| | Genomic DNA template is amplified in a 10ul PCR reaction using the parameters listed below. A subset of the resulting PCR products is then checked for amplificiation visually via gel electrophoresis. Prior to sequencing, PCR products are subject to enzy |
| | matic clean-up using SAP/Exo I digestion. Purified product is then sequenced via ABI Big Dye-Terminator |
| | DNA sequencing using ABI 3730XL. These are candidate (unvalidated) SNPs detected using automated scoring with no manual review. Two automated methods were used to detect SNPs: PolyPhred (Nickerson et al., Nucleic Acids Res. (14): 2745-51) and a novel al |
| | gorithm, PolyDahn (Richter et al in preparation). A SNP was considered a candidate if it was detected |
| | by both methods or by one method in two reads. Using this criteria, over 3,000 novel SNPs have been discovered (see http://www.hapmap.org/downloads/encode1.html.en for details) and 2,500 SNPs already in dbSNP were rediscovered in 2.5 Mb of sequence. At |
| | the time of submission, a subset of these SNPs were tested by independent genotyping, revealing a false positive rate of 7.3% for this method. Over the course of the project, all SNPs will be genotyped for validation and all genotype data will be depos |
| | ited for all SNPs at www.hapmap.org. |
| | PARAMETER: |
| | Reaction Volume:20 ul reaction volume |
| | Template:100 ng cDNA |
| | Buffer:1X PCR Buffer II (Applied Biosystems Inc.), 1.5 mM MgCl2 |
| | dNTPs:100 uM |
| | Polymerase:1 U AmpliTaq Gold |
| | Primers:0.125 uM mixed primers |
| | Thermal Cycler:MJ Research 96-well |
| | PCR Conditions:96C for 10 min ; 35 cycles of: 96C for 30 sec, 59C for 30 sec, 72C for 1 min ; 72C for 1 min ; 4C forever |
