An Introduction to PCR

1. An Introduction to PCR Dave Palmer, Byotix, Inc.

2. Byotix, Inc. 1260 S. 47th St Richmond, CA

3. Review: The structure of DNA Helix Complementary Base Pairing

4. Review: The structure of DNA Unzipping Antiparallel Strands

5. The Problem... How do we identify and detect a specific sequence in a genome?

6. The Problem... (How do we identify and detect a specific sequence in a genome?) • TWO BIG ISSUES: • There are a LOT of other sequences in a genome that we’re not interested in detecting. (SPECIFICITY) • The amount of DNA in samples we’re interested in is VERY small. (AMPLIFICATION)

7. Review: Genome Sizes • Pine: 68 billion bp • Corn: 5.0 billion bp • Soybean: 1.1 billion bp • Human: 3.4 billion bp • Housefly: 900 million bp • Rice: 400 million bp • E. coli: 4.6 million bp • HIV: 9.7 thousand bp http://www.cbs.dtu.dk/databases/DOGS/abbr_table.txt

8. Just How Big Is 3.4 Billion? • Human genome is 3.4 B bp • If the bases were written in standard 10-point type, on a tape measure... • ...The tape would stretch for 5,366 MILES )8633 Km! • Identifying a 500bp sequence in a genome would be like finding a section of this tape measure only 4 feet long...

9. How many molecules do we need to be able to see them? • To be visible on an agarose gel, need around 10 ng DNA • For a 500-bp product band, weighing 660 g/mol.bp, therefore need 10e-9 / (500*660) = 3.03e-14 moles • Avogadro’s number = 6.02e23 • Therefore need 1.8e10 copies! • In other words, to “see” a single “gene”, the DNA in a sample of 100 cells would have to be multiplied 180 million times!!!!!

10. The Problem... • How do we identify and detect a specific sequence in a genome? • TWO BIG ISSUES: • There are a LOT of other sequences in a genome that we’re not interested in detecting. • The amount of DNA in samples we’re interested in is VERY small. PCR solves BOTH of these issues!!! SPECIFICITY AMPLIFICATION

11. PCR History In what has been called by some the greatest achievement of modern molecular biology, Kary B. Mullis developed the polymerase chain reaction (PCR) in 1983. PCR allows the rapid synthesis of designated fragments of DNA. Using the technique, over one billion copies can be synthesized in a matter of hours. PCR is valuable to scientists by assisting gene mapping, the study of gene functions, cell identification, and to forensic scientists in criminal identification. Cetus Corporation, Mullis' employer at the time of his discovery, was the first to commercialize the PCR process. In 1991, Cetus sold the PCR patent to Hoffman-La Roche for a price of $300 million. It is currently an indispensable tool for molecular biologists and the development of genetic engineering.

12. Some Uses of PCR • Forensic DNA detection • Identifying transgenic plants • Detection of viral infection • Cloning • Detection of ancient DNA

13. Mr. PCR: Kary B. Mullis (1944 - ) The inventor of the DNA synthesis process known as the Polymerase Chain Reaction (PCR). The process is an invaluable tool to today's molecular biologists and biotechnology corporations. Mullis, born in Lenoir, North Carolina, attended the University of Georgia Tech for his undergraduate work in chemistry, and then obtained a Ph. D. in biochemistry from Cal Berkeley. In 1983, working for Cetus Corporation, Mullis developed the Polymerase Chain Reaction, a technique for the rapid synthesis of a DNA sequence. The simple process involved heating a vial containing the DNA fragment to split the two strands of the DNA molecule, adding oligonucleotide primers to bring about reproduction, and finally using polymerase to replicate the DNA strands. Each cycle doubles the amount of DNA, so multiple cycles increase the amount of DNA exponentially, creating huge numbers of copies of the DNA fragment. Mullis left Cetus in 1986. For his development of PCR, he was co-awarded the Nobel Prize in chemistry in 1993. Mullis is currently doing HIV and AIDS research.

14. The Invention of PCR The process, which Dr. Mullis conceptualized in 1983, is hailed as one of the monumental scientific techniques of the twentieth century. A method of amplifying DNA, PCR multiplies a single, microscopic strand of the genetic material billions of times within hours. Mullis explains: "It was a chemical procedure that would make the structures of the molecules of our genes as easy to see as billboards in the desert and as easy to manipulate as Tinkertoys....It would find infectious diseases by detecting the genes of pathogens that were difficult or impossible to culture....The field of molecular paleobiology would blossom because of P.C.R. Its practitioners would inquire into the specifics of evolution from the DNA in ancient specimens....And when DNA was finally found on other planets, it would be P.C.R. that would tell us whether we had been there before." http://www.osumu.org/mu/events_lectures1b.htm

