures is the notion of gender inversion and homoeroticism. Much has been speculated with reference to Bram Stoker's Dracula, as to the autobiographical nature of the work considering the connection of the Oscar Wilde trial (with which Bram Stoker was quite close) and Dracula's creation. Considering the nearly simultaneous occurrence of the two, it seems logical to examine the vampire as a possible figure of homosexuality. Following Freud's theories, some have argued for the vampire as a sort of infant, stuck permanently in the oral phase and forced to attempt adult sexuality through it.

Images of incest are another theme of criticism of vampires in literature. In Ken Gelder's Reading the Vampire, he explains this psychosexual reading as an "ambivalent impulse of the child towards its mother" (Gelder 68). If one accepts the idea that Bram Stoker's Dracula can be interpreted through a reading of the psychosexual, the interpretations made by critics such as Twitchell, Astle, and Jackson all support the "re-enactment of that killing of the primal father who has kept all the women to himself" (Gelder 68). In Phyllis A Roth's piece "Suddenly Sexual Women in Bram Stoker's Dracula," this intepretation is extended to include what Roth explains as "the hatred of the mother"-"the Oedipal rivalry among sons and between the son and the father for the affections of the mother" (Roth 60).

All three themes reiterate one central theme-the vampire as highly sexual. In terms of Jane Eyre, one can see how Bertha's description as vampire-like makes sense considering her punishment for "uncivilized" and unladylike sexuality. In Wuthering Heights also, as a symbol of the undead, the vampire image appears in reference to Catherine, the ghost who haunts Heathcliff for his passionate cruelty.
Vampires in Modern Fiction

The vampire continues to be a popular image in fiction. The popularity of Anne Rice's Vampire Chronicles have spawned a group of modern day vampire groupies who gather near Rice's home in New Orleans. In relation to gender, Rice interprets the vampire as a definite figure of homosexuality. Within a personal and bibliographical context, it has been said that Rice's Vampire Chronicles were the result of the loss of a child. In dealing with her grief, she became obsessed with ideas of the undead and of the possibilities of eternal life. In Salem Lot, Stephen King continues the vampires in literature tradition. In King's work, though, he acknowledges the current popularity of the horror genre by telling the tale of a group of boys in modern America. As a small American town, King uses Salem's Lot as a site of particular vulnerability to the threat of vampires. Its isolation from larger society makes it an easy target.
Conclusion

As one uncovers the web-like variety in vampire related criticism, it becomes more and more clear that the reason the vampire image has remained so powerful is in it's ability to transcend a single meaning. In this way, the vampire's fluidity and multifaceted nature allows malleability in interpretations that fit all different sorts of critical thought. Most recently, in the wake of the AIDS epidemic and the gay rights movement, vampires have symbolized (for some) homosexual desire at its most potentially dangerous. Yet, when analyzed within the larger framework of vampire criticism, this seems to be only a part of a developmental and historical process of naming the vampire as symbolic of a variety of societal issues. It continues to be unknown what shape vampire criticism will manifest itself next-and it is this difficulty (or ability) of the vampire figure which continues to keep interest alive (or in vampiric terms, to remain undead).
Bibliography

