Transcript Single Strand Conformational Polymorphism (SSCP)

Single Strand Conformational
Polymorphism (SSCP)
• DNA polymorphism produced by differential
folding (intra-molecular interaction) of singlestranded DNA harboring mutations (Orita et
al. 1989).
• Assays are performed using heat-denatured
PCR-amplified DNA on non-denaturing DNA
sequencing gels.
A
B
Wild
allele
PCR products
Mutant A’
B’
allele
Denaturation
G
A
C
B
A’
A
T
B’
Electrophoresis on a non
denaturing polyacrylamide gel
A
B
single-stranded DNA
fragments fold into a
three-dimensional shape
according to their primary
sequence
A’
B’
Wild type
Mutant type
SSCP
PCR
Low DNA
concentration to
prevent reanealing
SSCP
The electrophoretic mobility of separation is a function
of the shape of the folded, single-stranded molecules
(Hongtrakul et al. 1998)
Features of SSCPs
• Efficient screen for DNA polymorphism
• Monomorphic markers are often times
polymorphic with this assay
• Mutant bands separated from wild-type can be
isolated for analysis
• Co-dominant or dominant
• Locus-specific
• Less applicable to DNA with unknown
• sequence
Heteroduplex Analysis
• DNA polymorphism produced by separating
homoduplex from heteroduplex DNA using nondenaturing gel electrophoresis (White et al. 1992)
or partially denaturing HPLC
• Single mismatches between genotypes produce
heteroduplexes - presence of DNA polymorphism
• Heteroduplexes have an altered electrophoretic
mobility relative to the homoduplex
G
Wild allele
(Reference)
C
PCR on test samples using same primers as in
reference and denaturation of PCR products
Add denaturized PCR product of wild allele
(reference) to each test sample and allow re-anealing
G
C
G
C
Homoduplex
G
A
Heteroduplex
Features of HA
• Efficient screen for polymorphism
• Efficient tool to identify SNP
Diversity Arrays – DArT - Triticarte
Differences
between two rice
cultivars (IR64 green and Millin red)
Diversity Arrays – DArT – Triticarte
• 2,500 markers per sample
• 94 samples - ~$4,500
• ~ 2 cents per datapoint
http://www.diversityarrays.com/
Features of DArT
•
•
•
•
•
•
Very high multiplex ratio
Very high throughput
Bi-allelic
Dominant
Requires substantial investment
Fairly reproducible
Single Nucleotide Polymorphism (SNP)
• DNA sequence variations that occur when a single
nucleotide (A, T, C, or G) in the genome sequence is
altered
Allele 1
Allele 2
Allele 3
Allele 4
Concensus
...
...
...
...
...
ctgaGagat
ctgaAagat
ctgaAagat
ctgaAagat
ctgaNagat
...
...
...
...
...
atgaAaatc
atgaAaatc
atgaGaatc
atgaCaatc
atgaNaatc
...
...
...
...
...
