1. Chapter 4 Recombinant DNA Technology

2. 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)

3. Recombinant DNA Technology

4. 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

5. 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’

6. Cleavage by EcoRI (cohesive ends)

7. Cleavage by HindII (blunt ends)

8. 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

9. 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

10. Number of Restriction enzymes
>3000 enzymes from 10,000 species
4-Base Cutters
Sau3AI, HaeIII
6-Base Cutters
EcoRI, BglII, PvuII
8-Base Cutters
NotI, Sbf1

11. 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)

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

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

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

15. 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.

16. 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