amplicor technology detection of specific pcr products based on reverse hybridization uses amperase...
Post on 22-Dec-2015
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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
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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PCR
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