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

TLDR

The study aims to enable amplification of 10–40 kb DNA fragments, extending PCR’s utility to genomic mapping, sequencing, and molecular genetics. The authors optimized long‑PCR by raising pH, adding glycerol and DMSO, shortening denaturation, lengthening extension, and employing a proofreading thermostable polymerase. They successfully amplified up to 42 kb from lambda DNA, 22 kb from human beta‑globin, and 91 human genomic inserts (9–23 kb) while preserving specificity through reduced polymerase, temperature, and salt conditions.

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.