Hemi-stranded SSCP analysis of SNPs in short sequence-tagged sites.

SNPs are bi-allelic genetic markers abundantly distributed throughout the human genome. Millions of SNPs have been submitted to public databases and constitute a source of markers for the genotyping of individuals or for the estimation of allele frequencies within populations to identify the genes responsible for diseases using, for example, association studies. Such studies often involve the characterization of many marker SNPs so that a cost-effective and robust method for their analysis is desirable. We have previously developed such a method, post-PCR fluorescence labeling of DNA fragments and analysis using automated capillary electrophoresis under SSCP conditions (PLACESSCP) (1,5,6). The procedure is suitable for examining many SNPs without initial setup for dedicated machines (8,12), because it does not require costly reagents such as fluorescence-labeled primers, and can be performed with widely accessible DNA sequencer as a major instrumentation. PLACE-SSCP analysis with longer PCR products is more efficient in searching for new SNPs. However, shorter products usually give better separation of alleles in the analysis and are more suitable for typing known SNPs by comparing electropherograms. There is also less chance of confusion in interpreting the data by the occurrence of more than two SNPs (or more than three alleles) in the same product. Thus, we designed primers that amplify short DNA fragments (50–70 bp), each of which contained only one SNP, and analyzed them by PLACE-SSCP. Although such fragments should display at most two alleles, SSCP patterns were often complicated by the presence of extraneous peaks that are detected in both of the fluorescent colors and migrated within the range of the SSCP peaks. We regarded these spurious peaks to be annealed DNAs, such as homo-/heteroduplexes or nonspecific aggregates. The dsDNAs of this size range (50–70 bp) are known to have mobility similar to their single-stranded counterparts in native PAGE (10). We believe that the complex of primers and complementary strands is unlikely to be formed in the present system because the labeling procedure involves treatment with Klenow fragment of DNA polymerase, which efficiently degrades unincorporated, unpaired primers (1). In any event, these spurious peaks should be able to be eliminated by removing one of the strands of the PCR products. Lambda exonuclease is an enzyme that selectively digests duplex DNA in the 5′→3′ direction from phosphorylated 5′-ends. This enzyme has been used to prepare ssDNA from PCR products amplified with one 5′-phosphorylated primer for sequencing (4), SSCP analysis (13), and the determination of fragment sizes by electrospray ionization mass spectrometry (11). The reported procedures included steps for product purification to remove salts and PCR buffer by, for example, phenol:chloroform extraction and ethanol precipitation or column filtration. We tried to simplify the procedures and developed the protocol presented below, in which all reactions are performed in the same tube. The addition of a high-pH buffer is critical to ensuring the complete removal of one strand because lambda exonuclease is fully active at a final pH of 9.2 (9). One of the primers in the amplification reaction was phosphorylated using T4 polynucleotide kinase, as described previously (2). PCR was performed in a 10-μL reaction mixture [10 mM TrisHCl (pH 8.3), 50 mM KCl, 2 mM MgCl2, 0.25 μM each primer, 200 μM each of four dNTPs, 50 ng genomic DNA, and 0.25 U AmpliTaq® DNA polymerase (Applied Biosystems, Foster City, CA, USA) premixed with 55 ng TaqStart Antibody (BD Biosciences Clontech, Palo Alto, CA, USA)]. The thermal cycling profile was 1 min at 94°C for initial denaturation, followed by 40 cycles of 15 s at 94°C and 5 s at 48°C. The choice of primer sequences and PCR conditions is discussed later. The PCR product was diluted with an equal volume of distilled water, and a 2-μL aliquot was added to 4 μL of the post-PCR labeling reaction buffer to fluorescently label one of the complementary strands, as described previously (7). The reaction buffer comprised of 15 mM Tris-HCl, pH 8.3, 15 mM MgCl2, 0.3 μM rhodamine-labeled dideoxynucleotides (Perkin Elmer Life Sciences, Boston, MA, USA) as indicated in Table 1, 0.3 U Klenow fragment (New England Biolabs, Beverly, MA, USA), and 0.3 U Thermo SequeDRUG DISCOVERY AND GENOMIC TECHNOLOGIES