POLYMERASE CHAIN REACTION

(PCR)

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

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

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

1. Denaturation (94 degrees, 1min)

2. Annealing (60 degrees, 1min)3. 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