利用限制酵素在 pUC載體作 DNA序列 之同向定序

Abstract

利用二種 restriction endonuclease : Rsa I，Taq I互相配合，在不同的反應時間控制下可以把載體為 pUC19 的 recombinant DNA 做同一方向（ unidirectional ）切割成大小不同的subfragments ，再經由 self - ligation , transformation 之後，可得到一 ladder 長短不同的 DNA f ragments （附圖五），以這個方法配合 double strand DNA sequencing method ( 23 )決定了一級鯉魚 r - crystallin 基因 ( 1 . 8 kb ) .

We used two different kinds of restriction endonucleases, Rsa I and Taq I, to digest a large DNA fragment that is inserted to pUC 19 vector in a unidirectional deletion manner. Through a time course (about 1 - 5 minutes), the recombinant DNA became different sizes of subfragments. Appropriate size of those deleted subclones were checked and picked out after transformation and mini preparation. These subclones together with the use of dideoxy chain termination sequencing method made an insert DNA with size 1 - 6 kb could be sequenced in a short period. We have exploited this strategy to decide the sequence of carp egg genome, and found out several γ- crystallin genes.