Determination of nucleotide sequences in DNA
Determination of nucleotide sequences in DNA
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DOI：
10.1007/bf01115145
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发表时间：
1981
影响因子：
4
通讯作者：
F. Sanger
中科院分区：
文献类型：
--
作者：
F. Sanger
其中，被测序的DNA上的区域（模板）。从相应的脱氧核苷三磷酸向引物的3 '端依次添加单核苷酸肽残基，从而形成模板DNA的互补拷贝。通过使用在cz位置含有32p的三磷酸，可以标记新合成的DNA。在早期的实验中，合成的寡核苷酸被用作引物，但在发现限制性内切酶后，使用它们作用产生的片段更方便，因为它们更容易获得。最初使用复制程序来制备标记DNA的短的特定区域，然后可以对其进行部分消化程序。DNA测序的困难之一是找到将其分解成小片段的特定方法。没有已知的合适的酶只能识别一种核苷酸。然而，贝格、范彻和钱伯斯在（7）中已经表明，在某些条件下，用DNA聚合酶将核糖核苷酸取代正常的脱氧核糖核苷酸掺入DNA链中是可能的。因此，例如，如果使用核糖CTP和其他三种脱氧核苷三磷酸进行复制，则可以建立其中C残基为核糖形式的链。在脱氧核苷酸键不断裂的条件下，核糖核苷酸键可以被碱打断，因此可以在C残基处获得特异性断裂。使用这种方法，我们能够在一定程度上扩展我们的测序研究（g）。然而，仍然需要进行广泛的分级和分析。
with, a region on the DNA being sequenced (the template). Mononucleotide residues are added sequentially to the 3'end of the primer from the corresponding deoxynucleoside triphosphates, making a complementary copy of the template DNA. By using triphosphates containing 32p in the cz position, the newly synthesized DNA can be labelled. In the early experiments synthetic oligonudeotides were used as primers, but after the discovery of restriction enzymes it was more convenient to use fragments resulting from their action as they were much more easily obtained.The copying procedure was used initially to prepare a short specific region of labelled DNA which could then be subjected to partial digestion procedures. One of the difficulties of sequencing DNA was to find specific methods for breaking it down into small fragments. No suitable enzymes were known that would recognize only one nucleotide. However, Berg, Fancher, and Chamber] in (7) had shown earlier that under certain conditions it was possible to incorporate ribonucleotides, in place of the normal deoxyribonucleotJdes, into DNA chains with DNA polymerase. Thus, for instance, if copying were carried out using ribo CTP and the other three deoxynucleoside triphosphates, a chain could be built up in which the C residues were in the ribo form. Bonds involving ribonucleotides could be broken by alkali under conditions where those involving the deoxynucleotides were not, so that a specific splitting at C residues could be obtained. Using this method we were able to extend our sequencing studies to some extent (g). However, extensive fractionations and analyses were still required.