Module 3 Section 4

Question 1

PCR reaction mixture requirements

Answer 1

• DNA sample containing segment to be amplified
• pair of synthetic oligonucleotide primers
• Deoxynucleoside triphsophates (dNTP)
• DNA Polymerase (that will not denature with heat)

Question 2

PCR steps

Answer 2

1. Denaturation
   - solution temp increased briefly
2. Annealing
   - mixture cooled so primers can anneal ([primers]»[DNA], so more likely for primer to anneal than DNA to reanneal
3. Elongation
   - temp increased slightly, complementary strand synthesized by thermostable DNA pol
4. Amplification
   - process repeated (mixture heated to denature dsDNA, cooled for primers to anneal, etc.
5. Repeat 25-30 times
   - synthesis rate~1000BP/min

Question 3

Tm for olglionucleotides

Answer 3

• 14-20 base pairs

- Tm=2˚Cx(%A+T)+4˚C(%G+C)

Question 4

Ta (annealing temp) in PCR

Answer 4

Ta=Tm-5˚C

Question 5

Gel electrophoresis steps

Answer 5

• used for visualising PCR products
  1. sample added to slot at one end of gel (matrix composed of agarose)
  2. voltage applied to gel, DNA/RNA migrate to cathode b/c of neg backbone
  3. larger mlc move more slowly than smaller (so larger are closer to top at end of process)

Question 6

qPCR Steps

Answer 6

• amount of DNA produced is quantified after every rxn cycle
• fluorescent dye (SYBR green) added to rxn mix which fluoresces 100x brighter when bound to dsDNA
  1. Denaturation (increase temp)
  2. Primer annealing (decrease temp)
  3. Extension (slight temp increase)
  4. record fluorescence

Question 7

Quantifying cDNA with qPCR

Answer 7

• as more double stranded products produced exponentially, enters exponential phase
• reaches plateau phase as one or more rxn components is exhausted
• Ct (cycle threshold) is cycle # at which the previously defined signal threshold is surpassed
• with more target cDNA present, Ct moves smaller

Question 8

RT-PCR (reverse transcriptase PCR)

Answer 8

• mRNA to cDNA through reverse transcription

- can use to replicate only the exons of a gene through the use of mature mRNA

Question 9

cDNA

Answer 9

dsDNA complementary to RNA sequences

Question 10

Optimizing PCR stringency

Answer 10

• increase annealing temp
• reduce salt []
• increased [MgCl2]=less stringent
• decreased [MgCl2]=more stingent
• Mg2+ important as a cofactor, orients dNTP

Question 11

Endpoint PCR vs. qPCR

Answer 11

Endpoint: only tells if DNA template is present, analyze at end of cycles
qPCR: tells how many copies of the DNA template there are at start of rxn, analyze after each cycle

Question 12

If treatment with drug X increases Ct for Gene Y by 4 cycles, what is the effect of drug X on the expression of gene Y?

Answer 12

• decreased expression by 16x

- 4 cycles more to get to Ct, 2^4=16x less template

Question 13

Melt curve

Answer 13

• used to assess specificity of rxn
• fluorescence vs. temp (increased temp=decrease fluorescence)
• if only a single curve, then you know you have product of interest.