1. SNP Discovery in the Human Genome C244/144 November 21, 2005

2. Human Genetic Variation Discovery of new variants –Sequence known –Sequence unknown Genotyping of known variants

3. Collins…. 1997

5. A second impetus is the development of improved methods for the discovery and genotyping of single- nucleotide polymorphisms (SNPs). Past methods for SNP discovery have depended primarily on gel-based sequencing of DNA from several individuals and have therefore been relatively slow and expensive. Recent novel approaches to assessing DNA sequence differences between individuals offer considerable promise for reducing the cost and increasing the rate at which large numbers of SNPs can be discovered. We anticipate that it should be possible to generate SNPs in several thousand genes per year at a roughly estimated cost between $100 and $1000 per SNP.

6. 1999

9. Discovery of targeted new variants Sequence unknown –DNA sequencing (1978), complete info, expensive –Denaturing gradient gel electrophoresis(DGGE) (1985), Gradient gel, non-denaturing to denaturing. Double stranded products slow down dramatically when partially denatured, mismatches in duplex DNA decrease the amount of denaturant needed to denature. Small fragments 300bp, GC clamp improves sens to 90%, –RNAse A cleavage (1985) Rnase A will not cleave RNA when annealed to DNA, run gel to detect patterns, difficult pattern recognition, small fragments –Chemical mismatch cleavage (1988) Modify mismatched bases and cleave specifically at the mismatches, up to 2kb length, noisy, difficult, slow –Single stranded conformation polymorphism (1991) <300bp, 90% accuracy. Prepare single stranded end labeled DNA, run on denaturing gel, secondary structure of SSDNA determines mobility on gel

10. Discovery of targeted new variants Sequence unknown –Cleavase Fragment Length Polymorphism Analysis(CFLPA) Cleaves dsDNA at annealed junctions. Poor sensitivity, difficult to maintain secondary structure under cleavage conditions –MutS binding: Recognizes and binds to mismatches in duplex DNA. Small fragments <200bp, noisy –Mismatch Repair Detection (1995). Large inserts into bacterial plasmid vector, if mismatch, then repairs by methyl directed MMR of E coli, can visualize by LacZ +/- status of bacterial clones. Cumbersome, false positive, don’t know where mm is. –T4 Endonuclease VII cleavage of heteroduplex DNA Cleaves at site of mismatch –Heteroduplex analysis: Non denaturing gel electrophoresis kinked hets migrate differently than non-kinked DNA, simple, small fragments, expensive gels

11. Discovery of targeted new variants Sequence unknown –Denaturing HPLC. Temperature dependent variation in migration of heteroduplex DNAs; need to predict optimum temp for discrimination. Limited throughput –Sequencing by hybridization Universal short oligo arrays for de novo sequencing –Direct DNA sequencing Now cheap enough, gives all info about exactly where DNA variant is

12. Sequencing by hybridization Non-gel based All possible 6mers, 4096 arrayed On flat surface, DNA sequence Labeled and hybridized, the set of all 6mers in theory can be determined That hyb with the arrayed oligos Each overlaps with the next oligo Over by 5 bases. read the sequence of the DNA fragment Only short fragments can be seq’d Due to confusion of assembly with Repetitive appearance of the same 6 mer

13. Discovery of targeted new variants Sequence known –All of the previous plus –Variant detector arrays (allele specific hybridization to short oligos)

14. Technologies for typing known variations