* Auerbach, Nina. Our Vampires, Ourselves. Chicago: U of Chicago P. 1995.
* Botting, Fred. Gothic. from The New Critical Idiom Series. John Drakakis, ed. London: Routledge, 1996.
* Carter, Margaret L.. ed. The Vampire in Literature: A Critical Bibliography. Ann Arbor: UMI Research Press, 1989.
* "Stoker's Vampire of the Mind." in Specter or Delusion? The Supernatural in Gothic Fiction. Ann Arbor: UMI Research Press, 1987.
* Clemens, Valdine. "The Reptilian Brain at the Fin de Siecle: Dracula" The Return of the Repressed: Gothic Horror from The Castle of Otranto to Alien. NY: State University of NY Press, 1999.
* Craft, Christopher. "Kiss Me With Those Red Lips: Gender Inversion in Bram Stoker's Dracula." in Dracula: Contemporary Critical Essays. Glennis Byron, ed. NY: St. Martin's P, 1999.
* Frayling, Christopher. "Haemosexuality" in Vampyres: Lord Byron to Count Dracula. London: Faber and Faber, 1992.
* Gelder, Ken. Reading The Vampire. London: Routledge, 1994.
* Glover, David. "Sexual Aeternitatis" in Vampires, Mummies and Liberals: Bram Stoker and the Politics of Popular Fiction. Durham: Duke U P, 1996.
* Hendershot, Cyndy. "Vampire and Replicant: The One Sex Body in a Two-Sexed World." Science Fiction Studies 22 (1995) 373-98
* Leatherdale, Clive. Dracula: The Novel and the Legend. A Study of Bram Stoker's Gothic Masterpiece. Northamptonshire: Aquarian Press, 1993.
* Putner, David. "Dracula and Taboo" in Dracula: Contemporary Critical Essays. Glennis Byron, ed. NY: St. Martin's P, 1999.
* Roth, Phyllis. "Suddenly Sexual Women in Bram Stoker's Dracula" in Dracula: Contemporary Critical Essays. Glennis Byron, ed. NY: St. Martin's P, 1999.
* Schaffer, Talia. "A Wilde Desire Took Me: The Homoerotic History of Dracula" ELH 61 (1994) 381-425.
* Senf, Carol. "Dracula: Stoker's Response to the New Woman" in Victorian Studies 26:1 Autumn 1982.
* The Vampire in Nineteenth-Century English Literature. Bowling Green: Bowling Green State U Popular Press, 1988.
* Showalterm Elaine. Sexual Anarchy: Gender and Culture at the Fin de Siecle. New York: Viking- 1990
* Spear, Jeffrey L. "Gender and Sexual Dis-Ease in Dracula" in Virginal Sexuality and Textuality in Victorian Literature. Lloyd Davis, ed.. NY: State Univ of NY Press, 1993.
* Wolf, Leonard. Dracula: The Connoisseur's Guide. NY: Broadway Books, 1997.

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Red Gold | PBS

biometric

Brain Fingerprinting - Ruled Admissable

Brain Fingerprinting - Ruled Admissable

Brain Fingerprinting Testing Ruled Admissible in Court

On March 5, 2001 Pottawattamie County, Iowa District Court Judge Tim O'Grady ruled that Brain Fingerprinting® testing is admissible in court. Dr. Farwell conducted a Brain Fingerprinting test on Terry Harrington, who is serving a life sentence in Iowa for a 1977 murder. The test showed that the record stored in Harrington's brain did not match the crime scene and did match the alibi. Harrington filed a petition for a new trial based on newly discovered evidence, including the Brain Fingerprinting test. On February 26, 2003 the Iowa Supreme Court reversed his murder conviction and ordered a new trial. The Iowa Supreme Court left undisturbed the law of the case establishing the admissibility of the Brain Fingerprinting evidence.

In a Brain Fingerprinting test, words, pictures or sounds describing salient features of a crime are presented by a computer, along with other, irrelevant information, that would be equally plausible for an innocent subject. Items are chosen that would be known only to the perpetrator and to investigators, but not to the public or to an innocent suspect. The subject is told which features he will see (e.g., the murder weapon), but is not told which item is correct (e. g, gun, knife, or baseball bat). When a subject recognizes something as significant in the current context, the brain emits a specific brain response. If the record of the crime is stored in the subject's brain, this response appears when the subject recognizes the correct, relevant items. If not, then the response is absent. A computerized mathematical analysis of the data determines whether or not the subject has knowledge of the salient details of the crime.

Just as a personal computer emits a characteristic sound whenever its central processing unit is transferring information to or from or the hard drive, the human brain emits a characteristic electrical brain wave response, known as a P300 and a MERMER (memory and encoding related multifaceted electroencephalographic response), whenever the subject responds to a known stimulus. The P300 electrical brain wave response, one aspect of the larger MERMER response discovered and patented by Dr. Farwell, is widely known and accepted in the scientific community. There have been hundreds of studies conducted and articles published on it over the past thirty-plus years. The MERMER, a longer and more complex response than the P300, comprises a P300 response, which is electrical events occurring 300 to 800 milliseconds after the stimulus, and additional data occurring more than 800 milliseconds after the stimulus. While a P300 shows only a peak electrical response, a MERMER has both a peak and a valley.

In order to be admissible under the prevailing Daubert standard, the science utilized in a technology is evaluated based on the following four criteria: (The Iowa courts are not bound by the Daubert criteria used in the federal courts, but they do use them when determining the admissibility of novel scientific evidence.)

1. Has the science been tested?
2. Has the science been peer reviewed and published?
3. Is the science accurate?
4. Is the science well accepted in the scientific community?

