Collecting BAC-length-spaced mouse SNPs: Chromosome 17
S. Palczewski, A. Robinson, L. Schalkwyk
SGDP Research Centre,
Institute of Psychiatry
De Crespigny Park
London SE5 8AF
A very extensive and high quality BAC end sequence database made available by TIGR and BAC mapping data from Cai et al and Marra M et al unpublished, as compiled by ENSEMBL www.ensembl.org/Mus_musculus have made it quite straightforward to select sequences to screen for SNPs. We are in our pilot study, 86% of primers pairs designed using primer3 function under standardised conditions and of these 80.5% showed polymorphism by DHPLC of which 23% were PWD-only alleles (summary of 80 primer pairs). Confirmation of these candidates by sequencing reveals that many have multiple, generally parallel, SNPs. 33 of these are in sequences placed on chromosome 17 in the Cai et al (2001) /ENSEMBL BAC framework.
We have extracted a tiling set of BACs for chromosome 17 from ENSEMBL, systematically favouring those BACs which have been 'claimed' for sequencing, on the rationale that these are the most likely to be further characterised, for example it will become clear if their mapping is incorrect. Primers were designed from the masked mouse BAC ends (ftp://ftp.tigr.org/pub/data/m_musculus/bac_end_sequences/mbends.masked) for the selected BACs, and a first-pass set of 300 pairs of primers have been synthesised and are being screened for SNPs.
In order to facilitate primer picking and polymorphism searching we have developed software to facilitate primer picking and DHPLC. Perl modules DHPLC.pm and DNA.pm will be made publically available along with the SNP data generated by this project. DNA.pm includes a primer picking routine which uses more accurate thermodynamic parameters for Tm prediction (SantaLucia 1998) than the traditional primer3 as well as a primer selection logic that maximises product length.
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Cai WW, Chow CW, Damani S, Gregory SG, Marra M, Bradley A. An SSLP marker-anchored BAC framework map of the mouse genome. Nat Genet 2001 Oct;29(2):133-4