EFFECTIVE AMPLIFICATION OF LONG TARGETS FROM CLONED INSERTS AND HUMAN GENOMIC DNA

被引：527

作者：

CHENG, S ^{[1
]}

FOCKLER, C ^{[1
]}

BARNES, WM ^{[1
]}

HIGUCHI, R ^{[1
]}

机构：

[1] WASHINGTON UNIV, SCH MED, DEPT BIOCHEM & MOLEC BIOPHYS, ST LOUIS, MO 63110 USA

来源：

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA | 1994年 / 91卷 / 12期

关键词：

COSOLVENTS; GENOME MAPPING; PCR; THERMOSTABLE 3'-TO-5'-EXONUCLEASE;

D O I：

10.1073/pnas.91.12.5695

中图分类号：

O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];

学科分类号：

07 ; 0710 ; 09 ;

摘要：

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 phage lambda DNA. We have also amplified 91 human genomic inserts of 9-23 kb directly from recombinant A 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.

引用

页码：5695 / 5699

页数：5