Chapter 4 Recombinant DNA Technology

Recombinant DNA Technology Generation of recombinant DNA molecule Cloning vector-insert DNA construct (DNA construct) Cut DNA from a donor organism Cloned DNA, insert DNA, target DNA, foreign DNA Ligation to a cloning vector DNA Transformation Introduction and maintain the DNA construct within a host cell Selection of transformed cells Production of the foreign protein in the host (optional)

Recombinant DNA Technology

Restriction Endonucleases Type I: recognizes a specific sequence but makes cut elsewhere Type II: makes cut only within the recognition site EcoRI E: the genus of the source organism co: the first two of the species of this organism R: the strain of origin (Capital, RY13) I: the order of discovery (Roman numerals) BglII B: Bacillus gl: globigii

Type II Recognition Sequences Cohesive ends (sticky ends, protruding ends) 5’ overhang: Major, EcoRI, BamHI, etc. 5’ G/AATTC 3’ 5’ G AATTC 3’ 3’ CTTAA/G 5’ 3’ CTTAA G 5’ 3’ overhang: PstI, KpnI 5’ CTGCA/G 3’ 5’ CTGCA G 3’ 3’ G/ACGTC 3’ G ACGTC 5’ Blunt ends (flush ends): SmaI, EcoRV 5’ CCC/GGG 3’ 5’ CCC GGG 3’ 3’ GGG/CCC 5’ 3’ GGG CCC 5’

Cleavage by EcoRI (cohesive ends)

Cleavage by HindII (blunt ends)

Palindromic sequence --- The two strands are identical when either is read in the same polarity (5’3’). e y e 스 위 스 소 주 만 병 만 주 소 다 시 합 창 합 시 다 과 학 은 좋 은 학 과 R a c e C a r M a d a m , I’ m A d a m I l o v e T e v o l i

Palindrome AD 79, Pompei S A iT O R A R E P O O P E R A R O iT A S “Sator Arepo Tenet Opera Rotas” S A iT O R A R E P O iTi E N E iT O P E R A R O iT A S

Number of Restriction enzymes >3000 enzymes from 10,000 species http://rebase.neb.com/rebase/rebase.html 4-Base Cutters Sau3AI, HaeIII 6-Base Cutters EcoRI, BglII, PvuII 8-Base Cutters NotI, Sbf1

Restriction Enzymes Isoschizomer Neoschizomer Isocaudomers Enzymes that recognize the same target DNA sequence and cleave it in the same way e.g. SphI and BbuI (CGTAC/G) Neoschizomer Enzymes that recognizes the same target DNA sequence but cleave at different points e.g. SmaI (CCC/GGG) and XmaI (C/CCGGG) Isocaudomers Enzymes that produce the same nucleotide extensions but have different recognition sites e.g. BamHI (G/GATCC) and Sau3AI (/GATC)

Restriction Mapping of DNA Cut DNA with various endonuclease Determination of the sizes of the restriction fragments by gel electrophoresis

Annealing --- results in a nick (broken bond site)

Ligation --- seals the nick by DNA ligase Ligation --- seals the nick by DNA ligase --- formation of phosphodiester bonds between 3’ OH and 5’ phosphate

Ligation Sticky-end ligation Blunt-end ligation at low temp for long period for base pairing Blunt-end ligation at room temp stable base pairing is not required Blunt-end ligations require 10 to 100 times more DNA ligase than sticky-end ligation.

Ligation Conditions Temperature DNA concentration Consider enzyme activity and base pairing of cohesive termini Cohesive ends: 4-15oC: ensure base pairing Blunt ends: 18oC, use 10 to 100 times higher concentration of T4 DNA ligase DNA concentration Dilute concentration favors circulization of linear fragment Insert : Vector = 2 : 1 molar ratio Phosphatase treatment Prevention of self ligation of vector