Alida Munir Thomas Burlingame-smith

PCR – WHAT? Polymerase Chain Reaction (PCR) is a process used in molecular biology to amplify a single or a few copies of DNA, without a cell, across several orders of magnitude in order to generate thousands to millions of copies of a particular DNA sequence. Reverse Transcriptase: an enzyme that catalyzes the formation of DNA from an RNA template in reverse transcription.

Our Technology PCR:- It is a fast and inexpensive way to amplify – copy – small segments of DNA. Sometimes called “molecular photocopying,” the PCR is recognized as one of the most important scientific advances in molecular biology. Because significant amounts of a sample of DNA are necessary for molecular and genetic analysis, studies of isolated pieces of DNA are nearly impossible without PCR amplification.

Kary B. Mullis (Nobel Prize for Chemistry) Kary Banks Mullis, born in December 28, 1944 (age 71) is an award-winning biochemist, author and lecturer. Born in North Carolina, he was interested in observing organisms (as he recalls) from an early age. He grew up in South Carolina. Got a Bachelor’s Degree in chemistry from Georgia’s Institute of Technology and earned a PHD in biochemistry from the University of California, Berkeley. For some time he wrote fiction but quit and turned his focus back to science.

Kary B. Mullis (Nobel Prize for Chemistry) One night, Mullis had the idea to use a pair of primers to bracket the desired DNA sequence and to copy it using DNA polymerase. Mullis soon took off his usual projects and concentrated on what would become one of his greatest accomplishments; the PCR.

Our Technology The sample of DNA is first heated so that the DNA denatures or separates into two single stranded DNA. Then an enzyme called Taq polymerase synthesizes two new strands of DNA using the original strands as templates.

‘Ingredients’ and General Steps Denaturation: The DNA is heated (usually at 95oC) to separate the double strands. – heating breaks the Hydrogen Bonds among the DNA. Annealing: the two primers bind the appropriate complementary strand. - PCR requires two different primers, one that can attach one each side of the DNA molecule. Extension (Elongation): Taq polymerase adds nucleotides to the annealed primer. - Taq polymerase, just like human polymerase can add nucleotides towards one direction. These steps are repeated 30-35 times. “Ingredients”: Template DNA PCR Primers Nucleotides Taq Polymerase

Pros And Cons Pros: Fast, easy and inexpensive way to amplify, or copy DNA. - Can make billions of copies in several hours. Very sensitive. The DNA of interest can be amplified by the DNA of just one cell and thus really small amount of starting material can be used. Cons: Very Sensitive. Very small amounts of contaminated DNA (from a different sample) can also be amplified, or copy and disrupt the results. Less effective with larger strains.

Bioethical Concerns Some people believe that genetical engineering is unethical altogether, and believe that DNA should be left alone to duplicate naturally in the cell. Most ethical concerns surround the testing of amniotic fluid of organisms still waiting to be born. - Some believe it can be damaging to the embryo. When the DNA is taken from an embryo, it can be replicated to identify potential diseases of birth defects. - Some people say this is unethical because they believe it can lead to abortion of a child with down syndrome.

Uses Most mapping techniques in the Human Genome Project relied on PCR. Detection of bacteria or viruses (particularly AIDS). Diagnose of inherited disorders. - Mutations producing single-gene disorders can be detected by several methods like gel electrophoresis and endonuclease. Crime Scenes - PCR can be used to get a possible lead on the killer, and can also be used to eliminate suspects. - A sample of blood/skin/hair, no matter how little, can be used to shed further light on the case. PCR can be used to amplify the cells and efficiently study them.

Practical Case Scene: A man is found dead on a deserted warehouse. Forensic experts identify strangling as a cause of death. The absence of bloody footprints/fingerprints or weapons means there is no obvious leads to the killer. The experts dust down the body and its surroundings for hair, skin or any other evidence – find little bits of skin and hair on the fingernail of the victim indicating struggle prior the murder. Use of PCR: Little evidence (like bits of skin and hair on fingernails) can make it hard to study a case and find a lead on the killer. PCR can be used to isolate desired DNA and can amplify the DNA to make it much easier to study.

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