Reliable and efficient direct sequencing of PCR-amplified double-stranded genomic DNA template. | Zendy

Martin Jung | Zendy; Anatoly Dritschilo | Zendy; Usha N. Kasid | Zendy

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Reliable and efficient direct sequencing of PCR-amplified double-stranded genomic DNA template.

Author(s) -

Martin Jung,

Anatoly Dritschilo,

Usha N. Kasid

Publication year - 1992

Publication title -

genome research

Language(s) - English

Resource type - Journals

SCImago Journal Rank - 9.556

H-Index - 297

eISSN - 1549-5469

pISSN - 1088-9051

DOI - 10.1101/gr.1.3.171

Subject(s) - subcloning , biology , sequencing by ligation , sequencing by hybridization , dna nanoball sequencing , dna sequencing , multiple displacement amplification , dna , digital polymerase chain reaction , genomic dna , computational biology , genetics , polymerase chain reaction , microbiology and biotechnology , genomic library , base sequence , plasmid , gene , dna extraction , dna sequencer

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 direct sequencing; and sequences of 300 nucleotides can be easily read even after a single loading. The successful use of the modified dideoxynucleotide chain-termination method for direct sequencing of both strands demonstrates the efficiency of this technique for removing sequencing artifacts and for producing reliable sequence data.

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