Jamie Huffman EDU 505 Spring I 2012

Guided Notes for PCR

PCR Process
Polymerase Chain Reaction (PCR) a technique that allows scientists to amplify a single copy of DNA into millions of copies in a short amount of time Step One: Set up the reaction tube (add all ingredients): Template DNA - the DNA that has been extracted from the source organism used as a blueprint during DNA replication 2 Primers (at opposite ends of the target sequence) - short (20 base pair) single stranded fragments of DNA used as a starting point for replication Nucleotides - the single units (A,T,C,G) used to construct DNA Taq Polymerase - a heat resistant enzyme that builds a new strand of DNA based on the sequence of a single-stranded template in the presence of free nucleotides

Step 2: Denaturation - increase the temperature to 95oC to separate the double-stranded DNA molecule into 2 single-stranded molecules (by breaking the hydrogen bonds) Step 3: Anneal - decrease temperature to 50oC to allow primers to bind to the single stranded DNA molecules (by reforming the hydrogen bonds) Step 4: Polymerization - raise the temperature to 72oC (temperature at which Taq Polymerase functions best) and allow Taq Polymerase to extend/replicate the DNA strands (doubles the number of copies of the original DNA) the polymerase stops when it runs out of template (the strand ends) Step 5: Cycling - repeat steps 2-4 approximately 30 times (there are more templates for replication after each cycle is completed short templates rapidly outnumber the long templates resulting from and including the original DNA) usually accomplished using a thermal cycler Thermal Cycler an automated machine that can be programmed to cycle between different temperatures at set time points

Important Cycles During PCR

During the third cycle of PCR, the first complete short double-stranded segments of the targeted sequence emerge By the fifth cycle of PCR, the short fragments outnumber the long ones

Jamie Huffman EDU 505 Spring I 2012

How can PCR be used for genetic engineering? The product of PCR can be inserted into a plasmid and cloned. Point mutations can be made during PCR, inserted into a plasmid if necessary, and cloned. Restriction sited can be added to a fragment of DNA, inserted into a plasmid, and cloned. Scientists can test for the presence of a specific sequence of DNA in a sample (use as a test to see if the DNA was truly incorporated into a new organism). Scientists can amplify a piece of recombinant DNA to use in sequencing reactions.

What are some other uses of PCR? Sequencing of DNA Paternity testing Forensic proof of presence at a crime scene Identifying the sequence of specific genes Observing the evolution of a disease-causing agent (or other organism) Identifying unknown samples Testing for the presence of bacteria or a virus (as a sign of infection in a patient) Testing for the presence of cancer cells Testing for the presence of genetic defects