The judge ruled that Brain Fingerprinting testing met all four of the legal requirements for being admitted as valid scientific evidence. The ruling stated: "The test is based on a 'P300 effect.'… "The P300 effect has been studied by psycho-physiologists…The P300 effect has been recognized for nearly twenty years. The P300 effect has been subject to testing and peer review in the scientific community. The consensus in the community of psycho-physiologists is that the P300 effect is valid…."

The judge also ruled that "The evidence resulting from Harrington's 'brain fingerprinting' test… is newly discovered" and "material to the issues in the case," and thus meets the standard for being considered in a petition for a new trial.

The Brain Fingerprinting test on Harrington showed that the record stored in his brain did not match the crime, and did match his alibi. This is similar a finding that Harrington's fingerprints or DNA did not match the fingerprints or DNA at the crime scene, and did match those at the scene of the alibi.

Dr. Farwell conducted two different analyses of the data on Harrington. Both yielded the same conclusion. He performed the test and the analyses in strict accordance with the P300 science that has been extensively researched and is well accepted in the scientific community. In another analysis, Dr. Farwell used more state-of-the-art techniques, including the MERMER, which, though arguably more accurate, do not yet have the same level of acceptance as the P300.

After obtaining the results of the Brain Fingerprinting test, Dr. Farwell located the only alleged witness to the crime, Kevin Hughes. When Dr. Farwell confronted him with the Brain Fingerprinting test results exonerating Harrington, Hughes admitted that he had lied at Harrington's trial. He stated under oath that he had made up the story about Harrington committing the crime to avoid being prosecuted himself. Harrington’s attorney used Hughes' recantation along with Brain Fingerprinting test findings as evidence in his post-conviction petition for a new trial.

The Harrington case was about as difficult a case as could be envisioned for the Brain Fingerprinting system. The day after the crime, the perpetrator knows all about the crime and an innocent suspect knows nothing. Scientists could have readily constructed a Brain Fingerprinting test to distinguish between the two, if the technique had been invented at the time. Later, in his trial, Harrington was exposed to extensive information about the crime. This made it difficult after the trial to produce salient features of the crime that he would know only if he had committed the crime. Twenty-three years after the crime was committed, through examination of court documents, police reports, witness interviews, crime-scene photos and an investigation of crime scene itself, Dr. Farwell was able to structure a Brain Fingerprinting test that tested Harrington's brain for evidence of salient features of the crime, features that he claimed not to know because he was not there. The test showed that Harrington's brain in fact did not contain a record of these salient features of the crime. A second test conducted by Dr. Farwell showed that Harrington's brain did contain the details of his alibi.

As with other scientific evidence, Brain Fingerprinting testing does not prove guilt or innocence per se. It provides information about what is stored in the suspect's brain. A judge or jury can utilize this information in making the legal determination of guilt or innocence. The weight of the Brain Fingerprinting test evidence will be evaluated along with other evidence by a higher court in their consideration of Harrington's appeal. If a higher court finds that this and other evidence probably would have changed the result of the original trial if it had been known at the time, then Harrington will be granted a new trial.

"I believe the court’s admission of Brain Fingerprinting test results into evidence is a landmark in forensic science," said Dr. Farwell. "Innocent persons have a new technology to aid in their exoneration, and law enforcement has a new weapon with which to convict perpetrators. In the future we can use this technology to find out the truth early in a case. This will save innocent suspects from a traumatic investigation and trial, and potentially from false conviction and punishment. By allowing law enforcement to focus on the actual perpetrators, it will also save time and money in law enforcement.”

"I believe that in time we will be able to virtually eliminate false convictions through Brain Fingerprinting tests and other scientific technologies such as DNA and fingerprints. This new scientific technology will also allow us to significantly increase the number of actual criminals brought to justice."

Since the hearing, Dr. Farwell and Sharon Smith of the FBI have published their research on the MERMER in the prestigious peer-reviewed Journal of Forensic Sciences, January 2001.

Hundreds of scientific studies on this technology from dozens of laboratories have been published in the peer-reviewed scientific literature. Dr. Farwell has applied the technique not only in rigorous laboratory studies but also in over 100 real-life cases. Dr. Farwell and then FBI scientist Dr. Drew Richardson used the Brain Fingerprinting system to detect with 100% accuracy which people in a group were FBI agents and which were not by measuring brain responses to items only an FBI agent would recognize. Brain Fingerprinting testing was also 100% accurate in three studies Dr. Farwell conducted for a US intelligence agency and for the US Navy.

