PED3014 L2 Methods for genotyping

what are the methods for genotyping for known polymorphisms

• PCR followed by a restriction digest which recognises one allele but not the other (PCR-RFLP)

• Allele specific PCR

• Primer extension methods e.g. Sequenom

what does the Taqman system involve as an example of automated allele-specific PCR

• uses primers and allele-specific oligonucleotide probes

• probe includes a fluorescent reporter and quencher

• fluorescence is only seen if the probe binds and the 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

describe genotyping by primer extension

• carry out a PCR reaction to give the product covering site of the polymorphism

• add a primer which binds adjacent to the site of polymorphism and extend this by several base pairs (minisequencing)

what can genotyping by primer extension determine

which base is incorporated at the site of polymorphism

how can genotyping by primer extension determine which base is incorporated at the site of polymorphism

• using a single nucleotide combined with dideoxynucleotides and analysing products using mass spectrometry

• sequential addition of different nucleotides (pyrosequencing) in real time using a camera

what does sequential addition of different nucleotides (pyrosequencing) in real time using a camera rely on

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

what are the reaction principles for sequenom genotyping

• need to know the polymorphism to be tested

• design a specific assay

• use normal dGTP complementary to C but other primers (A,C and T) are dideoxys which cant be extended

SNP genotyping by pyrosequencing method

• prepare PCR product labelled with biotin on one strand

• isolate biotin-labelled strand and add primer that binds adjacent to the site of polymorphism

• add DNA polymerase plus detection system involving luciferin

• add single DNA bases in turn- dATP, d CTP, dGTP and dTTP

• The base complementary to the site will bind and release PPI when it binds in real time

• PPI reacts with the detection system to produce light

• the camera measures light and sends signal to detector

• if heterozygous, there will be decreased signals for 2 bases

what are genome-wide association studies

• they genotype for 500,000 to 1000000 SNPs scattered throughout the human genome

• strong linkage disequilibrium in human genome allows connections with genes some distance away from the marker to be detected

what are the methods of genome-wide association studies

• affymetrix gene chip

• illumina bead chip

what does the illumina infinium assay need

a primer specific to each SNP

WHAT DO THE BEADS HAVE IN THE ILLUMINA INFINIUM ASSAY

specific primers attached

the input DNA is… in the illumina infinium assay

not labelled

describe the affymetric microarray

• oligonucleotides specific to particular DNA sequences are attached to quarts surface-gene chip

• detect the hybridisation of fluorescently labelled DNA

describe the typical GWAS result - Manhattan plot

• Y axis is a negative log of p value so the lower the p value the higher the number

• significance usually set at 0.05 ×10-6 to correct for multiple testing

• red dots are genome wide significant

• green dots are close to genome-wide significance

what are the DNA sequencing methods for detection of new polymorphisms

• manual methods

• automated methods

what can manual DNA sequencing either do

• sequence PCR products directly

• clone PCR product into plasmid vector and sequence individual clones

What can high throughput sequencing systems do

sequence fragmented DNA directly

• still involves DNA polymerase reaction

DNA fragments are chosen at random so the process needs to be repeated to ensure complete coverage for genome-wide sequencing

describe human genome sequencing of exons only

• exome sequencing

• used in clinical genetics

• led to new information on genetic disease

describe human genome sequencing of the whole genome

increasingly possible but still challenging to assemble and interpret data