Molecular genetic techniques and applications

Question 1

At what temperature does heat denaturation take place during PCR?

Answer 1

94 degrees C

Question 2

At what temperature does primer annealing take place during PCR?

Answer 2

54 degrees C

Question 3

At what temperature does primer extension take place during PCR?

Answer 3

74 degrees C

Question 4

How might you use a PCR assay to detect differing product sizes eg detecting base deletions/additions

Answer 4

Amplify with PCR then use gel electrophoresis to compare size of product to control.

Question 5

How does an oligonucleotide ligation assay work and what is it used for?

Answer 5

Allele-specific mutant detection (i.e. figuring out which specific mutation you have). A specific primer is designed that will only ligate to the mutant allele (because it ligates at the point mutation). People positive for the mutation will have ligation products, people negative for it will not.

Question 6

What is multiplex ligation-dependant probe amplification analysis?

Answer 6

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 7

What is Northern blotting used for?

Answer 7

RNA

Question 8

What is Western blotting used for?

Answer 8

Protein

Question 9

What is Southern blotting used for?

Answer 9

DNA

Question 10

What molecular genetic technique uses dideoxynucleotides?

Answer 10

Sequencing

Question 11

What DNA is sequenced by clonal sequencing?

Answer 11

PCR products. Panels of selected genes known to be mutated for a particular phenotype. Very large exome panels based on all coding genes in the human genome.

Question 12

When is it appropraite to use genetic testing?

Answer 12

Rarely to confirm/refute diagnosis.

Assess carrier status.

Prenatal diagnosis.

Predictive testing.

Family history of particular disease e.g. BRCA1.