5. PCR Amplification Flashcards
Optimal PCR annealing temperature?
0-5C BELOW the Tm of primers (therefore primer pairs should have similar Tm)
What is PCR processivity?
Nucleotide addition rate
High processivity = high rate of extension
Another name for template strand
Antisense strand
Major source of DNA contamination when it comes to PCR amplification?
Previously-amplified DNA
What defines the length of an amplicon?
5’ end of each primer (forward/reverse)
Primers sequences are IN each amplicon
Another name for sense strand
Coding strand
Taq extension temeprature
72C
To which end of a primer does Taq start adding nucleotides?
3’ end of primer
Because 5’-3’ activity
After primer design, what are the 2 primary determinants of amplification specificity and sensitivity
- Annealing temperature
- Mg2+ concentration
Primer that anneals to coding strand
Reverse primer
High PCR fidelity = ________ dNTP mis-incorporation
low
3 things that determine the number of cycles a PCR assay should have
- Number of starting copies of target
- Efficiency of amplification reaction (presence of inhibitors, competition for reagents..)
- Amount of DNA required for detection or further analysis
Denaturation temperature range
83 - 97C
Why should the annealing temperature be lower than that of DNA polymerase
So that primers can anneal before Taq starts working
Primer that anneals to antisense strand
Forward primer
What is PCR fidelity?
Nucleotide incorporation specificity
