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AMPLICOR technology Detection of specific PCR products based on reverse hybridization Uses AmpErase...
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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
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AMPLICOR technology
Blue complex
Yellow colorDetected withspectrophotometer
Probe on BSABSA on plastic
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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
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Cycle-Sequencing
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cycle sequencing
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Cycle-Sequencing
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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
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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
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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
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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
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DNA micro array with cDNA probes
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DNA micro array with cDNA probe
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DNA micro arrays
• Oligonucleotide arrays– Probes 20-25 bases, can be synthesized directly onto
the chip (glass slide)– Photolithography and oligonucleotide synthesis– see publication
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Oligonucleotide arrays
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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
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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...
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Oligonucleotide array GENE-EXPRESSION studies
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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
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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
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Oligonucleotide arrays
Determining the DNA sequence of a certain, or some genes
Detection of mutations, substitutions
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Oligonucleotide arrays
Detection of several alleles of certain DNA sequences
Polymorphisms of genes
SNP analysis and mapping
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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)
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ASO
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ASO
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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
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Oligonucleotide ligation assay OLA
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Penthaethylene oxide units
Fluorescent labelsFAMHEXTET
For each label (with possible different common probes) EACH selective probe must have a different length
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Oligonucleotide ligation assay OLA
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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
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In situ amplification (In situ PCR)
Formalin paraformaldehyde
Denhardt’s solution
Proteinase K
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Southern blottingAlso ASO probecan be used
Also non-radioactive probe can be used
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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
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RFLP
Detectie met DNA-probe specifiek voor -globine gen
GEEN ASO-probe
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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)
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Restriction mapping
Functional gene Pseudogene
2
1
1
1
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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
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Northern blotting