The Brain Fingerprinting system tests for knowledge of salient features of a crime stored in the brain. Scientists know that we don't remember everything, but we do remember significant features of major events — like committing a serious crime. By scientifically determining what is stored in a suspect's brain, Brain Fingerprinting testing provides evidence that can be used by judges and juries in making a determination as to whether the suspect committed the crime or not.

The History of Fingerprints

What is Higher Order Thinking?

Bloom's Taxonomy of Thinking Skills

Different learning styles

Problem Based Learning

Problem Based Learning: "Problem Based Learning"

Problem Based Learning

If asked, most educators would agree that one essential goal of education is the development of students who are effective problem solvers for the Information Literacy Age. Most reports, such as the national SCANS (Survey of Necessary and Comprehensive Skills) and Goals 2000 documents, recommend such instruction. Most school goal statements allude to the need for critical thinking and problem solving skills. Recent California Frameworks in Mathematics and Science reflect consensus on this educational goal. But often such instruction in problem solving takes the approach of teaching models to students to apply to neat case studies rather than the messy problems of a real world.

Research indicates that critical thinking and problem solving skills are not typically addressed in the classroom. A number of studies indicate that in the typical classroom, 85% of teacher questions are at the recall or simple comprehension level. Questions that elicit synthesis and evaluative skills of thinking are rarely asked. The media portrays teachers as asking such simple, mindless questions in movies such as "Ferris Bueller's Day Off" and "Dead Poet's Society".

In Problem Based Learning (PBL) environments, students act as professionals and confront problems as they occur - with fuzzy edges, insufficient information, and a need to determine the best solution possible by a given date. This is the manner in which engineers, doctors, and, yes, even teachers, approach problem solving, unlike many classrooms where teachers are the "sage on the stage" and guide students to neat solutions to contrived problems.

What is Problem Based Learning?

Problem Based Learning is a curriculum development and delivery system that recognizes the need to develop problem solving skills as well as the necessity of helping students to acquire necessary knowledge and skills. Indeed, the first application of PBL was in medical schools which rigorously test the knowledge base of graduates. PBL utilizes real world problems, not hypothetical case studies with neat, convergent outcomes. It is in the process of struggling with actual problems that students learn both content and critical thinking skills.

Problem based learning thus has several distinct characteristics which may be identified and utilized in designing such curriculum. These are:

1. Reliance on problems to drive the curriculum - the problems do not test skills; they assist in development of the skills themselves.
2. The problems are truly ill-structured - there is not meant to be one solution, and as new information is gathered in a reiterative process, perception of the problem, and thus the solution, changes.
3. Students solve the problems - teachers are coaches and facilitators.
4. Students are only given guidelines for how to approach problems - there is no one formula for student approaches to the problem.
5. Authentic, performance based assessment - is a seamless part and end of the instruction.

(Adapted from Stepien, W.J. and Gallagher, S.A. 1993. "Problem-based Learning: As Authentic as it Gets." Educational Leadership. 50(7) 25-8 and Barrows, H. (1985) Designing a Problem Based Curriculum for the Pre-Clinical Years.

Problem Based Learning assists students to solve problems by the process of continually encountering the type of ill-structured problems confronted by adults or practicing professionals. As with information literacy, PBL develops students who can:

* Clearly define a problem
* Develop alternative hypotheses
* Access, evaluate, and utilize data from a variety of sources
* Alter hypotheses given new information
* Develop clearly stated solutions that fit the problem and its inherent conditions, based upon information and clearly explicated reasoning

Students with such ingrained skill are well prepared for occupations which rarely have a supervisor who has time, inclination, or knowledge to tell the worker what to do. They are also well prepared for the explosion of knowledge which gluts the world today.

Stages in Problem Based Learning

In the PBL curriculum, one may note three distinct phases of operation by students. Whether gathering knowledge through a variety of sources on the Internet, through print sources, or by speaking with experts, these stages explicated below are characteristic of PBL. Each step in the process is "hot linked" to a sample lesson developed by a SCORE Teacher on Assignment.

Stage 1: Encountering and Defining the Problem

Students are confronted with a real world scenario through authentic looking correspondence. Students may be asked to present to the Ancient World Architectural Review Board regarding their perspective about how and why great ancient monuments were built. They may ask some basic questions such as :

* What do I know already about this problem or question?
* What do I need to know to effectively address this problem or question?
* What resources can I access to determine a proposed solution or hypothesis?

At this point, a very focused Problem Statement is needed, though that statement will be altered as new information is accessed and understood.

