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AMPLICOR technology

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

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AMPLICOR technology

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  1. 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

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

  3. 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

  4. Cycle-Sequencing 50.000 1

  5. Cycle-Sequencing

  6. 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

  7. 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

  8. 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

  9. 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

  10. DNA micro array with cDNA probes

  11. DNA micro array with cDNA probe

  12. DNA micro arrays • Oligonucleotide arrays • Probes 20-25 bases, can be synthesized directly onto the chip (glass slide) • Photolithography and oligonucleotide synthesis • see publication

  13. Oligonucleotide arrays

  14. 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

  15. 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...

  16. Oligonucleotide array GENE-EXPRESSION studies

  17. 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

  18. 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

  19. Oligonucleotide arrays Determining the DNA sequence of a certain, or some genes Detection of mutations, substitutions

  20. Oligonucleotide arrays Detection of several alleles of certain DNA sequences Polymorphisms of genes SNP analysis and mapping

  21. 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)

  22. ASO

  23. ASO

  24. 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

  25. Oligonucleotide ligation assay OLA

  26. Penthaethylene oxide units Fluorescent labels FAM HEX TET For each label (with possible different common probes) EACH selective probe must have a different length

  27. Oligonucleotide ligation assay OLA

  28. 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

  29. In situ amplification (In situ PCR) Proteinase K Formalin paraformaldehyde Denhardt’s solution

  30. Southern blotting Also ASO probe can be used Also non-radio active probe can be used

  31. 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

  32. RFLP Detectie met DNA-probe specifiek voor b-globine gen GEEN ASO-probe

  33. 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)

  34. Restriction mapping Pseudogene Functional gene 1 2 1 1

  35. 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

  36. Northern blotting

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