L5

Question 1

What is quantitative PCR?

Answer 1

• allows you to measure the relative amount of target DNA in a sample
• same principle as PCR, only 2 main differences:
  1. Addition of a dye that emits fluorescence when inserted into double-stranded DNA
  2. Real-time PCR reader. As amount of DNA increases with each cycle the fluorescent signal is captured by a fluorometer

Question 2

What is sanger sequencing

Answer 2

• Used to determine the sequence of a segment of DNA
• Similar to PCR as it follows the same steps for each cycle
  Main differences:
  1. only one primer that is complementary to one of the 2 DNA strands
  2. Addition of radioactively labelled-dideoxynucleotides (ddNTPs) to the reaction

Question 3

How are ddNTPs different from dNTPs?

Answer 3

• Lacks the ribose 3’-OH

- Insertion of ddNTP forces DNA synthesis to a stop

Question 4

Why does sanger sequencing only use 1 primer?

Answer 4

• Wouldn’t be able to differentiate which template strand the sequence came from if using two primers

Question 5

Automated Sanger Sequencing

Answer 5

• PCR with fluorescent, chain-terminating ddNTPs, size separation by capillary gel electrophoresis, laser excitation and detection by sequencing machine
• If SNP is read as two different nucleotides, then the individual is heterozygote

Question 6

Detecting Polymorphisms: PCR and gel electrophoresis

Answer 6

• Work for SSLPs and INDELs

Question 7

Detecting Polymorphisms: Sanger Sequencing and Whole Genome Sequencing

Answer 7

• Works for SNPs, SSLPs, INDELs, CNVs