Techniques used In Pharmacogenetics/genomics

Question 1

what does RFLP mean ?

Answer 1

Restriction fragment length polymorphism

Question 2

what is differerent is PCR-RFLP compared to normal PCR

Answer 2

in RFLP, PCR is followed by restiction digestion which recognises one allele but not the otheres

Question 3

why can PCR-RFLP be used for genetyping

Answer 3

Peticular SNP removes the restriction site therefore alters the length of DNA fragments after PCR then digestion

Question 4

what is an example of a SNP found in PCR-RFLP genotyping

Answer 4

NAT2 - shown in Isoniazid
- Mutant (SNPs) acetylate slowly therefore therefore experience toxicity
- Non SNPs acetylate quickly
- Polymorphism mutates cut site from T to C therefore cant be cut by Ddel

Question 5

What is Taqman ?

Answer 5

Allele specific PCR

Question 6

How does Taqman work ?

Answer 6

= Involves using primers and allele-specific oligonucleotide probes
= Probe includes fluorescent reporter and quencher
= Only see fluorescence if probe binds and reporter is released by 5’-nuclease activity of Taq polymerase as it extends
= Use different colour reporters for each allele
= Colour is detected in “real time” during PCR reaction

Question 7

what is an example of Primer extension methods

Answer 7

sequenom

Question 8

how does Primer extension methods work

Answer 8

= Carry out PCR reaction to give product covering site of polymorphism
= Add primer which binds adjacent to site of polymorphism and extend this by several base pairs (minisequencing reaction)

Question 9

how can base incorporation (at site of polymorphism) be determined

Answer 9

= Using single nucleotide combined with dideoxynucleotides and analysing product using mass spectrometry (Sequenom)
or
= Sequential addition of different nucleotides (Pyrosequencing) in real time using camera

Question 10

what does pyrosequencing rely on

Answer 10

detection of pyrophosphate release on nucleotide incorporation rather than chain termination with dideoxynucleotides

Question 11

how does sequenom genotyping work

Answer 11

• Base of intested is immediately adjacent to primer added
  -(PCR + add mixture of nucleotide)
• nucleotides can either terminate (ddA,C,TTP)
• dGTP is added other base after
• depending on ase you either get 24 base product (23 base + 1) or 25 base (if C then dGTP binds therefore 1 more is added)

Question 12

if the person was heterozygous what would be seen in sequenom

Answer 12

24 base products and 25 base products

Question 13

how does Pyrosequencing happen (preparation)

Answer 13

Prepare PCR product labelled with biotin on 1 strand. Isolate biotin labelled strand and add primer that binds adjacent to site of polymorphism. Add DNA polymerase + ‘detection system’ involving luciferin.

Question 14

How does Pyrosequencinjg happen (analysis)

Answer 14

The base complementary to site will bind and release PPi when it binds in real time. PPi reacts with detection system to produce light
and camera measures light and sends signal to detector.
When bases is encorporated =light is emmited

Question 15

how does genome wide associated studies work ?

Answer 15

= Genotype for 500,000 to 1,000,000 single nucleotide polymorphisms (SNPs) scattered throughout human genome
= Identify novel genes involved in disease or drug response
= Strong linkage disequilibrium in human genome allows connections with genes some distance away from the marker to be detected

Question 16

what are micro arrays

Answer 16

allows determine the genotype of hundred thousands potential SNPs in a single experiment

Question 17

what are the two types of micro arrays

Answer 17

affymetrix gene chip
Illumina bead chip

Question 18

how does affymetrix gene chip work

Answer 18

• oligonucleotides specific to particular DNA sequences are attached to quartz surface gene chip
• these detect hybridization of fluorescently labelled DNA.
• millions of sequences specific probes (each probing one SNP)
• sequence mismatch results in reduce binding

Question 19

how does Illumina bead chip work

Answer 19

Need primer specific to each SNP, meaning they can screen for 1,000,000 different variants simultaneously.
each bead has specific primers attched, input DNA not labelled
- one labelled base is added to template on every bead and detected siganl from that label (fluoresce)

Question 20

how in Illumina bead chip, can bases be distingished

Answer 20

signals varys (colour)

Question 21

what is the p value needs for the SNP to be significant

Answer 21

5 x 10^8
0.05 (normal) would mean loads of false positive

Question 22

what is sanger

Answer 22

DNA sequencing were the PCR product is cloned into plasmid vector and sequence individual clones
- 4 reactions each one with C,G,T or A
- On a gel its read bottom to top

Question 23

what types of labels are used in sanger

Answer 23

• radioactive (previously)
• fluorescents (nowadays)

Question 24

what is an automated methods example

Answer 24

High throughput sequencing systems and Illumina sequencing

Question 25

how does HTSS work and what does it do

Answer 25

Can sequence fragmented DNA directly. Still involves DNA polymerase reaction. DNA fragments chosen at random so need to repeat process a number of times (e.g. 30 +) to ensure complete coverage for genome-wide sequencing.
- can sequence whole genome

Question 26

how does Illumina sequencing work

Answer 26

Terminating sequencing reaction and then identify signal given off. Can undo termination, detect the nucleotide and then remove terminator. Reaction is continuous, in a tiny environment

Question 27

how does Human whole genome sequencing work

Answer 27

can either sequence exons only which is used in clinical genetics
or sequence the whole genome (possible but still hard to do)

Question 28

what are the limitation of phenotypic approaches

Answer 28

• Enzyme activity measurements problematic = Tissue needs to be accessible
• Studies on patterns of drug metabolites often difficult = Need a suitable probe drug or chemical and good analytical chemical techniques
• Particularly difficult in studies of drug receptors

Question 29

what are the limitation of genotypic approaches

Answer 29

• May need to relate polymorphisms to function which can be technically complex
  There are techniques and algorithms available which can predict functional effects on protein structure and activity

Question 30

what are the advantages of genotypic approaches

Answer 30

• Looking directly at gene of interest
• Can use blood sample, buccal cells or saliva as source of DNA (white blood cells give better quality DNA)