摘要:

ABSTRACTA procedure using a 96-well format for growth, template preparation, and quantification was optimized to facilitate high-throughput automated sequencing of double stranded plasmid DNA templates. Modification of the 96-well Miniprep Kit [Advanced Genetic Technologies (AGTC), Gaithersburg, MD] protocol combined with the DNA quantification with a Millipore Cytofluor 2350 (Millipore, Bedford, MA) provided high-quality DNA plasmid templates rapidly, efficiently, and inexpensively. We utilized this procedure to prepare more than 130,000 templates for a number of large-scale cDNA and genomic projects. Four specific projects were: human expressed sequence tag (EST) project (Adams et al., 1994) (70,080 templates),Haemophilus influenzae(Fleischmann et al., 1995) (22,656 templates),Mycoplasma genitalium(Fraser et al., 1995) (5760 templates), andMethanococcus jannaschii(22,175 templates). Data from these projects support a sequencing success rate of 89%–95%, with an average read length of 432 bases with<1% ambiguous base

ISSN:1070-2830    

DOI:10.1089/gst.1995.1.1

年代:1995

数据来源: MAL

摘要:

ABSTRACTA new approach to assembling large, random shotgun sequencing projects has been developed. The TIGR Assembler overcomes several major obstacles to assembling such projects: the large number of pairwise comparisons required, the presence of repeat regions, chimeras introduced in the cloning process, and sequencing errors. A fast initial comparison of fragments based on oligonucleotide content is used to eliminate the need for a more sensitive comparison between most fragment pairs, thus greatly reducing computer search time. Potential repeat regions are recognized by determining which fragments have more potential overlaps than expected given a random distribution of fragments. Repeat regions are dealt with by increasing the match criteria stringency and by assembling these regions last so that maximum information from nonrepeat regions can be used. The algorithm also incorporates a number of constraints, such as clone length and the placement of sequences from the opposite ends of a clone. TIGR Assembler has been used to assemble the complete 1.8 MbpHaemophilus influenzae(Fleischmann et al., 1995) and 0.58 MbpMycoplasma genitalium(Fraser et al., 1995) genomes.

ISSN:1070-2830    

DOI:10.1089/gst.1995.1.9

年代:1995

数据来源: MAL

摘要:

ABSTRACTThe first wave of practical products from the international effort to produce systematic maps of the human genome and improve DNA sequencing technologies is taking the form of new tools to predict the risk of specific diseases in individual patients. DNA-based tests for molecular mutations associated with clinical syndromes increasingly allow clinicians to detect disease processes and health risks before clinical problems occur, sometimes making prevention possible. The increasing predictive power of medical diagnostics, however, also poses serious health policy challenges at both the professional and societal levels. At the professional level, DNA-based health risk assessments challenge traditional ethical commitments to confidentially, informed consent, and nondirective genetic counseling. At the societal level, these tests challenge institutions and governments to clarify their policies regarding access to opportunities by defining fair uses of genetic health risk information about individuals outside the clinical setting. Underlying all these issues are basic questions about how we interpret the meaning of DNA-based diagnostic findings.

ISSN:1070-2830    

DOI:10.1089/gst.1995.1.21

年代:1995

数据来源: MAL

ISSN:1070-2830    

DOI:10.1089/gst.1995.1.xv

年代:1995

数据来源: MAL