1. Single Strand Conformational Polymorphism (SSCP)
DNA polymorphism produced by differential folding (intra-molecular interaction) of single-stranded DNA harboring mutations (Orita et al. 1989).
Assays are performed using heat-denatured PCR-amplified DNA on non-denaturing DNA sequencing gels.

2. Electrophoresis on a non denaturing polyacrylamide gel
Wild allele B
PCR products
Mutant allele A’ B’
Denaturation G A A A’ C T B B’
Electrophoresis on a non
denaturing polyacrylamide gel
single-stranded DNA fragments fold into a
three-dimensional shape according to their primary
sequence A A’ B B’

3. Low DNA concentration to prevent re-anealing
SSCP
Wild type
Mutant type
PCR
Low DNA concentration to prevent re-anealing
SSCP
The electrophoretic mobility of separation is a function of the shape of the folded, single-stranded molecules
(Hongtrakul et al. 1998)

4. 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

5. Heteroduplex Analysis
DNA polymorphism produced by separating homoduplex from heteroduplex DNA using non-denaturing 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

6. 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 G C C
Homoduplex G
Heteroduplex A

7. Features of HA Efficient screen for polymorphism
Efficient tool to identify SNP

8. Diversity Arrays – DArT - Triticarte
Differences between two rice cultivars (IR64 - green and Millin - red)

9. Diversity Arrays – DArT – Triticarte
2,500 markers per sample
94 samples - ~$4,500
~ 2 cents per datapoint

10. Features of DArT Very high multiplex ratio Very high throughput
Bi-allelic
Dominant
Requires substantial investment
Fairly reproducible

11. 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 ctgaGagat ... atgaAaatc ... Allele ctgaAagat ... atgaAaatc ... Allele ctgaAagat ... atgaGaatc ... Allele ctgaAagat ... atgaCaatc ... Concensus ... ctgaNagat ... atgaNaatc ...

12. 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

13. 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

14. 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

15. 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

16. 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)

17. Primer extension coupled with HPLC (Hoogendoorn et al. 1999)
SNP alleles are discriminated as length variants
5’- ... aaggctgaGagatgatggcaggaatgaaaatccag ’
3’-tctactaccgtccttacttt-5’
5’- ... aaggctgaAagatgatggcaggaatgaaaatccag ’
Primer Extension + dTTP + ddCTP
G Allele: Ctctactaccgtccttacttt nucleotides
A Allele: CTTtctactaccgtccttacttt nucleotides

18. 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

19. Mini-sequencing coupled with gel or capillary electrophoresis (Pastinen et al. 1997)
SNP Alleles are discriminated as color variants
5’- ... aaggctgaGagatgatggcaggaatgaaaatccag ’
3’-tctactaccgtccttacttt-5’
5’- ... aaggctgaAagatgatggcaggaatgaaaatccag ’
ddATP + ddTTP + ddGTP + ddCTP
G Allele: Ctctactaccgtccttacttt - blue 21 nt
A Allele: Ttctactaccgtccttacttt - orange 21 nt

20. 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

21. High Throughput Gel-Free Systems

22. 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

23. 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)

24. TaqMan Assay – Applied Biosystems
(de la Vega et al. 2005)

25. Invader Assay - Third Wave Technologies
Assay can be perfomed on genomic DNA sample
(Oliver 2005)

26. Affymetrix GeneChips (Lipshutz et al. 1999) (Cutler et al. 2001)
Feature size
No. Markers
50 um
Up to 4,000
20 um
Up to 25,000
2 um
Up to 2.5 x 106
(Lipshutz et al. 1999)
(Cutler et al. 2001)

27. 10,000 to 100,000 SNP assay on an Affymetrix GeneChip 10K or 100K
Hyb.,
wash, and
scan
(Matsuzaki et al. 2004)

28. Oligo Ligation Assay (OLA)
(McDonald et al. 2005)

29. GoldenGate Assay - Illumina
DATA
1,536 SNP markers
per sample
192 samples - ~$12,000
~4 cents per datapoint

30. Infinium Assay - Illumina
300,000 to 500,000 SNP Assay
Fan et al. 2006

31. Molecular Inversion Probes (MIP) - Affymetrix
10,000 SNP Assay
(Syvanen 2005)