GENOMICS REVISION PART 2

Question 1

What is the difference between a missense and a nonsense variant?

Answer 1

A missense variant is when there is a base substitution where you replace one amino acid with a different one which may or may not produce a malfunctioning protein. A nonsense variation is where the amino acid is replaced with a stop codon and so the aa chain is stopped prematurely.

Question 2

Which are the most dangerous/ pathogenic mutations (null mutations)?

Answer 2

Nonsense variation and frameshift variants

Question 3

What is the difference between a variant, mutation and polymorphism?

Answer 3

Variant- any frequency, no assumption of clinical significance.
Mutation- rare, disease causing?
Polymorphism- common, benign?

Question 4

Explain the difference between PCR, Sanger sequencing and next generation sequencing (NGS)

Answer 4

PCR is used to amplify large quantities of a specific sequence of DNA from an initially minute sample. Firstly head to separate stands then cooled back down for primers to attach and Taq polymerase enzyme used and for this is heated back up (75o) for Taq to function.

Sanger sequencing- Where dideonucleotides are used instead of deoxyribose so ends the chain. 4 PCR mixes are then used to wuth one dideo on all of either A,T,G or C. so it then stops at each point and you can match it up.

Next generation sequencing- while the sanger method only sequences a single DNA fragment at a time. NGS is massively parallel, sequencing millions of fragments simultaneously per run.

Question 5

Out of single gene sequencing, gene parallel sequencing, whole exome sequencing and whole genome sequencing what is the difference between them and when are they used?

Answer 5

single gene sequencing- When you’re sure or expecting an exact gene mutation usually done by sanger sequencing. Cheap and quick

gene parallel sequencing- e.g. lynch syndrome 1. MLH1 2. MLH2 3. MLH6 4. PMS2 so you basically just test for the genes you are expecting. Good read depth

whole exome sequencing- sequencing just the exons advantages over whole genome sequencing: cheaper and has a better reading depth so has more detail than breath so can help to detect mosaicism

whole genome sequencing- use NGS whereas in in whole exome sequencing you use a technique called capturing. Has poor read depth but can detect structural re-arrangements.

Question 6

What are the two types of genetic testing pre-natally?

Answer 6

Chorionic Villi Sampling
Chorionic villi sampling involves removing a sample of the chorionic villus (placental tissue) via a tube inserted through the cervix
 It can be done at ~11 weeks of pregnancy with a slight risk of inducing miscarriage (~1%)

Amniocentesis
Amniocentesis involves the extraction of a small amount of amniotic fluid (contains fetal cells) with a needle
 It is usually conducted later than CVS (~16 weeks of pregnancy) with a slightly lower risk of miscarriage (~0.5%)