FRSC-3000: Lecture 4

What are the major advantages of PCR?

• Very small amount of template can be used

• Can used degraded DNA samples

• Multiplexing ability

• Contaminant DNA will not amplify due to human-specific primers

• Commercial kits available

What are the major disadvantages of PCR?

• Target template may not amplify due to inhibitors

• Amplification may fail due to mutations in the primer-binding region

• Contamination from other human DNA is possible

What are the three steps in the PCR cycle?

• Denaturation of template

• Primer annealing

• DNA synthesis by a thermostable polymerase

At what temperature does denaturation occur?

95-98 Celsius

At what temperature does primer annealing occur?

45-65 Celsius

Between primers and target DNA, which component must be at a much higher concentration?

Primers

At what temperature does polymerase synthesize DNA?

72 Celsius

What is special about Taq polymerase?

It is thermostable and can withstand the high temperatures required for the PCR reaction.

What characteristics are required of primers in the PCR reaction?

• Specific to target region

• Similar annealing temperatures for forward and reverse

• Do not interact with each other or form hairpin structures

The reverse primer is what relative to the forward primer?

The reverse complement.

What is the role of magnesium ions during DNA amplification in PCR?

• They are a cofactor in the enzymatic reaction of DNA polymerase; activates Taq

• They neutralize repulsion between negatively-charged DNA strands and stabilize primer-template binding

What is the function of BSA in PCR amplification?

It binds potential PCR inhibitors such as proteins and phenol. It can act as a chelating agent when in excess.

What are some solutions to PCR inhibition?

• Dilute the template DNA sample to dilute the inhibitor

• Add more Taq DNA polymerase

• Add optional additives such as BSA

• Purify/wash the sample to remove inhibitors

Why is annealing time kept short throughout the PCR cycles?

Allows primers to bind in the correct position while limiting the chance of nonspecific binding.

What is the general length of time for extension in PCR?

~ 1 minute

What is the purpose of adding an additional A onto the end of PCR products?

Makes scoring easier and prevents incomplete adenylation or split peaks.

What is touchdown PCR?

~50 cycles run at incrementally lower temperatures so that undesired amplification is low.

What is hot start PCR?

Taq is added at a higher temperature to minimize the effects of mis-priming.

What is Hot Start Taq?

DNA polymerases like AmpliTaq Gold which only activate after a heat shock of 95 Celsius.

What are the three types of PCR controls?

• Negative control

• Positive control

• Primer control

Product in the negative PCR control indicates:

Contamination

No product in the positive PCR control indicates:

• Problem with reaction mix

• PCR inhibitors

What is the purpose of the PCR primer control?

A troubleshooting tool to determine if faulty results are due to the primers used or another reagent.