L5 - Clinical applications of PCR

Question 1

How many PCR cycles is optimal?

Answer 1

30-35

Any more & the enzyme becomes less efficient so gets broken down

Question 2

What are the 3 steps of PCR?

Answer 2

DENATURE
95 degrees

ANNEAL
50-60 degrees

EXTENSION/ELONGATION
75 degrees

Question 3

Why is PCR valuable?

Answer 3

Sensitive 
Specific 
Cheap 
Rapid 
Robust

Question 4

Common uses of PCR in determining diagnosis & prognosis

Answer 4

Genotyping the patient

Genotyping the pathogen

Phenotyping the disease

Question 5

Genotyping the patient

Answer 5

– Diagnosis of genetic traits
– Detection of carriers of genetic traits
– Tissue matching – HLA typing
– Predicting responses to drugs – pharmacogenetics

Question 6

Genotyping the pathogen

Answer 6

– Diagnosis of species & strain of infecting pathogen

Question 7

Phenotyping the disease

Answer 7

– Measuring disease progression

– Measuring disease severity

Question 8

Sources of DNA when genotyping the patient?

Answer 8

• Blood
• Hair
• Buccal smear
• Cells from amniotic fluid

Question 9

What are the 2 PCR-based techniques for genotyping an individual?

Answer 9

PCR-RFLP – Restriction Fragment Polymorphism

ARMS-PCR – Amplification Refractory Mutation System

Question 10

What is an allele?

Answer 10

Any of the alternative forms of a gene that may occur at a given locus

Question 11

What is a restriction enzyme?

Answer 11

An enzyme that digests (cuts) DNA at a highly specific site

Question 12

PCR-RFLP – Restriction Fragment Polymorphism

Answer 12

The first step in a PCR-RFLP analysis is amplification of a fragment containing the variation

This is followed by treatment of the amplified fragment with an appropriate restriction enzyme

Since the presence or absence of the restriction enzyme recognition site results in the formation of restriction fragments of different sizes, allele identification can be done by electrophoretic resolvement of the fragments

Question 13

Advantages of PCR-RFLP

Answer 13

• Cheap
• Easy design
• Applied to microindels & SNPs
• Simple resources
• Commonly used technique

Question 14

Disadvantages of PCR-RFLP

Answer 14

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

Question 15

ARMS-PCR - Amplification Refractory Mutation System

Answer 15

Detects allelic variants using allele-specific primers

Involves manipulation of the primer for the allele of interest

Question 16

RFLP vs ARMS

Answer 16

Both relatively cheap

PCR-RFLP
• Uses locus specific primers
• Relies on the presence or absence of a restriction site to distinguish between variants

ARMS-PCR
• Uses allele specific primers
• Relies on the stringency of the PCR to distinguish between alleles
• Alternative is Tetra Primer ARMS-PCR which uses additional non-allele specific primers

Question 17

Sources of DNA when genotyping the pathogen?

Answer 17

DNA has to of been exposed to pathogen

• Blood
• Sputum
• Urine
• Faeces
• Skin swab
• Tissue biopsy

Question 18

What will the information from genotyping a pathogen influence?

Answer 18

Patient management – eg. choice of treatment

Infection control measures

Question 19

Advantage of PCR over some other methods of microbial diagnosis

Answer 19

Sensitive – can detect single copy of genome

Specific – can identify species & strain

Sensitivity means no need for culture
PCR takes a few hours

Detects DNA/RNA therefore not dependent on the immune response

Question 20

How does phenotyping the disease help us?

Answer 20

Can help us to tell:
• How severe is the disease?
• How is the disease likely to progress?

Question 21

What is the key technique in disease phenotyping?

Answer 21

Quantitative PCR – measures the abundance of DNA or RNA in a clinical sample

To measure the level of infectious pathogen in a sample

To measure the level of expression of a gene (RT-PCR)

Question 22

How do you measure the level of expression of a gene?

Answer 22

Using RT-PCR

To measure RNA by PCR, it must first be converted to cDNA by reverse transcriptase

The amount of DNA product after each cycle is proportional to the amount of RNA initially present

Question 23

What are the 4 PCR techniques?

Answer 23

PCR-RFLP

ARMS-PCR

RT-PCR – PCR from RNA

Real-Time PCR – quantitative PCR