The present invention provides a method for preparing a DNA fragment, in which a desired double-stranded DNA fragment having a sticky end is directly and easily obtained from an amplification product (an amplified fragment) after PCR without a restriction enzyme digestion. The method for preparing a DNA fragment having a sticky end of the present invention comprises: (i) a step of performing a PCR reaction using a template DNA and specific primers to obtain an amplified DNA fragment; and (ii) a step of performing a prescribed treatment on the amplified DNA fragment to dissociate a protecting group from the fragment. Herein, the above-mentioned specific primers are composed of a complementary DNA portion consisting of a nucleotide sequence complementarily binding to an amplification target region in a template DNA and a non-complementary DNA portion consisting of a nucleotide sequence that links to the 5′ end of the complementary DNA portion but does not complementarily bind to the amplification target sequence, and at least a base corresponding to the 3′ end in the nucleotide sequence of the non-complementary DNA portion is modified with a protecting group capable of terminating the progression of DNA replication catalyzed by a DNA polymerase.
本发明提供了一种制备DNA片段的方法,其中在PCR后,无需限制性内切酶消化,即可直接轻松地从扩增产物(扩增片段)中获得具有粘性末端的所需双链DNA片段。本发明的制备具有粘性末端的DNA片段的方法包括:(i)使用模板DNA和特异性引物进行PCR反应,以获得扩增的DNA片段;(ii)对扩增的DNA片段进行规定的处理,以使保护基从片段中解离。此处,上述特异性引物由互补DNA部分和非互补DNA部分组成。互补DNA部分包括与模板DNA中扩增目标区域互补配对的核苷酸序列,而非互补DNA部分包括连接到互补DNA部分5'端的核苷酸序列,但不与扩增目标序列互补配对,而且至少有一个与非互补DNA部分核苷酸序列中3'端相对应的碱基被保护基修饰,该保护基能够终止由
DNA聚合酶催化的DNA复制的进展。