Stage 2: Accessing, Evaluating and Utilizing information

Once they have clearly defined the problem, students might access print, human, or electronic information resources. In the case of the Southern Illinois Medical School, professors may be interviewed or medical texts examined. In the case of a city plan, calls to human resources such as the town manager or staff engineers might be of use. The Internet can be a focal point of research when a problem is constructed with that purpose. In the case of the sample problem, students may find a rich diversity of perspectives and resources preparatory to phase 3. Part of any problem is evaluation of the resource. How current is it? How credible and accurate is it? Is there any reason to suspect bias in the source? When utilizing the information, students must carefully appraise the worth of the sources they have accessed. If evaluating sites which theorize about these monuments and how and why they were built, students must carefully note and evaluate the accuracy and credibility of information posted at that site.

Stage 3: Synthesis and Performance

In this stage, students construct a solution to the problem. Students may create a multi-media production, a presentation to a body such as the U.N. Commission on Human Rights or the Ancient World Architectural Review Board, or a more traditional written paper focused around an essential question. In all cases, the students must re-organize the information is new ways. This is unlike an assignment which asks them to " make a report about the Palestinians and Israelis." This latter leads to use of the Internet as if it were a giant cyberspace encyclopedia. An assignment which asks students to propose a solution to the conflict between the Palestinian people and the Israelis involves a question which forces re-organization of information and consideration of perspectives.

Problems in Implementation

Cultural change is required to implement PBL. Students trained in the more traditional model of teaching, which features the teacher as "sage on the stage" and disseminator of knowledge, will experience culture shock of a sort. Students will wish to know expectations for a high grade. Though constructing a rubric with a teacher may allay fears, there is initial suspicion of the new approach.

Students must also learn to be part of the group. As with real life tasks, one person cannot conduct all research and make the entire presentation of the problem solution. Complaints about "hitchhikers" (those in the group who do not pull their own weight) will be heard from hard working students and their parents.

Teachers also experience major adjustments. More preliminary work must be done to design the problem and to ensure that there are enough materials available (in print, online, and through human resources) for this resource's ravenous approach. They must learn to construct problems that assist students to learn appropriate skills and knowledge. And they must learn to facilitate, rather than direct, student learning.

The Rewards

Though change from a teacher-centered to a problem and project based environment causes discomfort, those that have made the transition speak of new energy and enthusiasm for their classes. Students praise challenging tasks that prepare them for learning. For more information, see the Problem Based Learning online resources below:

* The University of Delaware has numerous articles about PBL including teaching art, science, and other courses. A good teacher resource. http://www.udel.edu/pbl/
* Howard Barrows, Southern Illinois School of Medicine (A medically focused analysis of PBL.) http://www.pbli.org
* Illinois Math and Science Academy (Includes K-12 applications in various disciplines.) http://www.imsa.edu/

If you have further questions about PBL, please email Bob Benoit of the Butte County Office of Education at bbenoit@bcoe.butte.k12.ca.us. Bob has directed a PBL project which included six high schools and 30 teachers over the last four years.

A Selected Problem Based Bibliography
Books:

* Barrows, H. (1994) Practice-Based Learning: Problem-Based Learning Applied to Medical Education. Springfield, Il: Southern Illinois University School of Medicine
* Barrows, H. (1985) Designing a Problem Based Curriculum for the Pre-Clinical years. New York: Springer Publishing Company.
* Boud, D., Felleti, G. (1991) The Challenge of Problem-Based Learning. London: Kogan.
* Woods, Donald R. (1994). Problem-Based Learning: How to Gain the Most from PBL. Hamilton, Ontario, Canada. Donald R. Woods, Publisher.


Selected Articles

* Barrows, Howard. See Southern Illinois University School of Medicine Homepage for an extensive list of articles published in medical journals.
* Gallagher, S., Rosenthal, H., and Stepien, W. (1992) "The Effects of Problem-Based Learning on Problem Solving. Gifted Child Quarterly, 36(4), 195-200.
* Knoll, Jean W. (1993). "An Introduction to Reiterative PBL." Issues and Inquiry in College Learning and Teaching. Spr/Smr. 19-36
* Stepien, W. and Gallagher, S., and Workman, D. (1993) "Problem-Based Learning for Traditional and Interdisciplinary Classrooms." Journal for the Education of the Gifted, 16(d4), 338-357.
* Stepien, W. and Gallagher, S.A. (1993). "Problem-based Learning: As Authentic as it Gets." Educational Leadership. 50(7), 25-8

DNA Extraction from vege and humans

Nexus Research Group - FUN Science and home experiments...: "To isolate plant DNA

* blend 50g of Cauliflower in 200 mL of water for 30 seconds to get single cells.
* Strain through muslin cloth. Pour 15 mL of liquid into a screw cap tube with 3 drops of detergent and shake gently for 5 seconds.
* Carefully layer to top of tube with cold alcohol (or meths). Use a hooked glass rod to spool out strands of DNA at the interface.



