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)
eye
스위스소주만병만주소 다시합창합시다 과학은좋은학과
Madam , I’m Adam I l ov e Tevol i
R ace Car Palindromic sequence
--- The two strands are identical when either is
read in the same polarity (5’3’).
Palindrome
AD 79, Pompei
“Sator Arepo Tenet Opera Rotas”
S A
iT O R A R E P O
i
T
iE 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
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
--- formation of phosphodiester bonds
between 3’ OH and 5’ phosphate
Ligation
Sticky-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
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
