16.童耕雷老师的要点.docVIP

六章 Restriction enzymes, also called restriction endonucleases,限制性核酸(内切)酶 recognize specific base sequences in double-helical DNA and cleave both strands of the duplex at specific places these exquisitely precise scalpels (marvelous gifts of nature) are indispensable for analyzing chromosome structure, sequencing very long DNA molecules. discovered by Werner Arber, Hamilton Smith, and Daniel Nathans in the late 1960s. The cells own DNA is not degraded because the sites recognized by its own restriction enzymes are methylated甲基化. Many restriction enzymes recognize specific sequences of four to eight base pairs and hydrolyze a phosphodiester bond in each strand in this region. A striking characteristic of these cleavage sites is that they possess twofold rotational symmetry双重旋转(轴相)对称. In other words, the recognized sequence is palindromic回文 and the cleavage sites are symmetrically positioned. A restriction fragment containing a specific base sequence can be identified by hybridizing it with a labeled complementary DNA strand The positions of the DNA fragments in the gel are preserved in the nitrocellulose sheet where they can be hybridized with a 32P-labeled single-stranded DNA probe. Autoradiography then reveals position of the restriction fragment with a sequence complementary to that of the probe. A particular fragment in the midst of a million others can readily be identified in this way, like finding a needle in a haystack. 大海捞针 This powerful technique is known as Southern blotting (DNA印迹法) because it was devised by E.M. Southern. 特定限制片段的鉴定: 混合限制片段(双链)经agarose分离(用NaOH将双链DNA变性为单链) →由胶转移至硝酸纤维素膜 →与标记的(与特定片段互补的)探针杂交 →放射自显影定位(确定此“特定片段”) DNA is Usually Sequenced by Controlled Termination of Replication (Sanger Dideoxy Method) 复制受控终止法即Sanger双脱氧法In fact, this is now the method of choice because of its simplicity. DNA polymerase I is used to copy a particular sequence of a single-stranded DNA . The synthesis is primed by a complementary fragment, which may be obtai