To isolate your own human DNA

(an original protocol independantly developed by Nexus, please acknowledge our contribution if using this protocol for teaching purposes)


* To a 1/2 cup of water dissolve about 1/2 teaspoon of salt and add a squirt of dish-washing detergent. Save this for step 3
* Swirl about 25 mls of water around your mouth for 30 seconds. This removes some cheek cells. Spit into a disposable cup
* Add about 2cm of the fluid to a test-tube (or a Fuji film cannister) and add about 1cm of the saline/detergent solution. Invert gently 3 or 4 times to mix well (but you don't want a lot of froth). This will break open the many cheek cells you spat out, releasing the DNA message that each cell must carry.
* Layer on top some ice cold ethanol (or methanol). Strands of DNA will be seen where the two layers meet. Hook out the strands of DNA that form with a glass hook (or one made from a plastic twist-tie) by slowly dipping up and down through the two layers.

CHEMICAL TESTS TO CONFIRM THE PRESENCE OF DNA:-

The following chemical tests can be used to check you actually have DNA:

* Test for purines: Add excess 2M ammonia solution and a few drops of 0.1M silver nitrate to 1 mL of DNA extract. A white precipitate indicates the presence of purines.
* Test for phosphate: Add 1 mL of 0.2M ammonium molybdate (39.02g/L) to 0.5 mL of extracted DNA and warm gently at 60-70oC. DO NOT BOIL. Yellow colour indicates presence of phosphate.
* Test for deoxyribose: Add 2 mL of Disches reagent to 1 mL of extracted DNA. Boil in water bath for 15 minutes. Green-blue colour indicates presence of deoxyribose. Disches reagent: 486 mL glacial acetic acid, 14 mL conc. sulphuric acid, 5g diphenylamine. Stir well and add 500 mL distilled water"

Biomeda - sells enzymes, antibodies etc

biomeda - Antibodies, Detection Kits, ELISA and Serums for Immunochemistry and Flow Cytometry

For the last 22 years, Biomeda has consistently developed, manufactured and sold the highest quality immunochemicals. Starting with our full range of detection kits and followed by a diversified line of monoclonal and polyclonal antibodies, Biomeda came to be recognized as a leader in the life sciences industry.

At Biomeda.com, you will find all our product information, up to date pricing, and an online order interface to streamline your purchasing experience. We are committed to quality products and meeting the progressing needs of our customers.

Genscope-free and fun genetics manipulative program

Mama Ji's Molecular Kitchen

AE Mystery Spot

Food Forensics: A Case of Mistaken Identity

Food Forensics: A Case of Mistaken Identity

Food Forensics: A Case of Mistaken Identity

By Michael Grupe

ABSTRACT
This lesson is a summary of a mini-unit put together by myself and five other St. Louis area teachers during a summer internship in immunology. The entire packet with complete preparation instructions and student sheets can be obtained from the St. Louis Mathematics and Science Education Center, 8001 Natural Bridge Road, St. Louis, MO 63121.

This lesson is designed to serve as an introduction to the immune system. It can stand alone or it can lead into further studies of the immune system. The primary focus of this inquiry-based lesson is antigen-antibody specificity. After focusing the students' attention on allergic reactions, two hands-on experiments allow students to explore the specific reaction between an antigen and the antibody that recognizes it. Students carry out an exploratory experiment leading to the concept of specificity. A second experiment allows students to apply the techniques and concepts learned in the first activity and subsequent discussion to solve a mystery. Follow-up discussion and problems apply the concept of specificity to related topics.

This lesson can be used with high school students and can be adapted to any level (biology or advanced biology) in association with the study of biochemistry, cell biology, health, or physiology. It can be used in association with topics such as allergies, food safety, or antigen-antibody precipitation. As a result of completing this lesson, students will be able to answer questions like these: "Why am I allergic to some things but not to others?" or "Why does clotting occur when incompatible blood types are mixed?"

