Molecular Genetic Techniques (3)

Question 1

What is PCR?

Answer 1

In vitro synthesis of large amounts of DNA by copying from small starting quantities

Question 2

What are oligonucleotides?

Answer 2

Small synthetic primers that define the boundaries of synthesis

Question 3

Dna is synthesised by DNA

Answer 3

‘polymerase’ enzymes from ‘monomers’ (deoxy-ribonucleotides)

Question 4

Gel electrophoresis

Answer 4

Separates the PCR product, stained DNA visualised by UV light, determines size of product (oligonulecotide ligation assay/direct sequencing)

Question 5

What does PCR do?

Answer 5

Determines the presence/absence of product (allele-specific PCR)

Question 6

Steps of PCR

Answer 6

1) Heat denaturation (94 degrees)
2) Primer annealing (55 degrees)
3) Primer extension (72 degrees)

Question 7

What do you need for PCR?

Answer 7

• An excess of primer, nucleotides and enzyme

- Priming sites

Question 8

CF mutation F508del

Answer 8

• 3 bp deletion eliminates single aa from CFTR (75% alleles)

- Cystic fibrosis genotype assay

Question 9

Most common inherited cause of deafness

Answer 9

Connexion 26 (GJB2) Δ35G - single base deletion

Question 10

Multiplex Ligation-dependent Probe Amplification (MLPA) analysis

Answer 10

• Multiplex PCR method for detecting chromosomal DNA copy number changes in multiple targets
• Two adjacent probes are designed that contain the forward and reverse primer sequences for PCR
• Forward primer is fluorescently labeled so that the amplicons can be visualized using gel electrophoresis
• Length of “stuffer” sequence can be varied depending on the experiment
• Probes are hybridized against the target DNA and subsequently ligated
• If ligation occurs then a functional PCR strand is created, so that amplification only happens if the target DNA is present in the sample
• Multiple probe pairs can be pooled and amplified with the same primer pair

Question 11

Difficulties in mutation analysis

Answer 11

• Gene might be too big for PCR
• Fragile X mutation in FMR1 gene is repetitive tract of (CGG)n sequence
• GC-rich regions are difficulty to PCR
• Can use Southern blotting instead

Question 12

Northern blotting

Answer 12

RNA

Question 13

Southern blotting

Answer 13

DNA

Question 14

Western blotting

Answer 14

Protein

Question 15

DNA sequencing/Sanger method

Answer 15

PCR style with fluoresecently marked ddNTP

Question 16

ddNTP

Answer 16

Lacks 3’OH group that is needed to form a phosphodiester bond with next nucleotide phosphate (OH > H)

Question 17

Sequencing gel

Answer 17

Use of electric field, largest negative > positive smallest

Question 18

Mutations in MEGF10

Answer 18

Regulator of satellite cell myogenesis > early onset myopathy, arflexia, respiratory distress and dysphagia (EMARDD)

Question 19

Colonal sequencing

Answer 19

• PCR product longer/cover part or whole of a gene
• Panels of selected genes known to be mutated for particular phenotype
• Large ‘exam’ panels, based on all coding genes of human genome of clinical relevance

Question 20

Why DNA test?

Answer 20

• Confirm/refute clinical diagnosis
• Assess carrier status
• Prenatal diagnosis
• Predictive testing