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Gene Cloning

Presentation · January 2015


DOI: 10.13140/RG.2.1.4008.9367

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Mohammed Nader Shalaby


Suez Canal University
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Gene Cloning

Dr.Mohammed Nader Shalaby


Review
Commonly used tool enzymes includes:
Restriction endonuclease (RE): ‘cut’ the phosphodiester bond
Ligase: form phosphodiester bond
DNA polymerase
DNA polymerase I: replicate and help repair DNA in vivo
Taq DNA polymerase: a thermostable DNA polymerase
Terminal transferase: adds a number of nucleotides to the 3’
end of linear single or double-stranded DNA or RNA
Alkaline phosphatase: remove phosphate group from 5’ end
nucleic acid
√ hybridization: to detect nucleotides and proteins
√ DNA sequencing
√ PCR: to amplify specific DNA segment
Gene cloning
——the way to manipulate gene

•A short overview: Gene cloning


•The process of Gene cloning
1.Overview: Gene cloning
Clone: a set of absolutely identical molecules,
cells or even organisms produced by
asexual propagation

molecule

DNA clone Cell colony organism

Gene cloning: the way to get a DNA clone


Gene cloning:

Isolating a gene and joining it to vector DNA,


called recombinant DNA molecules which are then
propagated in a host cell which produced a clone
that contain a single fragment of the target DNA.
Genetic engineering:
collection of applications
of DNA cloning

Gene study Protein study biotechnology Engineering medicine


Sequence Sequence Pharmaceutic organism
Function Function Animals Gene diagnosis
al pr.
mutation mutation plants Gene therapy
Tool Enzymes
Restriction Endonuclease (RE)
recognition sequences are palindromes;highly specific.
Cut the phosphodiester bond (sticky ends, blunting ends ).

Ligase
forming phosphodiester bond
2.Gene cloning process
Prepare target gene
Prepare vector

Construct recombinant DNA


1. cut
2. ligation
Introduce DNA into host cells
transformation
Select cells containing
recombinant DNA
selection
Uses of recombinant DNA
Amplification, expression etc
1.Get target DNA
Blood sample
•From genomic library
Gene library: a collection of clones
containing all genes of an organism
DNA
•From cDNA library
cDNA library: a collection of clones
containing all cDNAs reverse transcribed
from total mRNAs of an organism
•By PCR or RT-PCR
Part of DNA or its protein sequence is known
•By chemical synthesis in vitro
Target DNA sequences is known
PCR is to amplify specific DNA
DNA template thermal cycler

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 is a region with multiple restriction


enzyme sites enable the convenient
insertion of target DNA into a vector
Basic elements required as a
recombinant vector

MCS

Origin: to allow vector


autonomously replicate
independent of host’s
genome. Species-specific
Basic elements required as a
recombinant vector

MCS

Selective marker: to allow


select cells containing
vectors or recombinant
DNA. Species-specific
Types of vectors
Functional classification

•Cloning vectors: allowing the exogenous DNA


to be inserted, stored, and manipulated at DNA
level.

•Expressing vectors: allowing the exogenous


DNA to be inserted and expressed.
prokaryote expression vectors
eukaryote expression vectors
Cloning vector

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

--Ligase covalently join


DNA molecules

--Happened in tubes
(in vitro)
Types of ligation

Single RE prepared More efficient


sticky ends ligation Bidirectional
Vector
More religation

double RE prepared More efficient


sticky ends ligation
Vector directional

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

*Religated vector Vector


--Occurs on single RE prepared
Ligation
cohensive ends ligation or blunt
ends ligation
EcoRI EcoRI EcoRI EcoRI
EcoRI
*linear vector and DNA insert
-- fail to be ligated
Religated Recombinant Recombinant
X
vector Vector A X
Vector B
How to reduce the chance of vector self-ligation?......
# double RE to prepare both vector and insert

RE1

RE1
RE2

RE2

One orientation ligation


no vector recirculation
How to reduce the chance of vector self-ligation?......
# The use of alkaline phosphatase OH P
P OH

To remove the phosphate Vector


groups from the 5’-ends of the
RE linearized vector DNA
OH
OH
Linearized vector DNA could
not be ligated P OH Vector
OH P

Only foreign DNA inserted Ligation


nick
vector can be ligated
OH
nick
OH
Recombinant
vector
How to reduce the chance of vector self-ligation?......
# The use of alkaline phosphatase

What about the nick?


