PCR Polymerase Chain Reaction Flashcards
What is PCR?
quick and easy method for generating copies of any DNA fragment
why is it a good forensic investigative/medical diagnosis tool?
dont need a lot of DNA
What are the 4 components of PCR?
- template DNA
- DNA polymerase
- the four deoxynucleotides triphosphates
- two primers
Define denaturation
solution containing double-stranded DNA which is heated to separate the two strands
define annealing
the solution is cooled and the two primers anneal to the complementary sequence strands on template duplex
define extension
DNA polymerase synthesizes new DNA strands
how long are the PCR primers (typically)
20-30 nucleotides
how many strands are produced after the second cycle of PCR?
four
what is the equation of melting temp for primers?
Tm = [4(G or C) + 2(T or A)] - 5°C
What are the three key factors in PCR popularity?
- availability of DNA polymerase
- automation of temperature control heat blocks
- drastic decrease in the cost of thermocyclers and heat tolerant DNA polymerases
What is the standard temperature for E. coli?
~37°C
What is Taq polymerase?
thermus aquaticus a bacteria from hot springs, it is used as a DNA polymerase due to its high heat tolerance
Where would a large DNA fragment travel in gel electrophoresis?
the positive pole
How do you tell the concentration of DNA in gel electrophoresis?
Brighter fluorescence
What does RT-PCR mean?
reverse transcriptase polymerase chain reaction
What is the point of RT-PCR?
to synthesize RNA as if it were DNA since there is no enzyme that can synthesize RNA directly from RNA templates
How does RT-PCR work?
uses reverse transcriptase that uses an RNA template to make a DNA strand
What does qPCR stand for?
quantitative PCR
what is qPCR?
Measures the amplification in real time during amplification
How does qPCR measure gene expression?
flourescence is monitored and then used to determine the starting concentration of the mRNA of interest
If you increase the number of regions investigated you:
- increase the variability
- decrease the probability of finding another segment with the same combination of repeats
