Allele specific PCR or ARMS test Amplification Refractory Mutation System Used to detect point-mutations and small deletions, differentiates between DNA-sequences differing only in 1 nucleotide PCR-primers with 3’ terminus directed against the mutation site to be tested correct 3’ base pairing of a primer is required in order to produce a PCR product Can also be used to detect SNP’s See example of cystic fibriosis + exercise

Allele specific PCR or ARMS test

Pharmacogenetics versus pharmacogenomics Pharmacogenetics: 1 gene versus 1 drug Pharmacogenomics: which drugs for what disease

Real-time PCR or Quantitative PCR End point detection of PCR products to determine the amount of starting material is unreliable, since PCR products exponentially accumulate to a certain plateau level figure In Real-time PCR, the amount of PCR product is determined after each cycle. Here we will discuss the two most common formats of the technique The quantitation is RELATIVE, household genes (differences in mRNA extraction, cDNA synthesis...)

Real-time PCR with SYBR-green SYBR-green binds double stranded DNA and has a higher fluorescence intensity when bound

Real-time PCR with SYBR-green Selectivity only due to primers (check with PCR reaction and gel-detection if PCR reaction is specific) Important disadvantage: all produced double stranded DNA molecules produce a signal also the PCR-primer artefacts  higher detection limit Remark: sequence differences (additions, deletions, polymorphisms, point- mutations...) located in between the primers will not be detected

Real-time PCR with the TaqMan-probe Probe can not be extended at the 3’ end (dideoxy nucleotide) Förster Resonance Energy Tranfser = FRET

Real-time PCR with the TaqMan-probe More expensive and difficult in set-up than the SYBR-green method Enhanced specificity due to the extra selectivity of the probe Fluorescent signal is only generated by specific amplification of the target sequence  lower detectionlimit Due to the additional specificity of the probes mutations or polymorphisms can be detected

C T- value “cycle treshold” The C T -value is the fractional cycle number where the fluorescent signal reaches a certain treshold Plotting the C T -value in function of the log copy number gives a lineair relationship (standard curve) CTCT

Real-time PCR TaqMan with multiple colors Some equipment allows for the simultaneous detection of multiple colors: detection of mutations, polymorphisms, internal standard (house hold gene) in 1 reaction ALSO ARMS-assay is possible

Scorpion technology

Important with real-time PCR Good controles, besides positive en negative controles (controles with known copy number…) “standard curve”, dilution serie of a known amount of copy numbers For each sample the result should be in reference to a gene with constant expression (household gene) to normalize for differences in mRNA extraction and cDNA synthesis Or genomic DNA has to be removed from the RNA-extract before cDNA-synthesis or the probe/primer comination has to be based on the presence of introns in the genomic DNA

Special precautions to be taken when performing PCR See previous lesson Use positive displacement pipets or barrier pipet tips Separate workareas for handling pre- and post PCR samples Oneway trafficing from samples and materials from pre- to post PCR area.

Preventing PCR-product carry-over using AmpErase Replace dTTP by dUTP during PCR- reaction Treat all samples with Uracil N-glycosylase before PCR amplification

Preventing PCR-product carry-over using AmpErase Uracil N-glycosylase First PCR cycle Non active above 55°C

The line-probe assay (LiPA) Technique for sequence specific detection of PCR products based on reverse hybridization Strip with  probes for known mutations or polymorphisms Advantage: 1 PCR reaction and hybridization gives multiple answers Lots of commercial kits available see transparancies

The line-probe assay (LiPA) Biotinilated primer

AMPLICOR technology Detection of specific PCR products based on reverse hybridization Uses AmpErase For the detection of the presence or absence of specific PCR products and indication of the amount of target present Is not being used for the detection of the presence of mutations or polymorphisms Commercial kits see transparancies

AMPLICOR technology Blue complex Yellow color Detected with spectrophotometer Probe on BSA BSA on plastic

Sequencing Cycle sequencing with fluorescently labeled dideoxy-nucleotide triphosphates and ONE primer Amplification is linear and NOT exponential  more starting material is needed Most convenient and polyvalent technique to diagnose mutations and polymorphisms Data interpretation is very time consuming Some kits are commercially available eg HLA typing

Cycle-Sequencing

Cycle-Sequencing

Separate fragments on a polyacrylamide (high resolution gel) Fragments of a certain length all end with the same label (nucleotide) unless polymorphisms are present (heterozygous) Detection with laser fluorescence-detection

DNA micro arrays Very recent technology Gene array, GeneChip (Affymetrix), genome chip Current problem: large quantities of RNA are necessary 2-5 µg mRNA; cells; 1 tot 10 mg tissue for gene expression analysis Based on reverse hybridization Non labeled probes immobilized on glas or membranes (nylon or nitrocellulose) in spots smaller than 200 µm 2 big technological variants (see publications) Not suited for de novo gene discovery

DNA micro arrays cDNA-probes –Probes 500 to 5000 bases, prepare cDNA libraries using PCR, PURIFY cDNA –Using spotting robot probes are immobilised on carrier –Disadvantage: long probes give rise to mismatch hybridisation, construction of arrays is very labour intensive –Advantage: can be “self”-assambled –See publication on gene expression

DNA micro arrays Oligonucleotide arrays –Probes bases, can be synthesized directly onto the chip (glass slide) –Fotolithography and oligonucleotide synthesis –see publication