Overview Of Genomics Technologies In Clinical Diagnostics

Question 1

What is PCR and what is it used for?

Answer 1

Polymerase chain reaction is used to amplify a specific region of DNA

Question 2

What are primers and what do they do?

Answer 2

Primers are complementary to regions we want to amplify in PCR. They flank the region.

Question 3

What are + explain the 3 steps in PCR?

Answer 3

Denaturation - 90 degrees - high temp permits DNA strand separation
Annealing - 50 - 65 degrees - cooler temp allows primers to attach to ends of target sequence
Extension - 72 degrees - allows thermostable polymerase to add nucleotides at 3’ end of primer

Question 4

What is fragment analysis?

Answer 4

PCR based assay

PCR followed by capillary electrophoresis - separates DNA in PCR product by size

Question 5

What is fragment analysis used for?

Answer 5

detect repeat expansion or other small changes e.g. if more than one product present when only 1 in specific amount should be

diagnosing repeat expansion disease

Question 6

Which disease is fragment analysis used for?

Answer 6

repeat expansion disease e.g. Huntington’s disease (neurodegenerative disorder)

Question 7

What is Huntington’s cause by?

Answer 7

CAG repeat expansion on Huntington (HTT) gene
- above certain level pathogenic copies (protein produced is basic, too much protein produced - accumulates in neurons causing cell death)

Question 8

What is sanger sequencing?

Answer 8

Cycle sequencing based off of PCR (sequences the PCR product)

Question 9

What is the mechanism of sanger sequencing?

Answer 9

Each of 4 nucleotides has diff dye so can detect nucleotide sequence

Question 10

What is sanger sequencing used for?

Answer 10

Sequencing single exons of genes (sequencing whole genome is costly)

• can identify SNPs or mutations (monogenic single gene mutations)
• detect mutations in family

Question 11

What is the cause of cutaneous vasiculitis?

Answer 11

Mutation in gene C3

Question 12

What is FISH?

Answer 12

Fluorescent in situ hybridisation

Question 13

When can FISH be attached to cultured cells?

Answer 13

Metaphase spread so chromosomes are separated

Question 14

What is the use of FISH and what does it detect? example

Answer 14

Detects large chromosomal abnormalities (can be seen under microscope)

• large deleted segments
• translocations
• extra chromosomes

e.g. trisomy 21 - down’s syndrome

Question 15

What are the steps of FISH?

Answer 15

1) design fluorescent probe to chromosomal region of interest
2) denature probe and target DNA
3) Mix probe and target DNA (hybridisation)
4) Probe binds to target
5) Target flurosces/lights up

Question 16

Give two examples of FISH uses?

Answer 16

Spectral karyotyping
Target specific FISH

both can be seen under miscroscope

Question 17

What is array CGH? and why is it used

Answer 17

Array comparative genomic hybridisation

detects sub microscopic chromosomal abnormalities - too small to be detected by FISH

Question 18

… green signal thing

Question 19

What is MLPA?

Answer 19

Multiplex ligation-dependent probe amplification

• variation of PCR allowing amplification of multiple targets

Question 20

What is MLPA used for?

Answer 20

Detects abnormal copy numbers at specific chromosome locations (not across entire genome)

• detects sub-microscopic gene deletions / partial gene deletions