PCR Flashcards
most commonly used polymerase in PCR reactions
Taq from Thermus aquaticus
Who developed PCR?
Kary Mullis and Michael Smith (1993)
Applications of PCR
- detection of DNA sequences
- molecular diagnosis of diseases
- DNA fingerprinting
- detection of viruses and bacteria
- phylogenetics
Two phases of PCR
- screening phase
- amplification phase
Give at least 5 considerations when designing a primer
- length should be 17-24 bases
- 40-60% GC content
- no formation of secondary structures
- should end with G or C but prevents runs of G and C
- annealing temperature should be the same for both primers and 5oC lower than the Tm
- 100% homology at 3’ end
- no self-hybridization
- %GC and Tm difference of 5 (or 2) between the two primers
- the %base composition should approximate that of the template
optimum length of primers
17-24 bases
what happens if the primer length is too short?
could anneal to more than one site
what happens if the primer length is too long
could not bind to any site
what happens if there are too much runs of Gs or Cs
mispairings
components of a PCR reaction mix
- buffer
- dNTP mix
- MgCl2
- primer pair
- DNA Pol (Taq pol)
- genomic DNA template
- sterile water
example of a PCR profile
Initial D - 95 - 5 mins
35 cycles
Denaturation - 95 - 1 min
Annealing - 55 - 1 min
Extension - 72 - 1 min
Final extension - 72 - 10 mins
Extended temp - 4
mechanism of RT-PCR
reverse transcription PCR
1. the poly-A tail of the mRNA is recognized by the oligo(dT) primer to form the mRNA/cDNA heteroduplex by reverse transcribing the mRNA using reverse transcriptase
2. RNase H or OH cleaves the mRNA, leaving the ss cDNA
3. DNA polymerase synthesized the complementary strand of the cDNA
difference between 5’ and 3’ RACE PCR
rapid amplification of cDNA ends PCR
for the 3’ RACE-PCR
- an oligo(dT) primer would anneal to the poly-A tail of the mRNA and will be reverse transcribed to form the cDNA
- a forward and reverse primer pair would be used to amplify part of the cDNA up to the poly-A tail, creating the second half of the cDNA
for the 5’ RACE-PCR,
- an anchor primer would anneal to the 5’ end of the cDNA after reverse transcription from the mRNA (and cleavage by the terminal transferase with oligo(dC)
- forward and reverse primers would amplify the part of the cDNA from the 5’ end, creating the first half of the cDNA
Joining of the products of these 2 RACE-PCR reactions can generate the full-length sequence of the cDNA
a dye used in qPCR that binds the minor groove
SYBR Green Dye
mechanism of the TaqMan probe
- the primer and the TaqMan probe anneal to the complementary strand following denaturation
- the intact TaqMan probe would have the reporter fluorophore and the quencher protein close, preventing emission of fluorescence
- after hybridization and during elongation, Taq pol cleaves the probe and separates the quencher and reporter, generating fluorescence
- detection of the fluorescence is used for quantification
