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

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cycle sequencing

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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 GENE EXPRESSION studies

• Used to study differences in gene expression between different cell populations (disease versus healthy state, with or without stimulus,...)

• RNA extraction, use oligo-dT primed cDNA synthesis with fluorescent labels Cye3-dUTP or Cye5-dUTP

• The problem of transcription bias during RT-PCR is solved by comparing the same DNA between two populations

• After hybridization scan the chip with a CCD camera

DNA micro array with cDNA probes

DNA micro array with cDNA probe

DNA micro arrays

• Oligonucleotide arrays– Probes 20-25 bases, can be synthesized directly onto

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

Oligonucleotide arrays

Oligonucleotide arrays

• Oligonucleotide arrays– Probes 20-25 bases, can be synthesized directly onto

the chip (glass slide)– Photolithography and oligonucleotide synthesis– Disadvantage: has to be made by company (custom

synthesis) Affymetrix– Advantage: more polyvalent, more specific by using

multiple short probes for a single gene, mismatch controles

– See publication

Oligonucleotide arrays

• Applications:– Gene-expression studies comparable with cDNA-

probe micro arrays• Multiple probes for each gene under study

• Perfect match – mismatch probes

• Chips can be purchased for several applications (see publication)

• DIAGNOSTIC USE:– Gene-expression studies for classification of cancers, predict

disease progression, predict sensitivity towards certain therapeutics...

Oligonucleotide array GENE-EXPRESSION studies

Oligonucleotide array GENE-EXPRESSIE studies

Typical result after data analysis by computer

Clustered gene expressie

Green means less fluorescence than the reference sample

Red is more fluoresence

Black is equal expression

Oligonucleotide arrays

• Applications:– Gene-expression– DNA-sequence information

• Test for known mutations in genes linked to disease

• Single nucleotide polymorphims

• Determine the DNA sequence of a certain gene

• Sequence information CAN ONLY BE OBTAINED by oligonucleotide arrays

Oligonucleotide arrays

Determining the DNA sequence of a certain, or some genes

Detection of mutations, substitutions

Oligonucleotide arrays

Detection of several alleles of certain DNA sequences

Polymorphisms of genes

SNP analysis and mapping

Allele specific oligonucleotide hybridization ASO

• Dot-blotting, apply and bind target DNA on membrane (nylon, nitrocellulose)

• Denature and hybridize labeled probe, wash and detect (radio-active or non radioactive techniques (see earlier))

• Can be used for the detection of point-mutations• ASO-probe 15-20 nucleotides, difference central in

probe• Also reverse dot-blot ASO, unlabeled probe is bound to

membrane, hybridize with labeled target (analogous to LiPA and AMPLICOR)

ASO

ASO

Oligonucleotide ligation assay OLA

• Test for known point mutations or polymorphisms

• Based on the ligation of two probes in case of exact complementarity

• Uses DNA-ligases: rTth, T4 DNA ligase• Allows for testing of 31 known mutations in one

single analysis• Detect fluorescently labeled probes with laser

fluorescence detection

Oligonucleotide ligation assay OLA

Penthaethylene oxide units

Fluorescent labelsFAMHEXTET

For each label (with possible different common probes) EACH selective probe must have a different length

Oligonucleotide ligation assay OLA

In situ amplification (In situ PCR)

• PCR in fixed tissues or cells, correlation of a PCR result with morphology

• More sensitive than in situ hybridization ISH

• Sufficiently permeabilize cell for PCR reagents, but keep cell structure sufficiently intact to maintain PCR products

• Study of gene expression and/or mutations in abnormal cells or tissues

In situ amplification (In situ PCR)

Formalin paraformaldehyde

Denhardt’s solution

Proteinase K

Southern blottingAlso ASO probecan be used

Also non-radioactive probe can be used

RFLP

• = Restriction Fragment Length Polymorphisms

• Based on southern blotting

• Typing of mutations which change a restriction site and big insertions or deletions between restriction sites

• Used for genotypical classification of virusses

RFLP

Detectie met DNA-probe specifiek voor -globine gen

GEEN ASO-probe

Restriction mapping

• Also based on Southern blotting• Detects gene-deletions, with intragene DNA-

probe (probe against sequence in the gene)• Small deletions (about 100 bases) give rise to a

shorter fragment• Large deletions yield no fragment if

homozygous, heterozygous deletions yield a less intense fragment compared to other band

• Vb 21-hydroxylase deficiency (deficiency in cortisol and aldosteron production)

Restriction mapping

Functional gene Pseudogene

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Northern blotting

• Variant of Southern in which the target is RNA in stead of DNA

• Study of expression pattern of a cloned gene in several tissues

• No restriction enzymes necessary

Northern blotting