第四章DNA纯化后的利用.pptVIP

Factors that influence restriction efficiency Factors that influence restriction efficiency Factors that influence restriction efficiency Sticky ends and blunt ends in vitro DNA synthesis using the Klenow fragment (DNA pol I) Experimental Uses: a. Fill-in reaction to label ends of DNA b. DNA sequencing Question 6 Sticky ends are easier, but blunt ends could be done too…. Restriction – ligation 4.3 连接 —— 将DNA分子连接在一起 连接重组DNA分子构建的最后一步是将载体分子和将要被克隆的DNA片段连接到一起，这一过程称作连接，而催化这一反应的酶称作DNA连接酶。 5’ protruding ends 3’ protruding ends 黏性末端（ Cohesive/sticky ends) In biology, sticky end (cohesive end) and blunt end are the two possible configurations resulting from the breaking of double-stranded DNA. If two complementary strands of DNA are of equal length, then they will terminate in a blunt end, as in the following example: 5-CTGATCTGACTGATGCGTATGCTAGT-3 3-GACTAGACTGACTACGCATACGATCA-5‘ However, if one strand extends beyond the complementary region, then the DNA is said to possess an overhang: 5-ATCTGACT-3 3-TAGACTGACTACG-5 If another DNA fragment exists with a complementary overhang, then these two overhangs will tend to associate with each other and each strand is said to possess a sticky end: 5-ATCTGACT + GATGCGTATGCT-3 3-TAGACTGACTACG CATACGA-5 which can form complementary base-pairs: GATGCGTATGCT-3 5-ATCTGACT CATACGA-5 3-TAGACTGACTACG 黏末端之间的碱基配对能够将DNA分子重新连接起来。不同的限制性内切酶可以产生出相同的粘性末端。Bam I（识别序列是GGATTC）和Bgl II （识别序列AGATCT）都能够产生GATC黏末端。相同的黏末端还能够被Sau3A产生出来，而它识别的序列是GATC，经这些酶切割的DNA片断，都能够通过互补的黏末端连接在一起。 Buffer composition: Different restriction enzymes have differing preferences for ionic strength (salt concentration) and major cation (sodium or potassium). but some enzymes are more fussy Battery of 4 commercial buffers overlaps whole spectrum 2. Temperature conditions best at 37C, but there a