An Introduction to PCR

Transcription

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2 An Introduction to PCR

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

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 8633 Km! 1m =.00062mi convert that to miles! 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!!!

11 PCR History Kary B. Mullis developed the polymerase chain reaction (PCR) in PCR allows the rapid synthesis of designated fragments of DNA. 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.

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

13 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."

14 Uses of PCR: Ancient DNA Archaeologists 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.

15 Uses of PCR: Ecology Gathering genetic info. 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 for classification. Estimate population size in a particular locale, or to determine the geographic range of a single animal, or a group of them. Studies of plants, for analysis of patterns of seed dispersal and the relative reproductive success of specific plants. To study badly damaged specimens such as roadkill, or the leavings of carnivores, where little-known vertebrates have been identified among the prey.

16 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.

17 Uses of PCR: Endangered Species Researchers have used the technique to aid in reducing illegal trade in endangered species, and products made from them. PCR 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.

18 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.

19 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.

20 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.

21 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 Although the tests were negative, he died of the disease in In 1994, researchers compared a 1976 tissue sample from his cancerridden 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.

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23 Homework: Research a study within one of the categories that PCR is used in. Write about WHEN it occurred, HOW PCR was used, and the resolution of the study.