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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
Probe on BSA
BSA on plastic
Yellow color
Detected with
spectrophotometer
Blue
complex
Sequencing
• Cycle sequencing with fluorescently labeled dideoxynucleotide 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
1200000
PCR
cycle sequencing
1000000
800000
600000
400000
200000
50.000
0
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5
10
15
20
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 cDNAprobe 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 labels
FAM
HEX
TET
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)
Proteinase K
Formalin
paraformaldehyde
Denhardt’s
solution
Southern blotting
Also ASO probe
can be used
Also non-radio
active 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 DNAprobe specifiek
voor b-globine gen
GEEN ASO-probe
Restriction mapping
• Also based on Southern blotting
• Detects gene-deletions, with intragene DNAprobe (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
Pseudogene
Functional gene
2
1
1
1
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