| Popis: |
Genetic variation and allelic complexity in the human population seem to play an enormous role in the genetics of complex traits, as well as in individual drug responses. The most common variations in the human genome, comprising ∼90% of all differences, are single-nucleotide polymorphisms (SNPs). Following the recent deciphering of the human genome by sequencing (Lander et al. 2001; Venter et al. 2001), the next step for gaining additional insight into the genetic contribution to the risk for complex diseases should be the analysis of genetic variations, such as SNPs associated with a special phenotype or disease. However, frequent failure of complex disease studies as well as the great body of controversial results in single SNP association studies indicate the importance of haplotype analysis, that is, the determination of the phase of SNPs on a single chromosome, over simply SNP genotyping (Davidson 2000; Drysdale et al. 2000; Hoehe et al. 2000; Maat-Kievit et al. 2001; Boldt and Petzl-Erler 2002). Furthermore, the finding that haplotypes occur in blocks of limited diversity (Daly et al. 2001) will considerably cut down the workload involved in large-scale haplotyping of individuals in the future. Current procedures for haplotyping, and thus gaining the maximal possible information from the data, are very labor-intensive and expensive. The most frequently used methods require either somatic cell hybrid technology, which converts a diploid cell into haploid cell lines (Douglas et al. 2001) or excessive statistical analysis (Rohde and Fuerst 2001; Sachidanandam et al. 2001). In a different approach, parental genotyping can be used to infer haplotypes in a family study (Tapadar et al. 2000). An interesting proof-of-principle for direct haplotyping of kilobase-sized DNA using carbon nanotube probes and multiplex detection of labeled probes by atomic force microscopy was presented recently (Woolley et al. 2000). Direct haplotyping can also be achieved by the newly published polony-technique (Mitra et al. 2003). This technique relies on embedding intact chromosomal DNA in polyacrylamide gels on a glass slide and subsequent PCR. Another method includes diluting the DNA sample to the degree that quasi-single molecule PCRs are performed (Ruano et al. 1990). A more recent development simply exploits the fact that in a good DNA preparation the average DNA is about 100–150 kb in length and that several short-range PCRs can be performed on a single molecule (Ding and Cantor 2003). A further procedure employs allele-specific PCR and relies on subsequent gel-based separation and fluorescence detection (Fugger et al. 1990). The combination of allele-specific PCR and mass spectrometry analysis of SNPs was achieved by Gut and coworkers (Tost et al. 2002). Here we present a method for fast and efficient molecular haplotyping that we have termed clone-based systematic haplotyping (CSH). In this method, large-insert cloning (insert size ∼40 kb) and subsequent screening of clone pools (each representing ∼10% of the genome) by PCR are coupled with a mass spectrometric procedure for SNP typing that we have termed the GOOD assay (Sauer et al. 2000a,b, Sauer and Gut 2003). |
