PCR -L3

Question 1

What are the uses of PCR in determining diagnosis and prognosis

Answer 1

Genotyping the patient, genotyping the pathogen, phenotyping the diseases

Question 2

What does genotyping the patient mean

Answer 2

Detects which alleles an individual in carrying for a specific gene

Question 3

What are the uses of genotyping the patient

Answer 3

Diagnosis of genetic traits, detection of carriers of genetic traits, tissue matching, predicting response to drugs

Question 4

What are the sources of DNA for patient genotyping

Answer 4

blood, hair, buccal smear, cells from amniotic fluid

Question 5

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

Answer 5

PCR-RFLP and ARMS-PCR

Question 6

What is PCR-RFLP?

Answer 6

Restriction fragment polymorphism.

Question 7

What does PCR-RFLP do?

Answer 7

Identifies allelic variants based on presence/absence of restriction site

Question 8

What are the steps in PCR-RFLP?

Answer 8

1. amplify the substrate, 2. add the RE and then analyse via electrophoresis

Question 9

What does it mean if RE is in both products?

Answer 9

Homozygous for the disease allele

Question 10

What does it mean if RE is in neither products?

Answer 10

Homozygous for the healthy allele

Question 11

What does it mean if RE is in one product?

Answer 11

heterozygous

Question 12

Give a clinical example of a diagnosis via PCR-RFLP

Answer 12

Sorsby’s Fundus Dystrophy

Question 13

What is the mutation in Sorsby’s Fundus Dystrophy

Answer 13

TIMP3 - premature stop codon.

Question 14

Advantages of PCR-RFLP

Answer 14

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

Question 15

Disadvantages of PCR-RFLP

Answer 15

Only possible if the 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 16

What is ARMS-PCR

Answer 16

Amplification refractory mutation system

Question 17

How does ARMS-PCR detect variation?

Answer 17

Detects allelic variants using allele-specific primers

Question 18

Give a clinical example of a diagnosis via ARMS-PCR

Answer 18

Cystic Fibrosis

Question 19

What is the mutation in CF?

Answer 19

CFTR gene = imbalance in Cl-

Question 20

Most common CF mutation?

Answer 20

F508

Question 21

What does primer A include in CF?

Answer 21

includes primer for mutant F508 deletion

Question 22

What does primer B include in CF?

Answer 22

includes primer for wt at this position

Question 23

Compare RFLP and ARMS

Answer 23

PCR-RFLP
* Uses locus specific primers (i.e. will amplify all variants of the chosen DNA
sequence)
* 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 24

How is genotyping used in relation to pathogen?

Answer 24

Identifies the species and strain of an infectious pathogen (bacteria, virus or
parasite) by isolating a specific gene/piece of DNA

Question 25

Where can DNA/RNA be obtained for pathogen genotyping

Answer 25

Blood, septum, urine, faeces, skin swab, tissue biopsy

Question 26

What areas of treatment can be impacted by the information obtained from genotyping the pathogen

Answer 26

Patient management and infection control measures

Question 27

What are the advantages of PCR > microscope

Answer 27

PCR: Sensitive – can detect single copy of genome Specific – can identify species and strain Microscope: Requires high levels of infecting organism Often difficult to distinguish different species Electron microscopy required to visualise viruses

Question 28

What are the advantages of PCR > culture

Answer 28

PCR: Sensitivity means no need for culture. PCR shorter (few hours) Culture: Some org = cannot be cultured, can take weeks and hard to be strain specific

Question 29

PCR vs Patient AB response

Answer 29

PCR: Detects DNA/RNA therefore not dependent on immune response Genotyping the pathogen Patient Ab response: Pathogen may not elicit a strong antibody response

Question 30

Give a clinical example of a diagnosis using pathogen genotyping

Answer 30

TB

Question 31

Most common gene used in TB?

Answer 31

IS6110

Question 32

What does diagnosis of TB depend on?

Answer 32

Conclusive diagnosis depends on detection of M. tuberculosis in sputum

Question 33

Why do we phenotype the disease?

Answer 33

Shows us how severe it is and how likely disease is to proggress

Question 34

What sort of PCR technique is used in phenotyping disease?

Answer 34

qPCR

Question 35

Why do we use qPmCR?

Answer 35

measures the abundance of DNA or RNA in a clinical sample

Question 36

What do we measure using qPCR?

Answer 36

to measure the level of infectious pathogen in a sample to measure the level of expression of a gene Phenotyping the disease – taking a “snapshot” in time

Question 37

What do we use to measure level of a gene exrpression?

Answer 37

RT-PCR

Question 38

True or false: DNA and cDNA can be accurately quantified by real-Time qPCR

Answer 38

True

Question 39

Give a clinical example of using RT-PCR

Answer 39

measurement of HIV viral load by quantitative RT-PCR