Effective amplification of long targets from cloned inserts and human genomic DNA.

Abstract:

:We have used the polymerase chain reaction (PCR) to amplify up to 22 kb of the beta-globin gene cluster from human genomic DNA and up to 42 kb from phaga lambda DNA. We have also amplified 91 human genomic inserts of 9-23 kb directly from recombinant lambda plaques. To do this, we increased pH, added glycerol and dimethyl sulfoxide, decreased denaturation times, increased extension times, and used a secondary thermostable DNA polymerase that possesses a 3'-to 5'-exonuclease, or "proofreading," activity. Our "long PCR" protocols maintain the specificity required for targets in genomic DNA by using lower levels of polymerase and temperature and salt conditions for specific primer annealing. The ability to amplify DNA sequences of 10-40 kb will bring the speed and simplicity of PCR to genomic mapping and sequencing and facilitate studies in molecular genetics.

authors

Cheng S,Fockler C,Barnes WM,Higuchi R

doi

10.1073/pnas.91.12.5695

subject

Has Abstract

pub_date

1994-06-07 00:00:00

pages

5695-9

issue

12

eissn

0027-8424

issn

1091-6490

journal_volume

91

pub_type

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