L6 - Genomic Technologies in Clinical Diagnostics

Question 1

How does PCR work?

Answer 1

DNA denaturation
annealing of primers
Extension of DNA strand

Question 2

What happens during fragment analysis and what can be diagnosed?

Answer 2

PCR is followed by capillary electrophoresis
The PCR product can be sized and can be used to detect repeat expansions or other small changes

It can be used to diagnose repeat expansion diseases like Huntingtons.

Question 3

What is Sanger sequencing?

Answer 3

cycle sequencing like PCR
Each of the 4 nucleotides has a different dye so we can determine the nucleotide sequence
slow and costly process

Question 4

What is FISH and what is it used for?

Answer 4

Fluorescent In situ Hybridisation

used to detect large chromosomal abnormalities, extra chromosomes, large deleted segments, translocation etc

Question 5

What is Array CGH?

Answer 5

Array comparative genomic hybridisation
For the detection of sub microscopic chromosomal abnormalities

Patient DNA is labelled Green and the control DNA is labelled Red

Question 6

What is MLPA?

Answer 6

Multiplex ligation dependent probe amplification.
It is a variation of PCR that permits amplification of multiple targets
Each probe consists of 2 oligonucleotides which recognise adjacent target sites on the DNA .
Used to detect abnormal copy numbers at specific chromosome locations
It can detect sub-microscopic gene deletions.
Fragment analysis can be performed
determines ploidy

Question 7

What is whole exome sequencing?

Answer 7

sequencing only the protein coding regions of the genome.
more efficient and costs less

Target enrichment is carried out whereby target regions are captured with baits

Question 8

What are some challenges of next generation sequencing?

Answer 8

results interpretation is challenging:

• 20,000 genetic variations in exome
• 3 million variants in a genome

Question 9

What are the ethical considerations of next generation sequencing?

Answer 9

patient consent process

reporting incidental findings

Question 10

How do we establish whether a mutation is pathogenic?

Answer 10

• mode of inheritance(does mutation follow the correct path of inheritance)
• genetic database(are there any published reports of the mutation)
• see what effects the mutations have (deletions, insertions or substitutions)