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Gene Cloning
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molecule
Ligase
forming phosphodiester bond
2.Gene cloning process
Prepare target gene
Prepare vector
Two primers
dNTPs
Taq polymerase
Buffer solution
2.Get the vector
• Depending on purpose, the size of insert, the
MCS etc, select vectors
Cloning & expressing Prokaryotic & eukaryotic
Plasmid & Virus
Whether the MCS contains suitable RE site for target
gene insertion
Replication
•Prepare vectors
Vectors
– Why do we need vectors?
Because our interest gene need to be stably maintained
and replicate in cells
– How do you stably maintain and replicate a foreign
DNA in cells?
– by hitching a ride on a stable replicon
Vector : --an autonomously replicating piece of DNA
--carry foreign DNA and go into host cells
where it would replicate independently
Basic elements required as a
recombinant vector
MCS
Selective
marker
Basic elements required as a
recombinant vector
MCS
Multiple cloning site
(MCS): To allow
foreign gene inserted
MCS
MCS
Host cells:
usually: E.coli
seldom: Eu. Cells
e.g yeast cells
features:
√possess basic elements
√ Make large copies
√ Easily isolated
Expressing vectors
Features:
√ not only possess basic elements
√ also possess elements for gene expression
Transcription elements
promoter
SD MCS terminator
Translational elements
*just like origin, these elements are
species-specific
√ make proteins Expression
Ampr
Host cells: vector
Prokaryotic expressing vectors
Eukaryotic expressing vectors
ori
Vectors
Cloning Plasmid
Vectors
λPhage
Cosmid
Prokaryotic- M13 phage
Expressing
YAC
Vectors
Eukaryotic- BAC
mammalian
Virus
Plasmid Vectors
→The most commonly used vectors
→Naturally occurring but engineered
small, stable extrachromsomal circular dsDNA,
replicate indepentdently
→Host cells:
E.coli : natural
Eukaryotic cells: natural
or artificial
→capacity: 5~10kb
→for cloning and /or expression
3.Construct recombinant DNA
--to insert a foreign DNA into plasmid
--RE cut plasmid and foreign
DNA to make their ends
compatible
e.g single RE or double RE
produced base-pairing sticky
ends, or blunt ends
--Happened in tubes
(in vitro)
Types of ligation
Less efficient
Blunt ends ligation
Vector Bidirectional
More religation
Ligation product is a mixture
EcoRI
EcoRI EcoRI
Vector
*Recombinant DNA molecules
--foreign DNA May be RE digestion
inserted with either orientation RE digestion
RE1
RE1
RE2
RE2
Transfection:
-- the take-up of DNA into eukaryotic cells
Transformation:
--the take-up of DNA into prokaryotic cells
Infection:
--the entry of virus into cells
Host cells
• where the vectors get maintained and/or
multiplied copies -crucial
* Grow faster
More product, less time 12 hours
Plant cells
Yeast cells
Mammalian cells
Gene Transfer - Transformation methods
Transformation: a process of
uptake of exogenous DNA by
competent cells.
P lacI IPTG
- galactosidase (-gal)
Blue colony
substrate a Separated in
subunit gene
X-GAL subunit
3.5 kb 4.0 kb
3.0 kb
2.0 kb
1.6 kb
1.0 kb
insert 0.8 kb
0.5 kb
DNA sequencing
Confirm
Summary of DNA cloning
Vector Target DNA
Host Cell
Cloning
Selection
Recombinant DNA
Cloned recombinant DNA
Polymerase Chain Reaction
聚合酶链式反应
Polymerase Chain Reaction, PCR
target sequence
3’ 5’
5’
5’
5’ 3’
PCR
5’ 3’
3’ 5’
PCR reaction system
√template: --dsDNA denatured into ssDNA by heating
--any source of DNA with some sequence
information known
√a pair of primers
--18 to 30 nt long (ssDNA) synthesized in vitro
--designed to anneal on opposite of the target
sequence
√dNTPs
√reaction buffer
PCR is to amplify specific DNA
DNA template thermal cycler
Two primers
dNTPs
Taq polymerase
Buffer solution
What happened in
thermal cycler?
Procedures:
1. 模板的变性
2. 引物的退火
3. 新链的延伸
Parameters:
Temperature, Time
Three different steps proceed in each PCR cycle
DNA template
③
Cycle
②
Round 3
Round 4
Round 5
Round 6
Several
minutes
size separation
2. DNA Sequencing
RT-PCR
5‘-Cap
mRNA
RT+PCR
AAA(A)n
(dT)12~18
primer anneal
5‘-Cap
3‘ 5‘
AAA(A)n
5‘-Cap
5‘
Regular
AAA(A)n PCR
cDNA:mRNA hybrid
Real-Time PCR
fluorescent is added into the PCR system, whose intensity
increases in proportion to the PCR product and could be
detected in each cycle.
Real-Time PCR result:
Quantification
Free discussion:
After cycle ___ are there any double strand DNA which
are the exact length of the region to be amplified.
A. 2 B. 3 C. 4 D. 5 E. 6
M
General rules for PCR Primer design