Showing papers by "Richard K. Wilson published in 1995"

An infrared fluorescent dATP for labeling DNA.

Abstract: Near-infrared fluorescence provides a nonradioactive method of detection with high sensitivity and low background. An infrared fluorophore has been attached covalently to the nucleotide deoxyadenosine triphosphate (dATP) to provide a reagent for enzymatic labeling of various types of DNA molecules and for facilitating their detection with an automated DNA sequencing and analysis system. DNA sequencing reaction products can be labeled internally by performing limited polymerization utilizing infrared-labeled dATP (IR-dATP) as the sole source of adenine deoxynucleotide prior to a dideoxy-specific termination reaction. PCR products can be labeled fluorescently by the addition of limited quantities of IR-dATP to the amplification reaction. This latter strategy has been utilized for detection of short tandem repeat polymorphisms (STRPs) which are useful for gene mapping, genetic diagnostics, forensic analysis, and paternity testing. Restriction fragments can be labeled also by fill-in reactions of appropriate 5' overhangs. Diminutive amounts of such fluorescently labeled DNA molecules can be visualized rapidly and conveniently using infrared detection technology.

Genomic DNA sequencing methods.

Abstract: Sequence analysis of cosmids from C. elegans and other organisms currently is best done using the random or "shotgun" strategy (Wilson et al., 1994). After shearing by sonication, DNA is used to prepare M13 subclone libraries which provide good coverage and high-quality sequence data. The subclones are assembled and the data edited using software tools developed especially for C. elegans genomic sequencing. These same tools facilitate much of the subsequent work to complete both strands of the sequence and resolve any remaining ambiguities. Analysis of the finished sequence is then accomplished using several additional computer tools including Genefinder and ACeDB. Taken together, these methods and tools provide a powerful means for genome analysis in the nematode.

The construction and analysis of M13 libraries prepared from YAC DNA

Abstract: Yeast artificial chromosomes (YACs) provide a powerful way to isolate and map large regions of genomic DNA and their use in genome analysis is now extensive. We modified a series of procedures to produce high quality shotgun libraries from small amounts of YAC DNA. Clones from several different libraries have been sequenced and analyzed for distribution, sequence integrity and degree of contamination from yeast DNA. We describe these procedures and analyses and show that sequencing at about 1-fold coverage, followed by database comparison (survey sequencing) offers a relatively quick method to determine the nature of previously uncharacterized cosmid or YAC clones.

Large-scale complementary DNA sequencing methods.

Abstract: Publisher Summary This chapter discusses the large-scale complementary DNA sequencing methods. Sequence analysis of cDNA clones is a useful means for characterizing genes in Caenorhabditis elegans and investigating gene expression at specific stages of development. To provide high efficiency when cloning cDNAs, bacteriophage lambda vectors are typically used. This chapter describes methods for PCR amplification and single-pass fluorescent sequencing of C. elegans cDNAs. To produce the highest-quality data while maintaining throughput at a maximum, the PCR templates are purified by polyethylene glycol (PEG) precipitation. Cycle sequencing with dye-labeled primers is the method of choice for sequencing. The chapter also describes the methods for complete sequencing of C. elegans cDNAs. The exonuclease III deletion method of Henikoff is especially useful for cDNAs in the size range 2 to 6 kb, and the multiple cloning sites of some of the newer lambda vectors typically provide an adequate choice of restriction enzymes for unidirectional deletion. A second method for complete sequencing of cloned cDNAs is the random of “shotgun” strategy. Although this may be generally preferred for cDNAs larger than 5 kb, it is possible to stitch several small cDNAs together prior to shotgun subcloning.