Polymerase Chain Reaction

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Presentation transcript:

Polymerase Chain Reaction PCR Polymerase Chain Reaction

What is PCR? PCR intro: https://www.youtube.com/watch?v=Nl6eLez3CNI

PCR requires the following -

Reaction Components Template DNA -

Reaction Components DNA primers In the cell (in vivo), primers are that serve as a starting point for DNA replication In PCR (in vitro), Primers are that exactly match the beginning and the end of the DNA fragment to be amplified Reverse Primer Forward Primer Target DNA length 3’ 5’ Genomic DNA

Reaction Components DNA polymerase -

Reaction Components DNA Polymerase Why?

Reaction Components dNTPs - A T G C

Reaction Components Buffer To work properly, Taq polymerase needs magnesium ions: * Too little: * Too much:

A three- step process Denaturation: Annealing: Extension:

How Does PCR Work? Step 1: Denaturation Double Stranded DNA target Denatured single strand Denatured single strand

How Does PCR Work? Step 2: Annealing - Primer Binding to Target also called ________________ 5’ 3’ 5” Template Strand #1 Reverse Primer Template Strand #2 Forward Primer

How Does PCR Work? Step 3: Extension 5’ 3’ Taq Template Strand #1 Template Strand #2

Equipment Thermal cycler Thermal cyclers have metal heat blocks with holes where PCR reaction tubes can be inserted. The thermalcycler then

PCR a CHAIN reaction At the end of the first PCR cycle, there are now

Exponential Amplification of the Target DNA Sequence

http://highered. mheducation http://highered.mheducation.com/sites/0072556781/student_view0/chapter14/animation_quiz_6.html

BEWARE ! Sources: VALIDATION Do a_______________________________to validate that the PCR product is amplified from the intended DNA, not some other source of DNA. ________________________________________________validates that the reaction conditions and thermal cycler work properly.

PCR: Analysis At the end of a PCR reaction, there is a A LOT more of your target DNA than before the reaction started…billions of copies! Now the sample is large enough PCR can identify PCR can

PCR A powerful, versatile tool There are many uses for PCR in a endless array of scientific fields: