A major focus of the Human Genome Project is the development of automated sequenc-ing technology that can accurately sequence 100,000 or more bases per day at a cost of less than $.50 per base. Specific goals include the development of sequencing and detection schemes that are faster and more sensitive, accurate, and economical. Many novel sequencing technologies are now being explored, and the most promising ones will eventually be optimized for widespread use.

Second-generation (interim) sequencing technologies will enable speed and accuracy to increase by an order of magnitude (i.e., 10 times greater) while lowering the cost per base. Some important disease genes will be sequenced with such technologies as (1) high-voltage capillary and ultrathin electrophoresis to increase fragment separation rate and (2) use of resonance ionization spectroscopy to detect stable isotope labels.

Third-generation gel-less sequencing technologies, which aim to increase efficiency by several orders of magnitude, are expected to be used for sequencing most of the human genome. These developing technologies include (1) enhanced fluorescence detection of individual labeled bases in flow cytometry, (2) direct reading of the base sequence on a DNA strand with the use of scanning tunneling or atomic force microscopies, (3) enhanced mass spectrometric analysis of DNA sequence, and (4) sequencing by hybridization to short panels of nucleotides of known sequence. Pilot large-scale sequencing projects will provide opportunities to improve current technologies and will reveal challenges investigators may encounter in larger-scale efforts.

Partial Sequencing To Facilitate Mapping, Gene Identification

Correlating mapping data from different laboratories has been a problem because of differences in generating, isolating, and mapping DNA fragments. A common reference system designed to meet these challenges uses partially sequenced unique regions (200 to 500 bp) to identify clones, contigs, and long stretches of sequence. Called sequence tagged sites (STSs), these short sequences have become standard markers for physical mapping.

Because coding sequences of genes represent most of the potentially useful information content of the genome (but are only a fraction of the total DNA), some investigators have begun partial sequencing of cDNAs instead of random genomic DNA. (cDNAs are derived from mRNA sequences, which are the transcription products of expressed genes.) In addi-tion to providing unique markers, these partial sequences [termed expressed sequence tags (ESTs)] also identify expressed genes. This strategy can thus provide a means of rapidly identifying most human genes. Other applications of the EST approach include determining locations of genes along chromosomes and identifying coding regions in genomic sequences.

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