4.Introduce recombinant DNA into cells

•Transfection vs transformation vs infection

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

• Bacteria is the E. Coli


commonest host cells
especially for initial
cloning
Advantage of using of bacteria
20-30min

* Grow faster
More product, less time 12 hours

*produce high copy of


plasmids

*simple and inexpensive 6-8 Billion


culture bacteria
• Other eukaryotic cells are used for
further complicated application

Plant cells

Yeast cells

Mammalian cells
Gene Transfer - Transformation methods

Competent cells: Treated E.coli


cells who are susceptible to
take up exogenous DNA

Transformation: a process of
uptake of exogenous DNA by
competent cells.

• Each cell in a given colony has the same plasmid


• Cells in different colonies have different plasmids
Gene Transfer -Transfection methods
More problematic, lower efficiency

Transiently or permanently existing in


the cells
5.Selection
--to select cells containing recombinant DNA
In E.coli: Plate selection
Detecting protein product
Hybridization to screen library
PCR screening
RE digestion

In Eukaryotes: Plate selection for yeasts


antibiotics or others for
mammalian cells
 Plate Screening
1. Antibiotics selective plate
*Culture plate contains appropriate antibiotics according
to the antibiotics resistance gene carried by vector

In most cases, cells


containing vector (self or
recombinant) will grow
Plate Screening
2. Blue-white screening substrat
e
*plate contains appropriate antibiotics, IPTG and x-gal
2nd selective marker is LacZ gene
a-complementation

LacZ+/+ : blue colony

LacZ -/- : while colony

if lacZ gene is interrupted by being inserted


with foreign gene, the colony will be white

*Selecting cells containing recombinant DNA


Background: Lac operon
Plac Olac
Lac I Lac Z Lac Y Lac A

P lacI IPTG
- galactosidase (-gal)
Blue colony

substrate a Separated in
 subunit gene
X-GAL subunit

Subunits a- Plasmid: a subunit


recombine complementation Host cell:  subunit
in the cells
 Colony and plaque hybridization
 RE digestion

Directly analyze vector DNA isolated from cells


Electrophoresis: recreated recombinant

•Size of the plasmids

•RE digestion product

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

The sequence of bases


A, C, G, T in the
recombinant DNA.

Blast the sequence:


DNA sequencing result
sequence of the target DNA

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

K. Mullis, Nobel Prize in Chemistry,1993

This technique is used to amplify a


specific sequence of DNA in vitro by
simulating the replication procedure of
natural DNA in vivo.
Produces copies of selected DNA sequence that
occurs between two primer-binding sites.

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

√DNA polymerase --thermostable e.g Taq DNA pol.

√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


Cycle

Repeated rounds of DNA duplication


pre-denaturation 95℃ for 5min;

Denaturation: 95℃ for 30~60s

Annealing: 37~68℃ for 30~60s 30 cycles

extension: 72℃ for 30s~2min

72℃ extension for 10 min


Accumulation of PCR product
Rate of PCR
2n
Round 1
Round 2

Round 3
Round 4

Round 5
Round 6

Number of DNA fragments

efficient amplification of DNA fragments


Repeated rounds of DNA polymerization
in an in vitro (test-tube) reaction result in
the exponential amplification of the
region of interest. M
Identification of PCR-amplified DNA fragments
1. Agarose Gel Electrophoresis
DNA PCR
Marker Product
PCR product

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

dNTP Reverse transcriptase

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

How to design PCR primers in your opinion now?

M
General rules for PCR Primer design

(1) specificity: conserved genomic region.


Specially, 8 bases at 3’end.
(2) specific sequence length: 18~24 base.
(3) G+C contents=45-55%. ATCG randomly distributed
(4) Avoid hairpin in each primer, specially at 3’end.
(5) Avoid complement between primers (>4bp), specially at
3’end.
(6) unique restriction enzyme sites can only be added to
the 5’ end of the primers.
Two software widely used for PCR primer design
1. Primer Premier --------To design

2. Oligo --------To analyze


Thank you!

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