The Use of Sequence Analysis for Homozygote and Heterozygote Base Variation Discovery
The fluorescent version of Sanger dideoxy-sequencing has been in use for 15 years for gene discovery and comparative sequencing projects. Only a decade ago, detection of single-base changes, additions or deletions within genes of a given species were only
- PDF / 3,799,166 Bytes
- 28 Pages / 439 x 666 pts Page_size
- 58 Downloads / 132 Views
8.1 Introduction The fluorescent version of Sanger dideoxy-sequencing has been in use for 15 years for gene discovery and comparative sequencing projects. Only a decade ago, detection of single-base changes, additions or deletions within genes of a given species were only possible provided that the organism was haploid or that the change occurred in the homozygous state. High background and the presence of significant peak height variation within bases of a given sequence read made it difficult to recognize when a base substitution occurred in the heterozygous state. In addition, the lack of efficient software to search for and call hetero- and homozygote single nucleotide polymorphisms (SNPs) provided a major obstacle to scaling up sequencing-based mutation/polymorphism discovery. However, with the advent of new sequencing chemistries, more accurate sequencing enzymes and automation-amenable SNP-scoring software, it is now possible to detect homozygous or heterozygous sequence variations with a high degree of sensitivity and accuracy. Thus, the combination of improved "wet lab" technology and sophisticated bioinformatics tools now allows for a more rigorous quantitative means of determining heterozygous and homozygous sequence polymorphisms. In the following, we will elucidate the progress made in sequencing technologies and the impact on the identification of polymorphisms for disease gene discovery and validation as well as for pharmacogenomics. Automated fluorescent sequencing as well as various polymorphism-scoring software tools are described. We will also review different strategies utilized during polymorphism discovery, such as direct PCR sequencing (Genome Therapeutics Corporation) and subclone-mediated SNP discovery (SNP Consortium).
8.2 Sequence Analysis: a Polymorphism Discovery Method One must distinguish a priori polymorphism detection, i.e., the discovery of unknown mutations or single nucleotide polymorphisms (SNPs) from Principles and Practice Molecular Genetic Epidemiology - A Laboratory Perspective Ian N.M. Day (Ed.) © Springer-Verlag Berlin Heidelberg 2002
184
Hans-Ulrich Thomann et al.
polymorphism screening methods, i.e., the search or confirmation of known SNPs. The classic polymorphism discovery method is DNA sequence analysis (Nichols et a1.l989; Tamary et al. 1994). However, during the last lOyears or so, enormous efforts have been made to search for alternatives to sequencing (reviewed in Landegren 1992; Cotton 1993, 1997; Taylor 1997) mostly due to the perceived high cost of this method and due to the lack of suitable automation. Methods which will detect sequence polymorphisms in specific regions but will not reveal their nature or exact position are hereby termed class A SNP discovery methods. Typical representatives are: enzymatic mismatch scanning (EMS), single-stranded conformation polymorphism (SSCP), denaturing gradient gel electrophoresis (dGGE), and denaturing high performance liquid chromatography (dHPLC). Methods we hereby label as class B are capable of detecting and ch
Data Loading...
