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f. Microsatellites or SSR Markers
In 1989, Weber & May and Litt & Luty discover microsatellite
sequences, demonstrate their high level of
polymorphism due to variations in the number of
tandem repeats (1 - typical heterozygosities in cattle),
abundance and even distribution across the genome.
Microsatellites are genotyped using the polymerase chain
reaction (1 ) using primers targeted to the unique
sequences flanking the microsatellite motif. PCR can
easily be semi-automated (1 )
The resulting PCR products are separated according to size
by gel electrophoresis using either agarose gels or
more commonly (because of their higher resolution)
denaturing polyacrylamide gels (PAGE) (1 ).
PCR products are visualized by:
Fig. Tri-nucleotide repeat microsatellite marker:
Expansion of the CGG triplet in the FMR-1 gene seen in the fragile X
syndrome. Normal individuals have from 6 to 54 copies of the CGG
repeat, while individuals from susceptible families display an increase
(premutation) in the number of repeats: normally transmitting males
(NTMs) and their daughters are phenotypically normal but display 50 to
200 copies of the CGG triplet; the number of repeats expands to some 200
to 1300 in individuals showing full symptoms of the disease.
PCR for semi-automated Microsatellite
genotyping by auto-radiography.
Denaturated Poly-acrylamide gel electrophoresis
(PAGE) for microsatellite genotyping
•Direct staining (ethidium bromide or silverstaining)(1 )
•Autoradiography:
•The PCR products are labelled either by incorporation of
[a -P 32 or 33] dNTPs (1 )or [a -S 35] dNTS during the PCR
amplification (1 )(labels the two strands), or by using one
end-labeled primer (labels one strand). Primers are endlabeled using [g -P 32 ]ATP & T4 polynucleotide kinase (1 )
•After gel electrophoresis, an X-ray film is exposed to the
gel revealing the position of the PCR products as black
spots. A photon of light or a b particle or g ray released from
a radioactive molecule "activate" silver bromide crystals on
the film emulsion. This renders them capable of being
reduced through the developing process to form silver
metal (a "grain"). The silver grains on the film form the
image.
•Co-amplification and/or co-loading of multiples
microsatellites allows for multiplex genotyping (2-4
systems).
Agarose gel
Electrophoresis
(A), (B), (C).
Staining by ethidium
Bromide (B),
Stained by
auto-radiograph (C).
Labeling of genetic markers
Labelling of dNTP by P32 radio-isotops in microsatellite tying by auto-radiogragy.
Mode of action of P32 labeling in PCR product.
•Fluorescence labelling:
•The PCR products are labeled either by using primers or dNTPs
which are tagged with an appropriate fluorophore, a chemical group
which fluoresces when exposed to a specific wavelength of light.
Popular fluorophores used in direct labeling include fluorescein, a
pale green fluorescent dye, rhodamine, a red fluorescent dye, and
amino methyl coumarin, a blue fluorescent dye. Fluorophoes are
characterized by their excitation and emission spectra.
•The PCR products are detected during migration using automatic
sequencers. (1 )
•Co-amplification and/or co-loading of multiples microsatellites allows
for multiplex genotyping (up to 20 systems). (1 )
•Software packages allow for semi-automated data capture (1 ).
Microsatellite profiles are often difficult to read due to artefactual
bands, which result from (1 ):
•the differential migration of the two DNA strands in denaturing
acrylamide gels
•the "+ A" activity of the Taq polymerase generating x + 1 bands
•slippage of the Taq polymerase during polymerization generating …
+4, +2, -2, -4, … "stutter" bands
•non denatured secondary structures adopted by the PCR products.
Microsatellite typing by automated DNA sequencer:
New era of molecular genetics: Using fluorescent labeled primers.(4Pictures of
ABI377 PerkinElmer DNA sequencers)
g.SNPs (Single nucleotide polymorphisms)
The difficulty to fully automate microsatellite genotyping has
revived interest in a new type of markers: single nucleotide
polymorphisms or SNPs.
Definition:
SNPs are polymorphisms due to single nucleotide
substitutions (transitions > transversions) or single nucleotide
insertions/deletions.
Abundance:
The average heterozygosity per nucleotide site, p , has been
estimated at approximately 1/1000 in man, 1/2500 in cattle.
Informativeness:
SNPs are virtually always biallelic markers. Their
heterozygosity is therefore limited at 50%.
Examples of SNP genotyping methods:
•1) Single Stranded Conformation Polymorphism (SSCP)(1 )
•2) Allele specific oligonucleotides (ASO)(1 )
•3) Single nucleotide polymorphic discrimination by an electronic dot
blot assay (ASO) on semconductor microchips (1 ; 1 )
•4) Reverse dot blot on DNA chips (1 )
•5) Dynamic allele specific hybridisation (DASH) (1 ; 1 )
•6) Allele-specific PCR (=amplification refractory mutation system or
ARMS test)(1 )
•7) Mutation detection the ARMS test in combination with the
TaqmanTM 5' exonuclease assay (exploiting the 5'->3' exonuclease
activity of Taq DNA polymerase).(1 )
•8) Minisequencing and analysis of the extension products by PAGE.
