Overview of Genomic Technologies in Clinical Diagnostics

Question 1

List current gene technologies used in diagnostics

Answer 1

• PCR
• FISH
• Array CGH
• Sanger Sequencing
• NGS

Question 2

Summarise how PCR works

Answer 2

primers flank target sequence
1. denaturation
2. annealing
3. extension = amplification of target sequence

Question 3

What are some downstream applications of PCR?

Answer 3

• capillary electrophoresis allows individual separation of different sizes of fragments and identifying repeat expansions
• Sanger sequencing allows detection of SNPs in exon sequencing

Question 4

What sort of diseases is Sanger sequences useful in detecting?

Answer 4

single monogenic disease

Question 5

Summarise the main principles of FISH

Answer 5

fluorescent in-situ hybridisation
1. produce fluorescent probe
2. denature
3. hybridisation of probe and target DNA
4. detect fluorescence

Question 6

What is a common application of FISH?

Answer 6

• spectral karyotyping
• detecting large chromosomal abnormalities (eg: Downs)

Question 7

Summarise the principles of Array-CGH

Answer 7

patient and control DNA have fluorescent tags.
patient = red
control: green
patient and control compute to hybridise with microarrays in wells
microarray senses fluorescent signals
software processes data and generates plot
yellow = equal hybridisation
red = dosage loss (detectable on plot)
green = dosage gain

Question 8

What is a useful application of Array-CGH

Answer 8

sub-microscopic chromosomal abnormalities

Question 9

What is MLPA?

Answer 9

• specific gene analysis
• looks for small deletions
  1. - oligonucleotides: forward and reverse
• hybridise probes ot DNA
  2. - ligation and join forward and reverse probes
  3. - PCR amplification of probe = provides library of target
• Fragment analysis / capillary electrophoresis = sizing products and dosing
• relative ploidy of specific targets

Question 10

State how NGS works and the potentials in working with it

Answer 10

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