Techniques used In Pharmacogenetics/genomics Flashcards
what does RFLP mean ?
Restriction fragment length polymorphism
what is differerent is PCR-RFLP compared to normal PCR
in RFLP, PCR is followed by restiction digestion which recognises one allele but not the otheres
why can PCR-RFLP be used for genetyping
Peticular SNP removes the restriction site therefore alters the length of DNA fragments after PCR then digestion
what is an example of a SNP found in PCR-RFLP genotyping
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
What is Taqman ?
Allele specific PCR
How does Taqman work ?
= 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
what is an example of Primer extension methods
sequenom
how does Primer extension methods work
= 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)
how can base incorporation (at site of polymorphism) be determined
= 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
what does pyrosequencing rely on
detection of pyrophosphate release on nucleotide incorporation rather than chain termination with dideoxynucleotides
how does sequenom genotyping work
- 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)
if the person was heterozygous what would be seen in sequenom
24 base products and 25 base products
how does Pyrosequencing happen (preparation)
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.
How does Pyrosequencinjg happen (analysis)
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
how does genome wide associated studies work ?
= 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
what are micro arrays
allows determine the genotype of hundred thousands potential SNPs in a single experiment
what are the two types of micro arrays
affymetrix gene chip
Illumina bead chip
how does affymetrix gene chip work
- 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
how does Illumina bead chip work
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)
how in Illumina bead chip, can bases be distingished
signals varys (colour)
what is the p value needs for the SNP to be significant
5 x 10^8
0.05 (normal) would mean loads of false positive
what is sanger
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
what types of labels are used in sanger
- radioactive (previously)
- fluorescents (nowadays)
what is an automated methods example
High throughput sequencing systems and Illumina sequencing
how does HTSS work and what does it do
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
how does Illumina sequencing work
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
how does Human whole genome sequencing work
can either sequence exons only which is used in clinical genetics
or sequence the whole genome (possible but still hard to do)
what are the limitation of phenotypic approaches
- 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
what are the limitation of genotypic approaches
- 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
what are the advantages of genotypic approaches
- 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)
