Recognize specific sequences of 4-8 base pairs in double-stranded DNA molecules

The cleavage sites of most enzymes are within or on both sides of the recognition sequence

Identify the cleavage sequence as a classic rotationally symmetric palindrome structure

Cleaves DNA to produce ends containing 5' phosphate and 3' hydroxyl groups

Offset cuts produce sticky ends, cuts along the axis of symmetry produce flat ends

Most type II enzymes require similar reaction conditions

Multi-enzyme combined enzymatic hydrolysis

Impurities: protein, phenol, chloroform, ethanol, EDTA, SDS, NaC, etc.

Approach

Certain restriction endonucleases require many times more enzyme to cleave supercoiled plasmid DNA or viral DNA than to digest linear DNA.

Some nucleic acid restriction endonucleases cut restriction enzyme sites at different positions, and their efficiency is also significantly different.

5'->3' DNA polymerase activity

5'->3' exonuclease activity

3'->5' exonuclease activity

Nick translation: The 5'->3' exonuclease and polymerase activities work together to advance the nick.

Preparation of 32P-labeled DNA probes

Source: Escherichia coli DNA polymerase Ⅰ is treated with subtilisin to obtain the N-terminal third of the large peptide segment, which is the Klenow enzyme

Has 5'->3' DNA polymerase activity Has 3'->5' exonuclease activity Loss of 5'->3' exonuclease activity

1||| Fill in 5' sticky ends generated by endonucleases

2||| Isotopic end labeling of DNA fragments

3||| cDNA second strand synthesis

4||| Determination of DNA sequence by dideoxy chain end termination method

① 5'->3' DNA polymerase activity ② 3'->5' exonucleolytic polymerase activity ③ Lack of 5'->3' exonuclease activity ④ In the absence of dNTP, exo-cleavage can be performed from any 3'-OH end ⑤ When there is only one kind of dNTP, exolysis stops when the complementary nucleotide is exposed. ⑥ When all four dNTPs are present, polymerization activity dominates

①Cut the 3' sticky end generated by endonuclease

② Isotopic end labeling of DNA fragments

① 5'->3' DNA polymerase activity ② 3'->5' exonuclease activity ③ Has the strongest processivity among known DNA polymerases ④ After modification, it becomes a sequencing enzyme for long fragment DNA using the dideoxy chain termination method.

Primer extension reactions for longer DNA fragments

Rapid end labeling via fill-in or exchange (displacement) reactions

Source: calf thymus

Template-free DNA polymerase randomly incorporates dTNP at the 3'-OH end

Add complementary homopolymer tails to vector or target DNA

Isotopic labeling of the 3' ends of DNA fragments

(1) cDNA synthesis guided by RNA

(2) Bidirectional excision of the RNA strand in the DNA-RNA hybrid strand (RNase H activity)

(3) DNA-guided DNA polymerization activity

AMV: a reverse transcriptase enzyme derived from purified avian myeloblastoma virus (functions at 42°C)

MMLV: An expression product of the reverse transcriptase gene of Moloney murine leukemia virus (MMLV) in E. coli (functions at 37°C)

Transcribe the RNA of eukaryotic genes into DNA and construct a cDNA library

Evaluate and label the 3' end of DNA fragments with 5' overhangs, and prepare probes

Replaces Klenow fragment for DNA sequencing

Remove the 5' end phosphate group of the vector DNA to prevent the vector from cyclizing itself and improve the recombination rate

Remove 5' phosphate groups from DNA and RNA, then end-label with T4 polynucleotide kinase

① Endocution of single-stranded DNA or RNA

② Double-stranded DNA or RNA with nicks or clefts within it

① Remove the single-stranded overhang of the DNA fragment and make it a blunt end ② Remove the hairpin structure formed during cDNA synthesis ③ Analyze the amount of mRNA through S1 nuclease protection experiment ④ Hybridization of mature mRNA and genomic DNA, combined with hydrolysis by this enzyme, can locate introns ⑤ Trim the ends of progressive deletion mutations