11 - Mutation Detection 1

Question 1

Steps of PCR

Answer 1

• Denaturing (separating double stranded DNA at 95ºC)
• Annealing (facilitating primer attachment to template strands at 55-65ºC)
• Elongation (synthesis of new DNA strands from primers by Taq polymerase at 72ºC )
• Repeat

Question 2

Real time PCR allelic discrimination

Answer 2

• Fluorescent molecule used to indicate replication of sequence during PCR
• Uses Taqman technology
• Different colours distinguish different variants

Question 3

Which strands do forward and reverse primers bind to

Answer 3

Forward: Template strand
Reverse: Coding strand

Question 4

Taqman technology

Answer 4

• Probe has a flourophore and a quencher bound
• Fluorescence only occurs when they are no longer in proximity

Question 5

Steps of Real time PCR (Allelic discrimination)

Answer 5

• Probe binds to target sequencing containing SNP
• During PCR, polymerase removes the fluorophore
• Fluorescent signal indicates SNP sequence
• If variant wasn’t present, probe would never bind and therefore wouldn’t fluoresce

Question 6

Advantages of real time PCR (Allelic discrimination)

Answer 6

• Discriminate between genotypes
• Multiple samples analysed simultaneously

Question 7

Melt curve analysis

Answer 7

• Changes in sequence lead to changes in Tm.
• Mutant sequences have different Tm (become ss at different temp to wild type)
• when dsDNA denatures to ssDNA, no more fluorescence (dye binds to dsDNA only)
• measured at 50/50 ratio

Question 8

Heteroduplex

Answer 8

Heterozygotes have differently shaped
melt curve (dip). Combination of mutant and wildtype

Question 9

Southern blot

Answer 9

• Digest DNA with restriction enzymes
• Seperate DNA fragments by gel electrophoresis
• Hybridise probe to complementary target sequence
• Enzyme conjugated to probe produces luminescent product

Question 10

RFLP

Answer 10

• Restriction Fragment Length Polymorphism
• Mutations can create or destroy restriction enzyme sites which are detected via southern blot.

Question 11

Disadvantage of RFLP

Answer 11

• Not all mutations will cause changes to create or destroy RE sites
• Next gen sequencing has issues with repetitive sequences, RFLP can be used

Question 12

Microarray

Answer 12

Fixed DNA sequences which hybridise with fragmented labelled target DNA. can look at many variants simultaneously

Question 13

Steps of microarray

Answer 13

• Oligonucleotide probes vary in sequence and immobilised on chip
• DNA sample is labelled by fluorescent label
• Fragmented DNA hybridises with immobilised probes

Question 14

Applications of microarray

Answer 14

• Comparative Genome Hybridisation
• Expression array (mRNA –> cDNA, cDNA binds to chip, shows what is expressed)
• SNP detection / mutation analysis
• common pathogenic SNPs

Question 15

Types of pathogenic variants

Answer 15

• Single gene (mandelian) disease (mutation in one gene responsible for disease phenotype. CF, Sickle cell)
• Multifactoral diseases (influenced by more than one gene, environmental causes, type 2 diabetes)

Question 16

WGS advantages

Answer 16

Don’t need to have target sequence, or even disease of interest to be able to identify mutant

Question 17

Ethical dilemmas of WGS

Answer 17

• Not testing for a specific mutation or disease
• Many revealed disorders will have no prevention or treatment (BRCA in infants)
• Variants of Uncertain Significance (VUS)