15. The Invention of PCR Mullis's little silver Honda Civic was purring through the vineyards and redwoods of the Anderson Valley; and his mind wandered. Life is sweet, he thought: 'I am a big kid with a new car and a full tank of gas. I have shoes that fit. I have a woman sleeping next to me and an exciting problem, a big one.' At mile-marker 46.58 on Highway 128 - he had both the presence of mind and the sense of history to note the exact spot, if not the month - the epiphany arrives. 'Holy s__,' Mullis cries out, and his girlfriend almost, but not quite, wakes up. He pulls the Honda to the side of the road to write down his ideas and check his calculations. Within feverish minutes, the problem is solved, and Mullis is left with the mop-up operation of getting PCR actually to work. This takes almost two years, and the original report was famously rejected by both Nature and Science. Mullis was not fazed: '"F___ them," I said’. http://www.lrb.co.uk/v21/n13/shap2113.htm

16. Kary Mullis Trivia • Mullis repeatedly asserts that he was ripped off financially by the greedy confederacy of dunces who were his colleagues at Cetus. He got a $10,000 bonus, and Cetus cleared $300 million when the patent rights to PCR were sold to Hoffman-LaRoche - possibly the most ever paid for a patent. (http://barometer.orst.edu/0102/02winter/020207/020207n6.html) • A few years ago he started up a company - GeneStones - that would copy the DNA in hair or skin samples of famous people, multiply it by PCR, and then implant it in artificial gemstones, where it would appear as 'a white, ethereal cloud'. Depending on the setting and production-run, $75 to $200 would get you John F. Kennedy, Napoleon or Marilyn Monroe on your finger. (http://barometer.orst.edu/0102/02winter/020207/020207n6.html) • His first published scientific paper, in the premier scientific journal Nature in 1986, described how he viewed the universe while on LSD - pocked with black holes containing antimatter, for which time runs backward. He has been known to show photographs of nude girlfriends during his lectures. (http://www.virusmyth.net/aids/data/cfmullis.htm) • We tortured the cows. We sliced apples and slipped them onto the electric fence that contained them in the newer parts of the pasture. Cows like apples and they kept trying. – K. Mullis,www.nobel.se/chemistry/laureates/1993/mullis-autobio.html

17. "Take all the MVPs from professional baseball, basketball and football. Throw in a dozen favorite movie stars and a half-dozen rock stars for good measure, add all the television anchor people now on the air and collectively we have not affected the current good or the future welfare of mankind as much as Kary Mullis." -- Ted Koppel, on ABC's "Nightline" Mr. PCR: Kary B. Mullis http://www.buzzle.com/editorials/3-29-2000-6929.asp

18. Uses of PCR: Ancient DNA Archaeologists have happily seized on PCR and are applying it in an amazing variety of ways. It is helping, for example, to launch a new chapter in the colorful and controversial story of the 2000-year-old Dead Sea Scrolls, which are written on parchment made out of skins from goats and gazelles. Researchers are analyzing the parchment fragments to try to identify individual animals they came from. The hope is that the genetic information will guide them in piecing together the 10,000 particles of scrolls that remain. http://www.faseb.org/opar/bloodsupply/pcr.html

19. Uses of PCR: Ecology With PCR, scientists can glean genetic information from the faintest traces of the shyest, rarest animal-urine, feces, scent marks, infinitesimal bits of hair or skin rubbed onto a tree as the elusive creature passes by. In addition to information that aids classification, individuals can be identified so as to estimate population size in a particular locale, or to determine the geographic range of a single animal, or a group of them. The technique can be adapted to similar studies of plants, for analysis of patterns of seed dispersal and the relative reproductive success of specific plants. Researchers have even used PCR to study badly damaged specimens such as roadkill, or the leavings of carnivores, where little-known vertebrates have been identified among the prey. http://www.faseb.org/opar/bloodsupply/pcr.html

20. Uses of PCR: Disease Detection PCR can also be more accurate than standard tests. It is making a difference, for example, in a painful, serious, and often stubborn misfortune of childhood, the middle ear infection known as otitis media. The technique has detected bacterial DNA in children's middle ear fluid, signaling an active infection even when culture methods failed to detect it. Lyme disease, the painful joint inflammation caused by bacteria transmitted through tick bites, is usually diagnosed on the basis of symptom patterns. But PCR can zero in on the disease organism's DNA contained in joint fluid, permitting speedy treatment that can prevent serious complications. http://www.faseb.org/opar/bloodsupply/pcr.html

21. Uses of PCR: Endangered Species Researchers have used the technique to aid in reducing illegal trade in endangered species, and products made from them. Because PCR is a relatively low-cost and portable technology, and likely to become more so, it is adaptable for field studies of all kinds in the developing countries. It is also a tool for monitoring the release of genetically engineered organisms into the environment. http://www.faseb.org/opar/bloodsupply/pcr.html

22. Uses of PCR: Forensic DNA The technique's unparallelled ability to identify and copy the tiniest amounts of even old and damaged DNA has proved exceptionally valuable in the law, especially the criminal law. PCR is an indispensable adjunct to forensic DNA typing-commonly called DNA fingerprinting. http://www.faseb.org/opar/bloodsupply/pcr.html

