Pan H Q, Wang Y P, Chissoe S L, Bodenteich A, Wang Z, Iyer K, Clifton S W, Crabtree J S, Roe B A
Department of Chemistry and Biochemistry, University of Oklahoma, Norman 73019.
Genet Anal Tech Appl. 1994;11(5-6):181-6. doi: 10.1016/1050-3862(94)90039-6.
The complete nucleotide sequence of the 16,009-bp SacBII Kan domain of the P1 pAD10-SacBII cloning vector and the sequences of three cosmid cloning vectors, pTCF (7941 bp), svPHEP (9201 bp), and LAWRIST16 (5194 bp) have been determined. A modified diatomaceous earth (Prep-A-Gene)-based procedure, which rapidly yields highly supercoiled double-stranded DNA from recombinant P1 and cosmid clones suitable for generating shotgun libraries, also has been developed. The isolated recombinant DNAs were physically sheared to generate 1- to 2-kb fragments that then were blunt-ended and subcloned into double-stranded pUC-based sequencing vectors. The double-stranded sequencing templates were isolated by an alkaline lysis method and subjected to Taq polymerase catalyzed fluorescent end-labeled primer cycle sequencing. After shotgun sequence assembly, contig gaps were closed and ambiguities were resolved via Sequenase catalyzed fluorescent dye-terminator sequencing.
已确定P1 pAD10 - SacBII克隆载体16,009 bp的SacBII Kan结构域的完整核苷酸序列以及三种黏粒克隆载体pTCF（7941 bp）、svPHEP（9201 bp）和LAWRIST16（5194 bp）的序列。还开发了一种基于改性硅藻土（Prep - A - Gene）的方法，该方法能从适合构建鸟枪法文库的重组P1和黏粒克隆中快速产生高度超螺旋的双链DNA。将分离出的重组DNA进行物理剪切以产生1至2 kb的片段，然后将这些片段平端化并亚克隆到基于双链pUC的测序载体中。通过碱性裂解方法分离双链测序模板，并进行Taq聚合酶催化的荧光末端标记引物循环测序。在鸟枪法序列组装后，通过Sequenase催化的荧光染料终止子测序封闭重叠群间隙并解决歧义。
