Avoiding strand reassociation in direct sequencing of double-stranded PCR products with thermolabile polymerases.
نویسندگان
چکیده
منابع مشابه
Sequencing double-stranded DNA by strand displacement.
Duplex probes with five base single-stranded overhangs can capture dsDNA targets from type IIS restriction nuclease digests. Ligation generates a predesigned nick site, where DNA polymerase can generate sequencing ladders by strand displacement or nick translation in the presence of trace amounts of dideoxynucleotides. This allows dsDNA targets to be captured from mixtures and directly sequence...
متن کاملReliable and efficient direct sequencing of PCR-amplified double-stranded genomic DNA template.
Modified PCR amplification and direct sequencing procedures for the double-stranded genomic DNA template are described. Advantages of the approach we describe are: background artifact bands previously observed using high-molecular-weight DNA as a template were eliminated by this protocol; no gel purification or subcloning of the PCR-amplified double-stranded fragment was required prior to direc...
متن کاملDirect sequencing of PCR products in agarose gel slices.
Direct DNA sequencing of amplified polymerase chain reaction (PCR) products offers several advantages over cloning of amplified DNA products. It is faster (1 day versus 3—5 days) and in DNA samples containing sequence polymorphisms both the normal and mutated sequence can be detected in the same sequencing reaction. The major problems encountered in direct sequencing of amplified DNA have been ...
متن کاملDirect sequencing of PCR products using unlabeled primers.
An improved protocol is described for using lambda exonuclease to directly sequence PCR products. It is important not to execute PCR cycles beyond the plateau of amplification. The asymmetric PCR and double-stranded DNA sequencing by a snap-cooling procedure were also performed using the same DNA samples and primers. The improved method was the most reliable and produced the best results.
متن کاملSequencing of Large Double - Stranded
The dideoxy sequencing technique has been applied to the d i rec t sequencing of large double-stranded DNA molecu les with a small single-stranded primer. For ins tance , the method was applied to the lambda genome, which contains 48 502 base-pairs (Sanger F, Coulson AR, Hong GF, Hill D & Petersen GB, 1982, J. Mol. Biol., in press), and the coding region for gene W identified. The procedure pro...
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ژورنال
عنوان ژورنال: Genome Research
سال: 1992
ISSN: 1088-9051
DOI: 10.1101/gr.1.3.193