Overview of genomic technologies in clinical diagnostics

Question 1

What is PCR?

Answer 1

Used to amplify a specific region of DNA

Primers flank the region you want to amplify

Each cycle doubles the amount of DNA

Aim is to amplify enough DNA molecules so that you have enough material to perform applications on

Question 2

What is fragment analysis?

Answer 2

PCR based assay which you will analyse

After PCR use capillary electrophoresis to separate DNA molecules by size

Can be used to detect repeat expansion or other small size changes

Used to detect Huntington’s disease

• Caused by CAG repeat expansion in the Huntington gene
• Expanded protein is toxic and accumulates in neurones causing cell death

Question 3

What is sanger sequencing?

Answer 3

Sequence them after PCR

Each of the nucleotides have different dye so can determine the sequence

Up to 800bp of sequence per reaction - used for exons of genes

Slow, low-throughput and costly to perform

Used to identify single nucleotide polymorphisms or mutations

Question 4

What is FISH?

Answer 4

Detects large chromosomal abnormalities by culturing cells and spreading them under a microscope

• Extra chromosomes
• Large deleted segments
• Translocations

1. Design fluorescent probe to chromosomal region of interest
2. Denature probe and target DNA
3. Mix probe and target DNA
4. Probe binds to target
5. Target will fluoresces

Question 5

What is array CGH?

Answer 5

For detection of sub-microscopic chromosomal abnormalities

• Too small to see with FISH

Patient DNA is green and control DNA is red - they are mixed

On a microarray you will see the signal detected

• Can see if dosage loss or gain

Increased green signal indicates a gain in the patient sample not present in the parents

Question 6

What is MLPA?

Answer 6

Is a variation of PCR that permits amplification of multiple targets

Used to detect abnormal copy numbers at specific chromosomal locations

Can detect sub-microscopic gene deletions/partial gene deletions

Each probe consists of two oligonucleotides which recognise adjacent target sites on DNA

• Forward primer
• Reverse primer
  
  • Only when both probe oligonucleotides are hybridized to their respective targets can they be ligated into a complete probe

From the amplified library perform fragment anaylsis - used to determine relative ploidy (how many chromosome copies) at specific locations

Question 7

What is next generation sequencing?

Answer 7

Wider range of tests in shorter time for less money

Current strategy: disease panels

• Sequence only known disease genes relevant to the phenotype
• Then expandable to include new genes as they are published
• Variants confirmed by sanger sequencing

Exome sequencing - only interested in this so more cost effective

• Using target enrichment - to capture target regions of interest with baits

100,000 genomes project - benefit of whole genome sequencing and geneticsto patients - personalised medicine

Question 8

What happens in the NHS diagnostic lab?

Answer 8

• Help consultants reach a genetic diagnosis for individuals to help guide treatment and clinical management
• Clinical validity - how well the test predicts the phenotype
• Clinical utility - how useful the test adds to the management of the patient
• Outcomes: pathogenic mutation, normal variation or novel variant (not clear on clinical significance)

Question 9

What is huntington’s disease?

Answer 9

Used to detect Huntington’s disease

Caused by CAG repeat expansion in the Huntington gene

Expanded protein is toxic and accumulates in neurones causing cell death

• deteteted by fragment analaysis