CMB2000/L04 Types of PCR

Question 1

Name the reactants of PCR. (6)

Answer 1

DNA template
Primers
Nucleotides
Taq polymerase
Buffer
MgCl2

Question 2

What is the role of MgCl2 in PCR?

Answer 2

Essential for Taq activity
[Mg2+] affects stringency of primer binding

Question 3

Give the 3 steps of a PCR cycle.

Answer 3

Add excess primers & heat to separate strands
Cool to anneal primers
Synthesise new DNA

Question 4

Give 3 common uses of PCR.

Answer 4

Genotyping the patient
Genotyping the pathogen
Phenotyping the disease

Question 5

Describe genotyping the patient.

Answer 5

Detects which alleles an individual is carrying for a specific gene

Question 6

Give 2 PCR-based techniques for genotyping an individual.

Answer 6

PCR-RFLP
ARMS-PCR

Question 7

What is PCR-RFLP?

Answer 7

Restriction Fragment Polymorphism
Identifies allelic variants based on presence/absence of a restriction site

Question 8

What are the 3 steps of PCR-RFLP?

Answer 8

Amplify
Cut PCR product with restriction enzyme R
Size-fractionate by gel electrophoresis

Question 9

What would occur in PCR-RFLP if one allele has a restriction site for a specific restriction endonuclease and another allele did not?

Answer 9

RE site produces 3 products
a+b, a and b

Question 10

What is the BamHI recognition site?

Answer 10

GGATCC

Question 11

What is Sorsby’s Fundus Dystrophy?

Answer 11

Degenerative eye disease leading to blindness
Autosomal dominant
Mutation in TIMP3 (tissue inhibitor of metalloproteinase 3) gene introduces premature stop codon

Question 12

Give 3 advantages of PCR-RFLP.

Answer 12

Cheap
Easy design
Applied to microindels and SNPs
Simple resources
Commonly used techniques

Question 13

Give 3 disadvantages of PCR-RFLP.

Answer 13

Only possible if site contains a known RE site
Some RE are expensive
Only possible if a single nucleotide variation
Hands on and time consuming
Not suitable for high-throughput

Question 14

Define genotyping the patient.

Answer 14

Determine which alleles an individual carries for a specific gene (or set of genes)

Question 15

What 2 PCR techniques are used for genotyping an individual?

Answer 15

PCR-RFLP
ARMS-PCR

Question 16

Describe ARMS-PCR (amplification refractory mutation system).

Answer 16

Simple method for detecting any mutation involving single base changes or small deletions present
OR
Absence of a PCR product diagnostic for the presence or absence of the target allele

Question 17

Describe the process of ARMS-PCR. (2)

Answer 17

Design allele-specific primers
PCR with ASP1 or ASP2 + conserved primer

Question 18

How are primers in ARMS-PCR different to normal PCR?

Answer 18

G-C at the end is not possible

Question 19

Define cystic fibrosis.

Answer 19

Mutation in CFTR gene leads to imbalances in Cl- transport across plasma membrane
F508 mutation most common cause

Question 20

Compare the primers used in PCR-RFLP and ARMS-PCR.

Answer 20

RFLP-PCR - uses locus specific primers
Relies on presence or absence of restriction site
ARMS-PCR - allele specific primers
Relies on stringency of PCR to distinguish between alleles

Question 21

Give an alternative to ARMS-PCR.

Answer 21

Tetra Primer ARMS-PCR
Uses non-allele specific primers

Question 22

Define genotyping the pathogen.

Answer 22

Identifies the species and strain of an infectious pathogen by isolating a specific gene/piece of DNA

Question 23

What does the pathogen genotype influence in healthcare? (2)

Answer 23

Patient management e.g., choice of treatment
Infection control measures

Question 24

Describe diagnosis of TB using pathogen genotyping.

Answer 24

Conclusive diagnosis depends on detection of TB in sputum
Previous depended on microscopy and culture
Can achieve same-day diagnosis using PCR

Question 25

How is quantitative PCR used?

Answer 25

To measure the abundance of DNA or RNA in a clinical sample

Question 26

Describe RT-PCR.

Answer 26

Must first be converted to cDNA by reverse transcriptase
Amount of DNA product after each cycle is proportional to amount of RNA initially present

Question 27

Describe Real-Time qPCR.

Answer 27

PCR product measured as it is produced
Cycle number at which fluorescence reaches threshold value is measured (Ct value)
Lower Ct value = greater quantity of DNA/cDNA in starting template