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.

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’

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)

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 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

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

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 ... ctgaGagat ... atgaAaatc ... Allele 2 ... ctgaAagat ... atgaAaatc ... Allele 3 ... ctgaAagat ... atgaGaatc ... Allele 4 ... ctgaAagat ... atgaCaatc ... Concensus ... ctgaNagat ... 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’ Primer Extension + dTTP + ddCTP G Allele: Ctctactaccgtccttacttt - 21 nucleotides A Allele: CTTtctactaccgtccttacttt - 23 nucleotides

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’ ddATP + ddTTP + ddGTP + ddCTP 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) (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)

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 1,536 SNP markers per sample 192 samples - ~$12,000 ~4 cents per datapoint http://www.illumina.com/

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

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