lecture 3

Question 1

what are some uses of PCR?

Answer 1

• genetic testing (prenatal, cancer)
• diagnosis
• personalised medicines
• forensics

Question 2

what has to be done before starting PCR with DNA?

Answer 2

nothing

Question 3

what has to be done before starting PCR with RNA?

Answer 3

need to carry out reverse transcriptase first to convert RNA to DNA

Question 4

what happens in reverse transcriptase PCR?

Answer 4

1. convert RNA (often mRNA) to cDNA (complementary DNA)
2. use reverse transcriptase to convert RNA to DNA
3. amplify with PCR

Question 5

what is the source of RNA?

Answer 5

gene expression (mRNA) + RNA virus

Question 6

what is used in reverse transcriptase?

Answer 6

-template RNA
-primer
-dNTPs
-reverse transcriptase
-buffer

Question 7

end point vs real time PCR (qPCR)

Answer 7

end point = cheap, sees results, plateau, for sequencing, genotyping and cloning

real time = more expensive, quantity of PCR proportional to amount of template, quantification of gene expression, quality control, genotyping, measures exponential phase

Question 8

when is threshold reached?

Answer 8

when fluoresce can be detected above background levels
Ct value
lower Ct value = grater initial amount of DNA

Question 9

what is the function of reference genes?

Answer 9

used as point of comparison
supports validity

Question 10

what is an example of a reference gene?

Answer 10

beta actin