PCR - L13

Question 1

What does it mean for PCR to be specific and selective?

Answer 1

It is specific where it will only get an amplification of your selected sequence.

It is selective where it can amplify a specific sequence from a mixture.

Question 2

Why is amplification of PCR exponential?

Answer 2

Because the number of molecules of DNA doubles in each cycle.

The number of molecules of DNA = 2 ^ number of cycles.

After a period of time, dNTPs primers run out and DNA polymerase can lose activity.

Question 3

Describe the steps in PCR?

Answer 3

1. Denaturation 95oC– Double stranded DNA dissociates into single-stranded DNA.
2. Primer Annealing 55-65oC – Primers bind to complementary sequence on ssDNA. Primer binding is antiparallel.
3. Primer Extension 68-72oC – DNA polymerase synthesizes new strands of DNA from the 3’end of the primers.

This is for one cycle which is repeated ~ 30 times.

Question 4

Name some important features of PCR primers.

Answer 4

• Must be specific to your template
• Primers come in pairs – one of each pair will bind top strand of DNA; one will bind to bottom strand. Bind opposite strands in opposite orientation.
• Will amplify region of DNA in between the primers.

Question 5

What does Tm mean and why is it important?

Answer 5

Tm = temperature at which the primer will dissociate from the DNA template - usually design primers to have a Tm of 60-64^oC.

IF Tm is…
too low: primers may bind to non-specifically to another DNA.

just right: primers will bind to your specific sequence.

too high: primers may not bind efficiently (or at all), reducing product yield.

Question 6

What is reverse transcription PCR?

Answer 6

1. Reverse transcription (RNA into cDNA (complementary DNA))
2. PCR as usual is used to amplify a specific cDNA sequence.

Uses

• Molecular cloning of a protein coding cDNA sequence.
• Analysis of mRNA expression

Question 7

How does quantitative PCR. vary from standard PCR?

Answer 7

Standard PCR just monitors amplification at the end. qPCR looks at exponential bases (i.e. in real time – when amount of PCR product crossed a threshold level.

•The qPCR data has a Ct (cycle threshold) = the point at which fluorescence exceeds background levels
A sample with more template, will need fewer cycle of PCR to exceed threshold = lower Ct value.

Question 8

How can qPCR product be measured?

Answer 8

• Fluorescent dye

- Fluorescent probes

Question 9

How do we calculate relative template amounts?

Answer 9

• Difference in Ct values is the basis of how we quantify relative template amounts in qPCR
• More accurately, you should use a reference (standard) – a “housekeeping” gene that is expressed the same in both samples