Improved double-stranded DNA sequencing using the linear polymerase chain reaction.
Improved double-stranded DNA sequencing using the linear polymerase chain reaction.
复制标题
使用线性聚合酶链式反应改进双链 DNA 测序。
作者：
V. Murray
与双链DNA测序相关的问题之一是大多数[5 ′-3 P]标记的寡核苷酸引物13不能被DNA聚合酶延伸。因此，大部分标记的引物被浪费，凝胶必须进行几天的放射自显影。在本文所述的方法中，几乎所有的标记引物都被DNA聚合酶延伸，因此需要较短的放射自显影曝光。该方法是线性聚合酶链反应（LPCR）的改良，其中仅存在一个o配体核苷酸13（1）。它是U363 Taq DNA聚合酶，并且由于聚合在72°C下进行，导致低水平的测序伪影。多个LPCR循环3还去除了测序伪影。使用低于正常浓度的dNTP浓度是实现以下目标所必需的：
One of the problems associated with double-3tranded DNA sequencing is that most of the [5'-3P]-labelied oligonuoleotide primer i3 not extended by the DNA polymerase. Hence, most of the labelled primer is wasted and gels have to be autoradiographed for several days. In the method described in this paper, almost all of the labelled primer is extended by the DNA polymerase and thus a shorter autoradiograph exposure is needed. This method Is a modification of the linear polymerase chain reaction (LPCR) where only one o ligonucleotide 13 present (1). It U363 Taq DNA polymerase and, since polymerisation is at 72°C, results in a low level of sequencing artifacts. Multiple LPCR cycle3 also removes sequencing artifacts. The use of lower than normal dNTP concentrations i3 necessary to achieve