BACKGROUND INFORMATION
Notes for the Teacher:

The major concept of this lesson is the specificity of the reaction between an antibody and an antigen. Antibodies are proteins produced by cells of the immune system in response to the exposure of an individual to a foreign substance (an antigen). This concept will be illustrated through the use of an experimental procedure called a double diffusion assay.

This assay is based on the formation of a precipitate (precipitin line) when an antibody reacts with its specific antigen. In this test, often called the Ouchterlony test, antibody and possible antigens are placed in wells in agar plates and allowed to diffuse toward one another. The antibody is placed in a center well and antigens (specific or nonspecific) are placed in surrounding wells. When an antibody and its specific antigen meet one another and are at the proper concentrations, the precipitate will form a visible white line between the two wells. This line is called a precipitin line.

In the diagram below, a precipitin line can be seen between the center well and wells 2 and 3. The fact that the line is continuous indicates that both wells contain the same antigen. Antibodies and antigens that are not complementary will diffuse past one another in the agar and will not form a precipitate.

The scenario for this lesson is centered around hypersensitivity to environmental antigens that are generally not particularly harmful (e.g., pollen, dust mite excrement, mold, drugs, food, etc.). In these situations the immune system reacts to these antigens by producing a type of antibody known as immunoglobulin E (IgE). IgE antibodies trigger the release of histamine by mast cells which then leads to typical allergic symptoms. An extreme response is called an anaphylactic response.

Materials for experiments 1 and 2 (per class and per team of students):

* Anti-chicken egg albumin (Sigma Chemical Co.)
* 2 - 1.5% agar plates
* 6 mm diameter soda straw
* Toothpick
* Glass marking pen
* Small quantities of:
o Raw egg white (diluted 1:625)
o Uncooked egg-enriched pasta (1:40)
o Uncooked egg-free pasta (1:40)
* Samples of various foods:
o some positives (egg-containing) like mayonnaise (1:10), custard (1:10), pasta (1:40), baked items (1:10), egg white
o some negatives (without egg) like sugar, salt, milk, beef broth, molasses, etc.
The dilution is not critical on negatives.
(Note: I clean out leftovers from my refrigerator)
(For dilutions use 1gm of solid foods or 1 ml of liquids in .85% saline. For egg white dilution, start with a 1:25 dilution and dilute first dilution again 1:25. For dilutions of solids, use only the supernatent. Sterile technique is not necessary and some inaccuracy in dilutions is allowable.)
* fine-tipped dropping pipettes (plastic)
* test tubes or flasks for food solutions

Preparation:

Most of these materials are cheap and easily obtainable. Dilutions can be made up days in advance and stored until needed. Plates should be made several days prior to use to allow proper drying. Antibody is the biggest expense but a little bit goes a long way. (2 ml supplies 50 teams of 2) Out-of-date antibody would be cheaper and would still work for these experiments. Antibody (Anti-Chicken Egg Albumin) from Sigma Chemical Co., P.O. Box 14508, St. Louis, MO 63178 Stock # C-6534 2 ml is about $50. Total prep time is about 2 hours.

Class Time Needed:

This lesson is designed for four 50-minute periods. Activities can be reorganized to fit your schedule.

Day 1:

* Introduction and exploration
* Class discussion of allergies
* Exploratory activity

Day 2:

* Gather/discuss data
* Observe, record, discuss results of experiment 1
* Concepts presented
* Mystery read by students

Day 3:

* Design experiment to solve mystery and set-up of experiment 2

Day 4:

* Gather/discuss data
* Follow-up
* Observe, record, discuss results of experiment
* Form conclusion (solve mystery)
* Return to day 1 questions
* Solve application problems

LESSON
Day 1:

A quick discussion during which students are questioned about their allergies and symptoms and how their allergies compare to the allergies of family members or friends. This discussion is not intended to result in answers, but rather to stimulate interest. Highlight that different people are allergic to different and specific substances. This will be explained later by antigen-antibody specificity.

Experiment 1:

Fooling with Food is a chance for students to explore the interaction between various foods (some negative and some positive) and Reagent A (the antibody). The figure below shows the relative position of wells to be cut in the agar plates with the straws. Toothpicks are good for removing the plugs from the wells. Extra plates and colored water can be used first for students to practice loading the wells. Only 1-2 drops with a fine-tipped pipettes is needed per well.

1. Cut wells in agar using a template (teacher prepared) under the plate as a guide.
2. Remove plugs and label wells and plate.
3. Students select six different foods to load in the six outer wells. Give students about 10 foods to choose from so there is variation in selections. Different pipettes should be used for each food and foods should not spill over edge of wells.
4. Reagent A is placed in the center well. (I suggest teacher does this because of expense.)
5. Plates can be stored overnight in a flat position at room temperature.

