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Year 2 EMS GiM > Molecular Genetics - techniques and applications > Flashcards

Molecular Genetics - techniques and applications Flashcards

1
Q

Which common technologies are based on DNA:DNA hybridisation? (2)

A
  • PCR

- Ligation assay

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2
Q

What are the main features of PCR?

A
  • Synthesis of large amounts of DNA by copying
  • Primers define boundaries
  • DNA polymerase = enzyme
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3
Q

Describe the basic process of PCR.

A

Heat denaturation (94)
» primer annealing (55)
» primer extension (72*)
» REPEAT

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4
Q

In what direction are nucleotides added in PCR?

A

5’ to 3’.

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5
Q

How are PCR products separated for analysis?

A

Gel electrophoresis.

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6
Q

What are the main functions of PCR?

A
  • Determine presence / absence of product

- Determine product size

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7
Q

What type of PCR determines if a product is present / absent?

A

Allele-specific PCR.

-must know disease-causing point mutation and normal allele

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8
Q

What is a common mutation in CF?

A

F508 deletion.

- 3bp from CTFR

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9
Q

What does cystic fibrosis testing on newborns involve?

A

Immunoreactive trypsin protocol.

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10
Q

What mutation is the commonest cause of inherited deafness?

A 
Connexin 26 (GJB2). 
-PCR size analysis can detect base deletion
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11
Q

What is ligation?

A

Joining of 2 DNA strand by phosphate ester linkage.

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12
Q

What happens in an oligonucleotide ligation assay if there is a point mutation at the ligation site?

A

No ligation product forms.

-ligation fails for both primers due to mis-match

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13
Q

What is the cystic fibrosis genotyping assay?

A

Quantitative in vitro test to genotype a panel of mutations in CFTR.
-used for carrier screening

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14
Q

What are the results of the cystic fibrosis genotyping assay not used for?

A
  • Foetal diagnosis

- Stand-alone diagnosis

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15
Q

What does MLPA analysis stand for?

A

Multiplex Ligation-dependent Probe Amplification analysis.

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16
Q

What is MLPA analysis?

A

Multiplex PCR for detecting chromosomal DNA copy number changes in targets.

17
Q

How does MLPA analysis work?

A

2 Probes contain forward and reverse sequences.

  • forward is fluorescently labelled
  • probes are hybridised against target DNA
  • ligation / amplification only occurs if target DNA is present
18
Q

Is MLPA analysis a qualitative or quantitative test?

A

Quantitative test.

-PCR product is proportional to amount of target DNA present

19
Q

When is PCR unsuitable?

A
  • Gene too big

- GC-rich regions

20
Q

Why is fragile X syndrome with a mutation in FMR1 gene unsuitable for PCR?

A

Repetitive CGG sequence.

-GC-rich regions hard for PCR

21
Q

What method can be used if PCR is unsuitable?

A

Southern blotting.

22
Q

What are the different blotting methods? (3)

A

Southern - DNA
Northern - RNA
Western - protein

23
Q

What is Southern blotting used for?

A

Detection of a specific DNA sequence in DNA samples.

24
Q

How is DNA sequenced?

A 
-DNA produced by PCR
>>use primers to anneal strands
>> strand synthesis using DNA polymerase and dNTPS
>> small amount of ddNTPS 
>> strands of different lengths produced
25
Q

What does dNTP and ddNTP stand for?

A

dNTP - deoxynucleotide

ddNTP - dideoxynucleotide

26
Q

How does ddNTP lead to the termination of strand synthesis?

A

ddNTP lacks 3’ hydroxyl group&raquo_space; phosphodiester bond not formed.

27
Q

At what end of does the strand terminate when ddNTPs are incorporated?

A

3’ end.

28
Q

Do the fragments move towards the positive or negative charge in gel electorphoresis?

A

Move towards the positive charge.

  • ‘pulls’ them towards it
  • smaller fragments travel further
29
Q

What disorder is MEGF10 screened for?

A

Muscular dystrophies.

30
Q

What is another name for clonal sequencing?

A

‘Next generation’ sequencing.

  • non-Sanger method
  • billions of DNA strands sequenced in parallel
31
Q

Describe the stages of clonal sequencing. (6)

A
  • Prepare DNA sample
  • Attach DNA to surface
  • Bridge amplification
  • Fragments become double stranded
  • Denature double strands
  • Complete amplification&raquo_space; millions of DNA clusters
32
Q

What DNA is sequenced by clonal sequencing?

A
  • PCR products
  • Panels of genes known to be mutated
  • Large ‘exome’ panels
33
Q

What is ‘1000 genomes’?

A

Genomes of 1092 individuals from 14 populations.

-helps show variation and understand genetics contribution to disease

Year 2 EMS GiM (12 decks)

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