I Recombinant DNA technology.ppt

I binant DNA technology
1 Restriction enzymes
2 Nucleic acid hybridization
3 DNA cloning
4 Viruses
5 DNA sequencing
6 Polymerase chain reaction
Overview
Restriction enzyme digestion
Nomenclature
Gel electrophoresis
Restriction maps
Restriction fragment length polymorphisms(RFLP)
Restriction enzymes
Overview
Restriction enzymes allow DNA to be cut at specific sites; Nucleic acid hybridization allows the detection of specific nucleic acid sequences; DNA sequencing can be used to easily determine the nucleotide sequence of a DNA molecule.
Restriction endonuclease(Restriction enzyme)
Bacterial enzymes which cut DNA into defined and reproducible fragments
Identified in the late 1960s
Key discovery which allowed the DNA cloning to e a reality
Restriction endonuclease(origination)
ponent of the bacterial restriction-modification system, a natural defense mechanism of bacteria to against the introduction of foreign DNA into the cell
Restriction endonuclease: recognize a short, symmetrical DNA sequence, and cut DNA backbone in each strand at a specific site within that sequence (kill foreign DNA)
Mythylase: methylates C or A of the cellular DNA
Types of Restriction endonuclease
Type I
Type II
Type III
Functions
Endonuclease & methylase
Endonuclease
Endonuclease
Conditions
ATP, Mb2+
Mg2+
ATP, Mg2+
Recognition sequences
EcoK: 6GTGC
EcoB: TGAN8TGCT
Palindromic(回文序列)
EcoP1:
EcoP15: CAGCAG
Cutting sites
At least 1000bp away
At or close to recog. seq
24-26 bp away
Restriction enzymes
Recognize 4-8 bp palindromic sequences. monly used enzymes recognize 6 bp which occurs at a rate of 46=4096 bp. (44=256 bp; 48=65536 bp)
Highly mercially available
Require Mg2+ for enzymatic patible ends from different enzymes,
5’ GAATTC 3’
3’ CTTAAG 5’
. EcoRI site:
Recognition sequences
5’ protruding ends
3’ protruding ends
5’-CCCGGG-3’
3’--5’
5’-CCC-OH
3’-GGG- p
p -GGG-3’
C-5’
+
SmaI
blunt ends
Cohesive/sticky ends
Restriction sequences
Restrict