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

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