11.2 PCR and qPCR

Question 1

Minimum requirements for DNA synthesis in vitro

Answer 1

A template strand
A primer
DNA polymerase
Deoxyribonucleoside triphosphates
Mg+ for polymerase

Question 2

Kary Mullins insights on PCR

Answer 2

Enzymatic copying of dsDNA using 2 primers that are complementary to the opposite strand could lead to exponential increase in amount of target sequence

Question 3

PCR denaturation

Answer 3

94-96 degrees C
dsDNA melts

Question 4

PCR annealing

Answer 4

50-65 degrees C (dependent on annealing/Tm of primers)
Primers bind to complementary sequences
Tm is dependent on length and base composition of primers

Question 5

PCR elongation/extension

Answer 5

72 degrees C
DNA polymerase (Taq) binds to the annealed primers and extends DNA at the 3’ end of the chain.

Question 6

Primer details

Answer 6

ssDNA (oligonucleotides) about 18-25 by long.
PCR product depends on how far apart the annealing sites of the 2 primers are, can be up to 40kb but are usually 2kb or less.

Question 7

Importance of primer length

Answer 7

Shorter primers are not specific enough and may make incorrect matches, longer primers are more costly to make and offer little increase in specificity.

Question 8

Applications of PCR

Answer 8

Detect:
- rare sequences
- bacterial contaminants (listeria, e. coli)
- pathogens or endosymbionts (HIV, covid)
- forensics (evidentiary DNA)
- environmental DNA (eDNA)

Question 9

PCR exponential phase

Answer 9

Production is only limited to the amounts in previous cycle.

Question 10

PCR linear phase

Answer 10

dNTPs are less abundant and DNA pol may wear out.

Question 11

PCR plateau phase

Answer 11

Growth slows down greatly as dNTPs and polymerases are exhausted.

Question 12

Estimating the starting amount using PCR

Answer 12

Use the log-linear phase

Question 13

Quantitative PCR

Answer 13

Growth is monitored using a reporter dye that fluoresces more when bound to dsDNA. Usually SYBR Green - binds in the minor groove.

Question 14

qPCR applications

Answer 14

Quantify the abundance of a particular sequence in a sample.
Measure the rate at which a particular gene is transcribed, convert mRNA to cDNA first.

Question 15

Covid PCR test technical issues

Answer 15

Chemical contaminants interfere with Taq DNA.
Degradation of DNA by DNAses in nasal mucous.
Absence of SARS-CoV-2 DNA in sample.
Sourcing critical materials needed for the test are in short supply.