Direct sequencing of lambda DNA from crude lysates using an improved linear amplification technique.
We describe an improved method for directly sequencing lambda (lambda) DNA that has been isolated from either crude cleared lysates or plate lysates. This protocol does not require that the DNA be obtained from bacteriophage particles that have been purified by caesium chloride centrifugation. Nanogram quantities of lambda DNA are unidirectionally amplified using a radioactively-labelled oligonucleotide primer, and Thermus aquaticus (Taq) DNA polymerase, in the presence of T4 gene 32 protein (gp 32). The amplification/sequencing reactions are then incubated with terminal deoxynucleotidyl transferase (TdT) and all four deoxynucleotide triphosphates to elongate any prematurely-arrested products. This procedure, which is a modification of a previously-published method, results in a significant improvement in the quality and amount of DNA sequence information that can be obtained from lambda templates. Although it was developed to sequence DNA directly from lambda EMBL3 recombinants, it can also be used with cosmid DNA, M13 and plasmid DNA, and polymerase chain reaction (PCR) amplification products, yielding excellent ladders in each case. In addition, our method resolves the nucleotide sequences of double-stranded plasmid templates that are difficult to determine by conventional dideoxynucleotide sequencing protocols because of 'stalling', in which bands appear at the same position in all four lanes.
Lasham
A
A
MRC Molecular Neurobiology Unit, MRC Centre, Cambridge, UK.
Darlison
M G
MG
D010582:Bacteriophage lambda / Q000235:genetics*; D003360:Cosmids; D004279:DNA, Viral / Q000235:genetics*; D004268:DNA-Binding Proteins; D004259:DNA-Directed DNA Polymerase; D005784:Gene Amplification; D015345:Oligonucleotide Probes; D010957:Plasmids; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA / Q000379:methods*; D019914:Taq Polymerase; D013698:Templates, Genetic; D014764:Viral Proteins
D004279:DNA, Viral; D004268:DNA-Binding Proteins; D015345:Oligonucleotide Probes; D014764:Viral Proteins; C060035:gp32 protein, Enterobacteria phage T4; D019914:Taq Polymerase; D004259:DNA-Directed DNA Polymerase