I Recombinant DNA technology 1 Restriction enzymes 2 Nucleic acid hybridization °3 DNA cloning ·4 iruses °5 DNA sequencing 6 Polymerase chain reaction
I Recombinant DNA technology • 1 Restriction enzymes • 2 Nucleic acid hybridization • 3 DNA cloning • 4 Viruses • 5 DNA sequencing • 6 Polymerase chain reaction
Restriction enzymes ● Overview o Restriction enzyme digestion ● Nomenclature ● Gel electrophoresis ● Restriction maps O Restriction fragment length polymorphisms(RFLP)
• 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
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 become a reality
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 become a reality
ol, chromosome potB dinB (a RecA exA b repressor Damaged DNA single-strand gap aanc.D CerA Replication porB dinB ZorB ∠A Inactivate 5零 activate Single-stranded DNA RecA complex a42 E
Restriction endonuclease (origination) One component 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
Restriction endonuclease (origination) • One component 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 ll Type l Functions Endonuclease Endonuclease Endonuclease methylase Conditions ATP Mb Mg ATP,Mg2+ Recognition EcoK: AACN6GTGC Palindromic(回文序列)EcoP1: AGACC sequences EcoB: TGAN8TGCT EcoP15: CAGCAG Cutting sites At least 1000bp away At or close to recog 24-26 bp away seq
Types of Restriction endonuclease Type I Type II Type III Functions Endonuclease & methylase Endonuclease Endonuclease Conditions ATP, Mb2+ Mg2+ ATP, Mg2+ Recognition sequences EcoK: AACN6GTGC EcoB: TGAN8TGCT Palindromic(回文序列） EcoP1: AGACC EcoP15: CAGCAG Cutting sites At least 1000bp away At or close to recog. seq 24-26 bp away
Recognition sequences Recognize 4-8 bp palindromic sequences. Most commonly used enzymes recognize 6 bp which occurs at a rate of 46=4096 bp. (44=256 bp 48=65536bp) 5′ GAATTC37 e.g. EcoRI site: 3′ CHAAG5′ Restriction enzymes 1. Highly specific 2. Commercially available 3. Require Mg2+ for enzymatic activity 4. Compatible ends from different enzymes
Restriction enzymes Recognize 4-8 bp palindromic sequences. Most commonly used enzymes recognize 6 bp which occurs at a rate of 46=4096 bp. (44=256 bp; 48=65536 bp) 1. Highly specific 2. Commercially available 3. Require Mg2+ for enzymatic activity 4. Compatible ends from different enzymes, 5’ GAATTC 3’ 3’ CTTAAG 5’ e.g. EcoRI site: Recognition sequences
Restriction sequences EcoRI Pst I T G C GA C GT C 5 protruding ends 3’ protruding ends PIAATT TGCA3OH HCT T AASP OH IG 3 5 protruding ends 3 protruding ends Cohesive/sticky ends 5-CCCGGG-3 Smal →5ccc0H+⑩GGG3 3-GGGCCC-5 3GG⑥ OH-CCC5 blunt ends
5’ protruding ends 3’ protruding ends 5’-CCCGGG-3’ 3’-GGGCCC-5’ 5’-CCC-OH 3’-GGG- p p -GGG-3’ OH-CCC-5’ + SmaI blunt ends Cohesive/sticky ends Restriction sequences
Restriction digestion Plasmid with B= BanHI gene x E- ECORI BamHI ECORI FEE Linear fragments Fig. 2. The digestion of a plasmid with two different restriction enzymes
Restriction digestion