Transcript End point

Allele specific PCR or ARMS test
• Amplification Refractory Mutation System
• Used to detect point-mutations and small
deletions, differentiates between DNA-sequences
differing only in 1 nucleotide
• PCR-primers with 3’ terminus directed against the
mutation site to be tested
• correct 3’ base pairing of a primer is required in
order to produce a PCR product
• Can also be used to detect SNP’s
• See example of cystic fibriosis + exercise
Allele specific PCR or ARMS test
Pharmacogenetics versus
pharmacogenomics
• Pharmacogenetics: 1 gene versus 1 drug
• Pharmacogenomics: which drugs for what
disease
Real-time PCR or Quantitative PCR
• End point detection of PCR products to
determine the amount of starting material is
unreliable, since PCR products
exponentially accumulate to a certain
plateau level figure
• In Real-time PCR, the amount of PCR
product is determined after each cycle.
• Here we will discuss the two most common
formats of the technique
• The quantitation is RELATIVE, household
genes (differences in mRNA extraction,
cDNA synthesis...)
Real-time PCR with SYBR-green
SYBR-green binds double
stranded DNA and has a higher
fluorescence intensity when
bound
Real-time PCR with SYBR-green
• Selectivity only due to primers (check with
PCR reaction and gel-detection if PCR
reaction is specific)
• Important disadvantage: all produced
double stranded DNA molecules produce a
signal also the PCR-primer artefacts 
higher detection limit
• Remark: sequence differences (additions,
deletions, polymorphisms, pointmutations...) located in between the primers
will not be detected
Real-time PCR with the TaqMan-probe
Probe can not be extended at
the 3’ end (dideoxy nucleotide)
Förster Resonance Energy
Tranfser = FRET
Real-time PCR with the TaqMan-probe
• More expensive and difficult in set-up than
the SYBR-green method
• Enhanced specificity due to the extra
selectivity of the probe
• Fluorescent signal is only generated by
specific amplification of the target sequence
 lower detectionlimit
• Due to the additional specificity of the
probes mutations or polymorphisms can be
detected
CT-value
• “cycle treshold”
• The CT-value is the
fractional cycle
number where the
fluorescent signal
reaches a certain
treshold
• Plotting the CT-value
in function of the log
copy number gives a
lineair relationship
(standard curve)
CT
Real-time PCR TaqMan with multiple colors
• Some equipment allows for the
simultaneous detection of multiple colors:
detection of mutations, polymorphisms,
internal standard (house hold gene) in 1
reaction ALSO ARMS-assay is possible
Scorpion technology
Scorpion technology
Important with real-time PCR
• Good controles, besides positive en negative
controles (controles with known copy number…)
• “standard curve”, dilution serie of a known
amount of copy numbers
• For each sample the result should be in reference
to a gene with constant expression (household
gene) to normalize for differences in mRNA
extraction and cDNA synthesis
• Or genomic DNA has to be removed from the
RNA-extract before cDNA-synthesis or the
probe/primer comination has to be based on the
presence of introns in the genomic DNA
Special precautions to be taken when performing
PCR
• See previous lesson
• Use positive displacement pipets or barrier
pipet tips
• Separate workareas for handling pre- and
post PCR samples
• Oneway trafficing from samples and
materials from pre- to post PCR area.
Preventing PCR-product carry-over using AmpErase
• Replace dTTP by dUTP during PCRreaction
• Treat all samples with Uracil N-glycosylase
before PCR amplification
Preventing PCR-product carry-over using AmpErase
Uracil N-glycosylase
Non active
above 55°C
First PCR
cycle
The line-probe assay (LiPA)
• Technique for sequence specific detection
of PCR products based on reverse
hybridization
• Strip with  probes for known mutations or
polymorphisms
• Advantage: 1 PCR reaction and
hybridization gives multiple answers
• Lots of commercial kits available see
transparancies
The line-probe assay (LiPA)
Biotinilated
primer
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
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
1200000
PCR
cycle sequencing
1000000
800000
600000
400000
200000
50.000
0
0
1
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
• Oligonucleotide arrays
– Probes 20-25 bases, can be synthesized directly
onto the chip (glass slide)
– Fotolithography and oligonucleotide synthesis
– see publication