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

CT-value

• “cycle treshold”

• The CT-value is the fractional cycle number where the fluorescent signal reaches a certain treshold

• Plotting the CT-value in function of the log copy number gives a lineair relationship (standard curve)

CT

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

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 colorDetected withspectrophotometer

Probe on BSABSA 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

0

200000

400000

600000

800000

1000000

1200000

0 5 10 15 20

PCR

cycle sequencing

50.000

1

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; 107-108 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 20-25 bases, can be synthesized directly

onto the chip (glass slide)– Fotolithography and oligonucleotide synthesis– see publication