Direct Colony Sequencing of Plasmid DNA by Dye Terminator Methods

Direct colony sequencing of plasmid DNA by dye terminator methods.

Direct colony sequencing of plasmid DNA is a powerful technique. Radioactive or fluorescent dye primer methods are the reported direct colony sequencing methods (2–4,6). Recently, dye terminator sequencing chemistry (7) has been improved by the introduction of a modified enzyme, AmpliTaq DNA polymerase FS (8), as well as a new d-Rhodamine and BigDye terminator (PE Biosystems, Foster City, CA,...

Anchored oligo(dT) primers for automated dye terminator DNA sequencing.

Direct sequencing of RAPD fragments using 3'-extended oligonucleotide primers and dye terminator cycle-sequencing.

Random amplified polymorphic DNA (RAPD) markers are used widely to develop high resolution genetic maps and for genome fingerprinting. Typically, single oligomers of approximately 10 nucleotides are used to PCR amplify characteristic RAPD marker fragments. We describe an efficient method for the direct end-sequencing of gel-purified RAPD fragments using one primer from a set of four 3'-terminal...

Direct neutralization of alkaline-denatured plasmid DNA in sequencing protocol by the sequencing reagent itself.

Denaturation of the double stranded DNA is required before sequencing by the dideoxy chain termination method (1). Good results are routinely obtained with the alkaline-denaturation method, which involves incubation of the DNA in the presence of 0.2 M NaOH for a few minutes at room or higher temperatures (2,3). The alkaline mixture is then neutralized by the addition of sodium acetate (0.3 M), ...

AmpliTaq DNA polymerase, FS dye-terminator sequencing: analysis of peak height patterns.

Taq DNA polymerases in which the phenylalanine is substituted by a tyrosine at position 667 (Taq F667Y) are members of a new class of DNA polymerases that incorporate chain-terminating dideoxyribonucleoside triphosphates (ddNTPs) much more efficiently than the wild-type Taq DNA polymerase. Improved incorporation of ddNTPs into DNA during cycle sequencing using AmpliTaq DNA polymerase, FS (Taq-F...