Features of SNP
•
•
•
•
Highly abundant (1 every 1,000 bp)
Locus-specific
Co-dominant and bi-allelic
Basis for high-throughput and massively
parallel genotyping technologies
• Phenotype due to SNP can be mapped directly
SNP Discovery and Assays
• Screen for SNPs
– Assay to detect heteroduplexes (HA)
– Sequencing
• Locus Specific Oligonucleotide Probe (LSOPs)
– Primers for amplifying the target DNA fragments
(locus)
• Allele specific oligonucleotide probes (ASOPs)
– Probes to discriminate SNP alleles
SNP
Assay Systems and Platforms
• Polyacrylamide Gel Electrophoresis
• Capillary Gel Electrophoresis
• High Performance Liquid Chromatography
• Matrix-Assisted Laser Desorption/Ionization
Time-Of-Flight (MALDI-TOF) Mass
Spectroscopy
• Various Gel-Free Systems
SNP
Assays Performed on Locus Specific PCR Products
• Mini-Sequencing (Kuppuswamy et al. 1991; Pastinen
et al. 1997)
• Primer Extension (Hoogendoorn et al. 1999)
• TaqMan (Livak et al. 1995)
• Molecular Beacons (Tyagi et al. 1998)
• Sequenom Assays
SNP
Assays Performed on Total Genomic DNA
• Invader Cleavase (Lyamichev et al. 1999)
• Array and microchip technologies (Matsuzaki
et al. 2004a, 2004b)
• Bead arrays (Fan et al. 2003)
• Review (Fan et al. 2006; Syvanen 2005)
Primer extension coupled with HPLC
(Hoogendoorn et al. 1999)
SNP alleles are discriminated as length variants
5’- ... aaggctgaGagatgatggcaggaatgaaaatccag ... -3’
3’-tctactaccgtccttacttt-5’
5’- ... aaggctgaAagatgatggcaggaatgaaaatccag ... -3’
3’-tctactaccgtccttacttt-5’
Primer Extension + dTTP +
ddCTP
G Allele:
nucleotides
Ctctactaccgtccttacttt
- 21
A Allele: CTTtctactaccgtccttacttt
nucleotides
- 23
Features of Assay
• LSOPs are used to PCR amplify target locus
• PCR products are extended using an ASOP and
the appropriate dNTPs and ddNTPs
• Specific dNTP-ddNTP mixtures are needed for
each allele pair
• Fragment separation through HPLC can be
automated
• Throughput is moderate
Mini-sequencing coupled with gel or capillary
electrophoresis
(Pastinen et al. 1997)
SNP Alleles are discriminated as color variants
5’- ... aaggctgaGagatgatggcaggaatgaaaatccag ... -3’
3’-tctactaccgtccttacttt-5’
5’- ... aaggctgaAagatgatggcaggaatgaaaatccag ... -3’
3’-tctactaccgtccttacttt-5’
ddCTP
ddATP + ddTTP + ddGTP +
G Allele:
Ctctactaccgtccttacttt
- blue 21 nt
A Allele:
Ttctactaccgtccttacttt
- orange 21 nt
Features of Assay
• LSOPs are used to PCR amplify the target DNA
fragment (locus)
• PCR products are extended using an ASOP from the
target amplicon using a mixture of all four ddNTPs
• Genotyping is performed using a ‘standard’ assay
protocol
• Genotyping assays are performed on DNA
sequencing systems
• Potential for very high genotyping throughput
High Throughput Gel-Free
Systems
SEQUENOM ASSAYS
• SNP genotyping platform
• Combines PCR and mass spectrometry detection
• Multiplex up to 40 SNPs in a single well (optimum ~30)
• Process up to 384 samples in parallel
• On demand: Different sets of SNPs can be used in
different runs
• Need to provide the DNA sequences of interest
Molecular beacons (Tyagi et al. 1998;
Cayouette et al. 1999)
• Beacons are added
prior to PCR
• PCR
• PCR amplicons
analyzed in
fluorescent plate
reader
(Mhlanga and Malmberg 2001)
TaqMan Assay – Applied Biosystems
(de la Vega et al. 2005)
Invader Assay - Third Wave Technologies
Assay can be
perfomed on
genomic DNA
sample
(Oliver 2005)
Affymetrix GeneChips
(Lipshutz et al. 1999)
Feature
size
No. Markers
50 um
Up to 4,000
20 um
Up to 25,000
2 um
Up to 2.5 x
106
(Cutler et al. 2001)
10,000 to 100,000 SNP assay on an Affymetrix
GeneChip 10K or 100K
Hyb.,
wash, and
scan
(Matsuzaki et al. 2004)
Oligo Ligation Assay (OLA)
(McDonald et al. 2005)
GoldenGate Assay - Illumina
DATA
http://www.illumina.com/
• 1,536 SNP markers
per sample
• 192 samples ~$12,000
• ~4 cents per
datapoint
Infinium Assay - Illumina
Fan et al. 2006
300,000 to
500,000
SNP Assay
Molecular Inversion Probes (MIP) - Affymetrix
10,000 SNP Assay
(Syvanen 2005)