Week 3

Question 1

Polymerase Chain reaction

Answer 1

Technique used to amplify a single copy or a few copies of a segment of DNA a massive amount of repeats. Can be used to investigate size of a region of DNA sequence.

Question 2

Sanger sequencing

Answer 2

Chain termination method, is a technique for DNA sequencing based upon selective incorporation of chain terminating dideoxynucleotides (ddNTPs) by DNA polymerase during in vitro DNA replication.

Question 3

Exome sequencing

Answer 3

1. Select only the subset of DNA that encodes proteins. These regions are known as exon - constituting 1% human genome.
2. Sequence the exonic DNA using any high-throughput DNA sequencing technology.

Question 4

Whole genome sequencing

Answer 4

Determining the DNA samples identity of the 3 billion nucleotides that compose the human genome. (it can identify indels,

Question 5

Targeted gene sequencing panels

Answer 5

• Analysing specific mutations in a given sample.
• Focused panels contain select set of genes with suspected associations with the disease or phenotype under study. Gene panels can be purchased with preselected content or custom designed to include genomic regions of interest.

Question 6

SNP array

Answer 6

Type of DNA microarray that constitutes a powerful tool for high-throughput analysis of thousands of SNPs in a single experiment to globally analyse the human genome for genetic alteration. Often used to identify disease causing genes.

Question 7

TaqMan chemistry

Answer 7

• Fast and simple way to get SNP genotyping results.
• Two primers and probes for each SNP.
• Specific and Sensitive.

Question 8

One SNP in gene X is associated with hypersensitivity with drug A. Which technique would not be appropriate to detect the variant?

• MLPA
• Allele specific PCR (TaqMans assay)
• Sanger sequencing
• Pyrosequencing

Answer 8

MLPA

Allele specific PCR would be used - quick, cheap, no specialist equipment needed. A targeted method.

Question 9

Why is MLPA appropriate to look at exon deletions?

Answer 9

• It can detect whole exon deletions and duplications in a quick assay.
• Southern blot could also be used but is more laborious and time consuming.
  Sanger and pyro-sequencing won’t detect deletions

Question 10

Why would next generations sequencing at specific custom panels be used to look for 290 SNPs in 15 genes, to eliminate people viable for an expensive drug?

Answer 10

• Expensive however so is the drug.
• Using a next gen panel will identify all variants in the genes in questions and additional genes can be added to the panel easily.
• Sanger and exome could also work but Sanger is inefficient for this number of genes, exome is overkill.
• ArrayCGH is used to identify chromosomal deletions and duplications.

Question 11

Why would BEAMing be used to find 5 SNPS in a hard to biopsy tumour?

Answer 11

Looking at circulating tumour DNA in a blood sample
Very high sensitivity would therefore be required. Next generation sequencing is over kill and slow with only 5 SNPs . Droplet digit PCR would also work.

Question 12

What does MLPA stand for?

Answer 12

Multiple ligation-dependent probe amplification

Question 13

What is MLPA used for?

Answer 13

Amplification of multiple target genes, detects whole exon deletions and duplications.

Question 14

What is target for gene sequencing panels?

Answer 14

It can identify SNPs, INDELs in specific regions.

Question 15

When testing 290 SNPs in 15 genes, what method would be best for an expensive drug?

Answer 15

Next generation sequencing, Exome is overkill

Question 16

Why would the Sanger sequencing method not be used for lots of SNPs in loads of genes?

Answer 16

Inefficient for this number of genes.