Sorge J A, Blinderman L A
Stratagene, La Jolla, CA 92037.
Proc Natl Acad Sci U S A. 1989 Dec;86(23):9208-12. doi: 10.1073/pnas.86.23.9208.
A method is reported for sequencing DNA based on exonuclease III digestion and strand protection by using modified nucleoside triphosphates. Up to 10 kilobases of sequence information may be obtained from each strand of a given template without subcloning. Prior knowledge of the restriction map is not important; prior knowledge of any of the sequence is not required. Nor are oligonucleotide primers needed. Double-stranded cosmids, plasmids, lambda phage, or linear molecules (including amplified molecules) may be used as starting material. The method creates a single-stranded template from these starting molecules, thus generating high-quality sequence ladders. Most commonly used DNA polymerases may be utilized, including reverse transcriptase and T7 DNA polymerase. The approach is "ordered", so little time is wasted on redundant sequencing.
报道了一种基于核酸外切酶III消化和使用修饰核苷三磷酸进行链保护的DNA测序方法。无需亚克隆，从给定模板的每条链中可获得多达10千碱基的序列信息。限制酶切图谱的先验知识并不重要；不需要任何序列的先验知识。也不需要寡核苷酸引物。双链黏粒、质粒、λ噬菌体或线性分子（包括扩增分子）可用作起始材料。该方法从这些起始分子创建单链模板，从而生成高质量的序列梯。可使用最常用的DNA聚合酶，包括逆转录酶和T7 DNA聚合酶。该方法是“有序的”，因此在冗余测序上浪费的时间很少。
