Overview Of Genomics Technologies In Clinical Diagnostics Flashcards

1
Q

What is PCR and what is it used for?

A

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

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2
Q

What are primers and what do they do?

A

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

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3
Q

What are + explain the 3 steps in PCR?

A

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

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4
Q

What is fragment analysis?

A

PCR based assay

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

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5
Q

What is fragment analysis used for?

A

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

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6
Q

Which disease is fragment analysis used for?

A

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

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7
Q

What is Huntington’s cause by?

A

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)

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8
Q

What is sanger sequencing?

A

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

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9
Q

What is the mechanism of sanger sequencing?

A

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

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10
Q

What is sanger sequencing used for?

A

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

  • can identify SNPs or mutations (monogenic single gene mutations)
  • detect mutations in family
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11
Q

What is the cause of cutaneous vasiculitis?

A

Mutation in gene C3

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12
Q

What is FISH?

A

Fluorescent in situ hybridisation

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13
Q

When can FISH be attached to cultured cells?

A

Metaphase spread so chromosomes are separated

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14
Q

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

A

Detects large chromosomal abnormalities (can be seen under microscope)

  • large deleted segments
  • translocations
  • extra chromosomes

e.g. trisomy 21 - down’s syndrome

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15
Q

What are the steps of FISH?

A

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

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16
Q

Give two examples of FISH uses?

A

Spectral karyotyping
Target specific FISH

both can be seen under miscroscope

17
Q

What is array CGH? and why is it used

A

Array comparative genomic hybridisation

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

18
Q

… green signal thing

A
19
Q

What is MLPA?

A

Multiplex ligation-dependent probe amplification

  • variation of PCR allowing amplification of multiple targets
20
Q

What is MLPA used for?

A

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

  • detects sub-microscopic gene deletions / partial gene deletions