(graphic)

Day 2:

1. Let students find precipitin lines. Tell them only that they may have to hold plates up to a light or toward a window. Faint white lines will be seen by someone. Then others will see.
2. Compile a list of positive foods and students will quickly see that all are egg-containing.
3. Teacher can now discuss specific interaction between antibody (reagent A) and antigen (albumin in egg). Terms can be presented at this time. Basic (forked) structure of antibodies that allows for cross-linkage and formation of precipitate can also be discussed.

Mystery:

Stan's Salad Saga
(Let students read this or assign)

As Stan lay in his hospital bed, red, swollen and gasping for breath, he agonized over the cause of the near life-threatening reaction he had suffered. All of his adult life he had known of his allergy to eggs. His physician had made abundantly clear to him the severity of the reaction that he could expect if he included eggs in his diet. Now he was suffering from the very symptoms that had been predicted. He wasn't allergic to lots of different things. Eggs were the only substance that could have brought him to this extreme condition. Now he faced a multi-thousand dollar hospital bill and his insurance agent was placing the blame on him. The company would refuse to pay if Stan was shown to have been negligent. He had been far too careful to have made a mistake on his own. He had to somehow convince his agent that he was not at fault. Someone else was responsible for his being here! For the benefit of both his insurance agent, Carl, and his allergist, Judy, he recapped the activities prior to this onset of anaphylactic shock.

It had been a typical day with the exception of his departure time for work. Running late, he had not had time to eat breakfast or make his lunch. He grabbed an apple on his way out the door. When the lunch hour came, he went to the nearest branch of a local grocery chain to get a salad bar. The pasta salad looked particularly appealing that day. Conscientiously, Stan asked the salad technician whether any eggs were used in the salad. He was assured that the salad was egg-free. Stan's decision was made. His wife would be pleased that he was avoiding his usual high cholesterol diet. Stan had walked to the park to eat his lunch and that was when the crisis began. After eating only three or four bites of lunch, he began to experience a burning sensation in his ears and had trouble breathing. A police officer who happened to be nearby noticed his difficulty and made a 911 emergency call. That is how Stan ended up in the hospital.

Knowing that Stan was not allergic to anything else that he had eaten, the contents of the pasta salad became the immediate focus of the allergist's attention. A sample had been brought into the hospital by an alert paramedic. In addition to the pasta, it had contained tomatoes, onions, black olives and an oil and vinegar dressing. Since all the other ingredients clearly did not contain egg, the only possible source of egg was the pasta itself. The salad technician had told Stan there was no egg in the salad. Had a mistake been made? Had egg-enriched pasta been used? Or had Stan eaten something else?

You are the lab technician asked to test for the presence of egg in the pasta. Your evidence might place responsibility on the grocery store, in which case the insurance company will pay Stan's medical bills. Or you will show no evidence of egg in the pasta and Stan will be handed the blame and will be forced to pay for his negligence.

Day 3:

Experiment 2:

Students (or teacher) can design a test to answer the question posed in the mystery. Make sure both positive and negative controls are included. With six wells you could test the unknown (egg-enriched pasta), egg-enriched pasta (+ control), egg-free pasta (- control), egg white - 1:625 dilution and 1:3125 dilution (+ controls that show a range of concentrations that will result in formation of a precipitin line), saline solution (- control that is used for all dilutions).

Experiment 2 is also a double-diffusion test as was experiment 1. Now, however, students know that they are putting different antigens in the outer wells and antibody in the center well. They know why there will be positive results and why there will be negative results so they can predict which wells will have precipitin lines. Again, store plates at room temperature until the following day.

Day 4:

Follow-up:

Results are observed and compared to predictions. A conclusion is formed about Stan's Salad Saga (of course, Stan is innocent).

Now you can refer back to your original discussion about allergies and answer some of the unanswered questions from day 1. Other uses of specificity can also be discussed at this time. Many home pregnancy tests use an antibody to detect the presence of human chorionic gonadotropin (HCG) that is present in a woman's urine during pregnancy. The test for HIV also involves formation of an antibody-antigen complex.

Challenge Problems:

I have not included these but they are reviews and extensions of the terms and concepts presented in this lesson. They are part of the entire packet, should you decide to decide to get the activity packet from the Mathematics and Science Education Center of St. Louis.