PCR Flashcards
What does PCR stand for?
Polymerase chain reaction
What is PCR?
PCR is a laboratory technique for rapidly producing millions to billions of copies of a specific segment of DNA, which can then be studied in greater detail.
Why do we want to increase the copy number of DNA?
To detect a sequence of DNA
What does PCR require?
PCR requires two primers – one that is complementary to each strand of DNA – and a DNA polymerase.
What are the three stages of PCR?
Denaturation, annealing and extension
What temperature is Denaturation held at?
Denaturation is carried out at 94-95 ºC
What happens during the denaturing phase?
Double-stranded DNA is separated into its 2 complementary single strands by heating
What temperature is Anneaing held at?
Typically a temperature of 45 – 60 °C is used
What happens during the annealing phase?
The single-stranded DNA is allowed to cool in the presence of the DNA primers.
Primers are complementary to the start of your specific target DNA sequence of interest.
Primers recognize and bind each target strand.
What temperature is Extension held at?
Taq polymerase has a temperature working optimum of 72-74 ºC
What happens during the Extension phase?
DNA polymerase attaches to a free 3′ end on each of the bound primers.
Enzyme uses dNTPs to synthesize a new DNA strand in a 5′ to 3′ direction.
Use thermostable DNA polymerase.
Does the PCR cycle just happen once?
No, it will be repeated over and over again
What usually goes in a tube for simple PCR?
It requires primers, template DNA, polymerase, nucleotides, water and a buffer.
It could also possibly include MgCl2 or DSA
What are some examples of PCR types?
Nested PCR, Reverse Transcriptase (RT) PCR, Quantitative/Real-Time PCR and Touchdown PCR
