Cheng S, Fockler C, Barnes W M, Higuchi R
Department of Human Genetics, Roche Molecular Systems, Inc., Alameda, CA 94501.
Proc Natl Acad Sci U S A. 1994 Jun 7;91(12):5695-9. doi: 10.1073/pnas.91.12.5695.
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.
我们利用聚合酶链反应（PCR）从人类基因组DNA中扩增出长达22 kb的β-珠蛋白基因簇，从噬菌体λ DNA中扩增出长达42 kb的片段。我们还直接从重组λ噬菌斑中扩增了91个9 - 23 kb的人类基因组插入片段。为此，我们提高了pH值，添加了甘油和二甲基亚砜，缩短了变性时间，延长了延伸时间，并使用了一种具有3'至5'核酸外切酶活性（即“校对”活性）的二级耐热DNA聚合酶。我们的“长PCR”方案通过使用较低水平的聚合酶以及特定引物退火所需的温度和盐条件，维持了基因组DNA中靶标的特异性。扩增10 - 40 kb DNA序列的能力将把PCR的速度和简便性应用于基因组图谱绘制和测序，并促进分子遗传学研究。
