Polymerase Chain Reaction (PCR)

U. S. Department of Energy Genomics:GTL Program http://www. ornl

Introduction to PCR Genome: composed of DNA, is our hereditary code (the “blueprint”) Molecular biology: the study of genes and the molecular details that regulate the flow of genetic information from DNA to RNA to proteins, from generation to generation. Biotechnology uses this knowledge to manipulate organisms’ DNA to help solve human problems.

History of PCR 1983 Kary Mullis (Cetus Corp) developed the molecular biology technique that has revolutionized genetic research: Polymerase Chain Reaction PCR quickly transformed molecular biology into a multidisciplinary research field.

Why PCR? With PCR, you can target and make millions of copies (amplify) a specific piece of DNA (or gene) out of a complete genome.

How PCR is used PCR impacted several areas of genetic research: PCR used as a medical diagnostic tool to detect specific mutations that may cause genetic disease PCR used in criminal investigations and courts of law to identify suspects PCR used in the sequencing of the human genome

DNA Basics Double Helix Ladder Sides = Phosphate/ Sugar backbone Rungs (steps) = Nucleotides A, T, C, G Also called bases DNA can only be viewed using special microscopes. It is easiest to envision if you think of a ladder. The sides of the DNA ladder are made of the phosphate/sugar backbone; the rungs or the steps of the ladder are made of nucleotides that bond together. A always bonds with T; C always bonds with G. In cells, the DNA is twisted into a double helix. http://www.pbs.org/wgbh/nova/genome/dna.html

PCR Amplification PCR makes use of the same basic processes that cells use to duplicate their DNA (replication) Complementary DNA strand hybridization DNA strand synthesis via DNA polymerase

Recipe for PCR Amplification DNA sample: containing the intact sequence of DNA to be amplified MASTER MIX: Free nucleotides (dNTPs): raw material of DNA (A,T,C,G) DNA polymerase (Taq polymerase): enzyme that assembles the nucleotides into a new DNA chain Primers: pieces of DNA complementary to the template that tell DNA polymerase exactly where to start Flourescent dye: “lights up” when it binds to complete DNA strands

PCR Equipment Thermocycler: A thermocycler is used to rapidly heat and cool DNA samples to facilitate DNA amplification.

Steps of PCR Denaturation (94 degrees, 1min) Annealing (60 degrees, 1min) Elongation (72 degrees, 2min)

Denaturation Heating phase (94°C) Causes the two strands that make up a piece of DNA to separate.

Annealing The temperature is dropped (52°C). Primers attach (i.e., anneal) to the single-stranded DNA.

Elongation Temperature is raised (72°C). DNA polymerase (Taq polymerase) takes free nucleotides and adds them to the end of the primer. A new double stranded piece of DNA is created that is identical to the original piece of DNA that you are trying to replicate.

Result of PCR ~30 cycles Amplified exponentially Results in 1.1x1012 sets of precise-length DNA

Information Resources http://www.dnai.org/d/index.html http://gslc.genetics.utah.edu/teachers/tindex/index.cfm?switch=single&unitid=basics http://www.bioteach.ubc.ca/TeachingResources/ http://genetics-education-partnership.mbt.washington.edu/ http://genome.pfizer.com/index.cfm http://www.bioteach.ubc.ca/MolecularBiology/DNAfingerprint/index.htm http://www.genome.gov/Pages/EducationKit/online.htm http://www.pbs.org/wgbh/harvest/engineer/ http://www.pbs.org/wnet/secrets/lessons/lp_virus.html http://www.ornl.gov/sci/techresources/Human_Genome/education/education.shtml http://www.forensic.gov.uk/forensic_t/index.htm http://gslc.genetics.utah.edu/features/forensics/ http://health.discovery.com/minisites/dna/zs_forensics.html http://www.denverda.org/legalResource/Overview.pdf