•9) Minisequencing and analysis of the extension products on DNA
chips
•10) Minisequencing and analysis of the extension products using
matrix-assisted laser desorption/ionization time-of-flight mass
spectrometry (MALDITOF)(1 )
•11) Pyrosequencing (1 )
•12) OLA (1 )
•13) Invasive clivage of oligonucleotide probes (Invader technology)
•1) Single Stranded Conformation Polymorphism (SSCP)(1
(Figure 1-3)
Detection and production of Allele specific Oligo-nucleotide (ASO)
Allele-specific oligonucleotide (ASO) dot-blot hybridisation can identify individuals with the sickle cell mutation.
The sickle cell mutation is a single nucleotide substitution (A to T) at codon 6 in the b -globin gene, resulting in a
GAG (Glu) to GTG (Val) substitution. The example shows how one can design ASOs: one specific for the normal
(b A) allele and identical to a sequence of 19 nucleotides encompassing codons 3-9 of this allele, and one specific
for the mutant (b S) allele, being identical to the equivalent sequence of the mutant allele. The labeled ASOs can
be individually hybridized to denatured genomic DNA samples on dot-blots. The b A- and b S-specific ASOs can
hybridize to the complementary antisense strand of the normal and mutatnt alleles respectively, forming perfect
19-bp duplexes. However, duplexes between the b A-specific ASO and the b S allele, or between the b S-specific
ASO and the b A allele have a single mismatch and are unstable at high hybridization stringency.
•2) Single nucleotide polymorphic discrimination by an
electronic dot blot assay (ASO) on semiconductor microchips
•Single nucleotide polymorphic discrimination by an electronic
dot blot assay (ASO) on semiconductor microchips
Dynamic allele-specific hybridization (DASH)
Figure 1: DASH assay principle.
Probe-target denaturation is
monitored over a wide temperature
range by measuring fluorescence
while heating. All tested sequences
thus yield a melting temperature
indicative of either perfect match or
single-base mismatch to the assay
probe.
Dynamic allele-specific hybridization
W. Mathias Howell et al.
Nature Biotechnology 17, 87 - 88 (1999)
Correct base-pairing at the 3' end of PCR primers is the basis of allele-specific
PCR.
The allele-specific oligonucleotide primers ASP1 and ASP2 are designed to be identical to
the sequence of the two alleles over a region preceding the position of the variant
nucleotide, up to and terminating in the variant nucleotide itself. ASP1 will bind perfectly to
the complementary strand of the allele 1 sequence permitting amplification with conserved
primer. However, the 3'-terminal C of the ASP2 primer mismatches with the T of the allele 1
sequence, making amplification impossible. Similarly ASP2 can bind perfectly to allele 2 and
initiate amplification, unlike ASP1.
The TaqmanTM 5' exonuclease assay (Mutation detection by ARMS)
In addition to two conventional PCR primers, P1 and P2, which are specific for the target sequence
(P1 being for instance allele specific), a third primer, P3 is designed to bind specifically to a site on
the target sequence downstream of the P1 binding. P3 is labeled with two fluorophores, a reporter
dye (R) is attached at the 5' end, and a quencher dye (D), which has a different emission
wavelength to the reporter dye, is attached at its 3' end. Because its 3' end is blocked, primer P3
cannot by itself prime any new DNA synthesis. During the PCR reaction, Taq DNA polymerase
synthesizes a new DNA strand primed by P1 and as the enzyme approaches P3, its 5'-> 3'
exonuclease activity processively degrades the P3 primer from its 5' end. The end result is that the
nascent DNA strand extends beyond the P3 binding site and the reporter and quencher dyes are no
longer bound to the same molecule. As the reporter dye is no longer in close proximity to the
quencher, the resulting increase in reporter emission intensity is easily detected.
Figure 1: Mass compression for a sixfold multiplex genotyping assay.
Loci 2, 3, 5, 7, 8, and 10 were amplified and typed using corresponding primers and
standard assay conditions. Arrows indicate corresponding primer-extended primer pairs.
Molecular weight assignments from two point calibration are given.
High level multiplex genotyping by MALDI-TOF mass spectrometry
Philip Ross et al.
Nature Biotechnology 16, 1347 - 1351 (1998)
Pyrosequencing™
is
sequencing by synthesis. ' A simple
to use technique for accurate and
consistent analysis of large numbers
of short to medium length DNA
sequences.
Step1.
A sequencing primer is hybridized to
a single stranded, PCR amplified,
DNA template, and incubated with
the enzymes, DNA polymerase, ATP
sulfurylase, luciferase and apyrase,
and the substrates, adenosine 5´
phosphosulfate (APS) and luciferin.
Polymorphism identification and quantitative detection of genomic DNA by invasive
cleavage of oligonucleotide probes
Victor Lyamichev et al. Nature Biotechnology 17, 292 - 296 (1999)
Figure 1: Schematic representation of the configurations assumed by
oligonucleotides with an overlap complementary to a DNA target.
The 3´ end of the upstream oligonucleotide (A) or the 5´ end of the
downstream oligonucleotide (C) can each be displaced by the other, with
both conformers interchanging through an intermediate form (B). The
FEN1 endonucleases used in this study cleave only the flap created by
displacement of the downstream segment (C).