文档介绍：binant DNA Technology
Dr. Hui LI
Office : S408
Tel: 26538722
Topic 4
Identification of binant clones
1. Screening
The process of identifying one particular clone containing the gene of interest from among the very large number of others in the gene library .
by insertion inactivation;
by plementation;
by new characteristics generated by inserted genes;
by different kinds of molecular hybridization;
by immunochemistry technology.
1. Insertion inactivation
vector carrying two or more antibiotic resistance genes
cloning sites within the one of antibiotic resistance gene
disruption of gene by insertion of the foreign DNA
pBR322
(1)Tetracyclin (四环素):
(2)Ampicilin(氨苄青霉素抗性基因):
Example: pBR322 plasmid
产生-内酰***酶,使氨苄青霉素转变为青霉***酸,使碘-青霉素指示液(I2-KI-Ampicillin)(蓝灰色)褪色。
抑制细菌生长,但不杀死细菌。
杀死生长的细菌,但不杀死停止生长的细菌。
(3) Cycloserine(环丝氨酸):
如果在Tetr上插入外源DNA,导致四环素抗性基因失活,可用四环素加环丝氨酸平板培养基选择重组克隆。
Tetr失活的菌生长被四环素抑制,不被环丝氨酸杀死,保留下来;
Tetr不失活的菌抗四环素,能分裂生长,反而被环丝氨酸杀死。
(1)四环素抗性插入失活
无环丝氨酸培养基
(2)氨苄青霉素抗性插入失活选择过程
如果Ampr上插入外源DNA,导致氨苄青霉素抗性基因失活,可用碘-青霉素指示液选择。
2. plementation
lications/images/
vector carrying lacZ gene – encodes for N-terminus of β-galactosidase (α-fragment)
host chromosome carrying C-terminal of β-galactosidase (ω-fragment)
IPTG (isopropyl-[beta]-D-thiogalactopyranoside) – inducer
X-Gal – substrate for the enzyme
cloning sites within the LacZ gene
disruption of gene by insertion
of the foreign DNA
blue – functional protein
white – non-functional protein