Automated fluorescent DNA sequencing of polymerase chain reaction products.
The methods described in this chapter provide some useful approaches for DNA sequencing of templates produced by PCR. These procedures have been employed successfully for large-scale DNA sequencing of cosmid fragments subcloned in plasmid or M13 vectors, and for sequence analysis of cDNAs cloned in bacteriophage lambda vectors. In addition, the method describing direct sequencing from PEG-precipitated PCR product has been used successfully for analysis of Caenorhabditis elegans genomic and cDNA sequences. It is important to reiterate that for every combination of amplification primer pair and target DNA, there is an optimal method for PCR amplification; the ability to sequence the products of any PCR experiment directly will also vary. A coupled PCR/DNA sequencing method that works well for one experimental system may work quite poorly with others. Hence, a few days or hours spent optimizing PCR amplification conditions and selecting the best DNA sequencing method for the target DNA of interest will be time well spent.
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Department of Genetics, Washington University School of Medicine, St. Louis, Missouri 63110.
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Wilson
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D000818:Animals; D001322:Autoanalysis / Q000379:methods; D001483:Base Sequence; D017173:Caenorhabditis elegans / Q000235:genetics; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004247:DNA; D004274:DNA, Recombinant / Q000302:isolation & purification; D016678:Genome; D007202:Indicators and Reagents; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D016133:Polymerase Chain Reaction / Q000379:methods*; D013050:Spectrometry, Fluorescence / Q000379:methods
D004274:DNA, Recombinant; D007202:Indicators and Reagents; D009838:Oligodeoxyribonucleotides; D004247:DNA