23. Uses of PCR: Proving Innocence DNA typing is only one of many pieces of evidence that can lead to a conviction, but it has proved invaluable in demonstrating innocence. Dozens of such cases have involved people who have spent years in jail for crimes they did not commit. One example is Kirk Bloodsworth. The Maryland waterman was wrongly imprisoned for almost nine years for the rape and murder of a 9-year-old girl, but was freed in 1993 with the aid of PCR. Even when evidence such as semen and blood stains is years old, PCR can make unlimited copies of the tiny amounts of DNA remaining in the stains for typing, as it did in Bloodsworth's case. http://www.faseb.org/opar/bloodsupply/pcr.html

24. Uses of PCR: Disease Detection The method is especially useful for searching out disease organisms that are difficult or impossible to culture, such as many kinds of bacteria, fungi, and viruses, because it can generate analyzable quantities of the organism's genetic material for identification. It can, for example, detect the AIDS virus sooner during the first few weeks after infection than the standard ELISA test. PCR looks directly for the virus's unique DNA, instead of the method employed by the standard test, which looks for indirect evidence that the virus is present by searching for antibodies the body has made against it. http://www.faseb.org/opar/bloodsupply/pcr.html

25. Uses of PCR: Ancient DNA Archaeologists are finding that PCR can illuminate human cultural practices as well as human biology. Analyzing pigments from 4000-year-old rock paintings in Texas, they found one of the components to be DNA, probably from bison. The animals did not live near the Pecos River at that time, so the paleo-artists must have gone to some effort to obtain such an unusual ingredient for their paint. Taking so much trouble suggests that the paintings were not simply decorations, but had religious or magical significance. http://www.faseb.org/opar/bloodsupply/pcr.html

26. Uses of PCR: Disease Detection PCR can even diagnose the diseases of the past. Former vice president and presidential candidate Hubert H. Humphrey underwent tests for bladder cancer in 1967. Although the tests were negative, he died of the disease in 1978. In 1994, researchers compared a 1976 tissue sample from his cancer-ridden bladder with his 1967 urine sample. With the help of PCR amplification of the small amount of DNA in the 27-year-old urine, they found identical mutations in the p53 gene, well-known for suppressing tumors, in both samples. "Humphrey's examination in 1967 may have revealed the cancerous growth if the techniques of molecular biology were as well understood then as they have become," the researchers said. http://www.faseb.org/opar/bloodsupply/pcr.html

27. Today’s PCR: Population Genetics Alu Insertion Polymorphism detects the presence or absence of a "jumping gene" on chromosome 16. This simple genetic system has only two alleles and three genotypes. Despite this simplicity, allele frequencies vary greatly in different world populations. Alternate explanations about the causes of this variation are consistent with opposing theories of the origins of modern humans http://www.geneticorigins.org/geneticorigins/

28. Today’s PCR: Population Genetics • Fossil evidence: • 5 million years ago: human lineage diverged from primates • 1.5 million years ago: early hominids migrated out of Africa to found populations in Europe, the Middle East, and Asia • Conflicting Theories: • Did these early groups develop independently, and in parallel, to become modern humans (multiregional theory)? • Did a common ancestor develop in Africa, then migrate worldwide and displace the early hominids (displacement theory)? http://www.geneticorigins.org/geneticorigins/

29. Today’s PCR: Population Genetics Alu is an example of a so-called "jumping gene" – a transposable DNA sequence that "reproduces" by copying itself and inserting into new chromosome locations. Alu elements are classified as SINEs, or Short INterspersed Elements. All Alus are approximately 300 bp in length and derive their name from a single recognition site for the restriction enzyme AluI located near the middle of the Alu element. Human chromosomes contain about 1,000,000 Alu copies, which equal 10% of the total genome. Once an Alu integrates into a new site, it accumulates new mutations at the same rate as surrounding DNA loci. Alu elements can be sorted into distinct lineages, or families, according to inherited patterns of new mutations. These studies suggest that the rate of Alu transposition has changed over time – from about one new jump in every live birth, early in primate evolution, to about one in every 200 newborns today. http://www.geneticorigins.org/geneticorigins/

30. Today’s PCR: Population Genetics Each Alu is the "fossil" of a unique transposition event that occurred only once in primate evolution. Thus, all primates showing an Alu insertion at a particular locus have inherited it from a common ancestor. This is called identity by descent. Most Alu mutations are "fixed," meaning that both of the paired chromosomes have an insertion at the same locus (position). However, a number of human-specific Alus are dimorphic – an insertion may be present or absent on each of the paired chromosomes of different people. These dimorphic Alus inserted within the last million years, during the evolution and dispersion of modern humans. These dimorphisms show differences in allele and genotype frequencies between modern populations and are tools for reconstructing human prehistory. http://www.geneticorigins.org/geneticorigins/

31. Today’s PCR: Population Genetics Today, we’ll isolate our own DNA from cheek cells, Amplify the PV92 region of our DNA with PCR, And determine (using a gel) if we have an Alu insertion there. +/+ or +/- or -/- The ratios of these genotypes, over a whole population, can help determine the relatedness to other populations. http://www.geneticorigins.org/geneticorigins/