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GeneCloning

PRESENTATION·JANUARY2015

DOI:10.13140/RG.2.1.4008.9367

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1AUTHOR:

MohammedNaderShalaby

SuezCanalUniversity

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Gene Cloning

Dr.Mohammed Nader Shalaby

√ hybridization: to detect nucleotides and proteins

√ DNA sequencing

√ PCR: to amplify specific DNA segment

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

Review

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

Cell colony organism

Gene cloning: the way to get a DNA clone

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.

Gene study Protein study biotechnology

Pharmaceutic

al pr.Gene diagnosis

Gene therapy

medicineEngineering

organismSequence

Function

mutation

Sequence

Function

mutation

Animals

plants

Genetic engineering:collection of applications

of DNA cloning

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

Introduce DNA into host cells

Select cells containing recombinant DNA

selection

transformation

Construct recombinant DNA

1. cut 2. ligation

Uses of recombinant DNA

Amplification, expression etc

Prepare vector

1.Get target DNA

•From genomic library

Gene library: a collection of clones

containing all genes of an organism

•From cDNA library

cDNA library: a collection of clones

containing all cDNAs reverse transcribed

from total mRNAs of an organism

Blood sample

DNA

•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

Two primers

dNTPs

Taq polymerase

Buffer solution

thermal cycler

2.Get the vector

• Depending on purpose, the size of insert, the MCS etc, select vectors

Cloning & expressing Prokaryotic & eukaryotic

Plasmid & Virus

•Prepare vectors

Replication

Whether the MCS contains suitable RE site for target gene insertion

– How do you stably maintain and replicate a foreign

DNA in cells?

Vectors

– Why do we need vectors?

Because our interest gene need to be stably maintained

and replicate 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

MCSMultiple 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

•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

Functional classification

Cloning vector

Host cells:

usually: E.coli

seldom: Eu. Cells

e.g yeast cells

features:

√possess basic elements

√ Make large copies

√ Easily isolated

Features:

√ not only possess basic elements

√ also possess elements for gene expression

√ make proteins

ori

promoter SD MCS terminator

Expression

vectorAmpr

*just like origin, these elements are

species-specific

Transcription elements

Translational elements

Expressing vectors

Host cells:

Prokaryotic expressing vectors

Eukaryotic expressing vectors

Cloning

Vectors

Expressing

VectorsEukaryotic-

Prokaryotic-

Plasmid

λPhage

Cosmid

M13 phage

YAC

BAC

mammalian

Virus

Vectors

Plasmid Vectors

→capacity: 5~10kb

→Naturally occurring but engineered

→Host cells:

E.coli : natural

Eukaryotic cells: natural

or artificial

→for cloning and /or expression

→The most commonly used vectors

small, stable extrachromsomal circular dsDNA,

replicate indepentdently

3.Construct recombinant DNA

--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)

--to insert a foreign DNA into plasmid

Vector

Vector

Vector

Blunt ends ligation

double RE prepared

sticky ends ligation

Single RE prepared

sticky ends ligation

More efficient

Bidirectional

More religation

More efficient

directional

Less efficient

Bidirectional

More religation

Types of ligation

Ligation product is a mixture

*Recombinant DNA molecules

--foreign DNA May be

inserted with either orientation

*Religated vector

--Occurs on single RE prepared

cohensive ends ligation or blunt

ends ligation

*linear vector and DNA insert

-- fail to be ligated

EcoRI EcoRI

Vector

RE digestionRE digestion

Ligation

Vector

EcoRI

Religated

vector

EcoRI

Recombinant

Vector A

EcoRI EcoRI

XRecombinant

Vector B

EcoRI EcoRI

X

How to reduce the chance of vector self-ligation?......

# double RE to prepare both vector and insert

RE1

RE1RE2

RE2

One orientation ligation

no vector recirculation

# The use of alkaline phosphatase

To remove the phosphate

groups from the 5’-ends of the

RE linearized vector DNA

Linearized vector DNA could

not be ligated

Only foreign DNA inserted

vector can be ligated

Ligation

Recombinant

vector

nick

nick

Vector

OH

P OH

P

OH

OH P

P Vector

OH

OH

OH

OH

How to reduce the chance of vector self-ligation?......

# The use of alkaline phosphatase

What about the nick?

How to reduce the chance of vector self-ligation?......

4.Introduce recombinant DNA into cells

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

•Transfection vs transformation vs infection

• where the vectors get maintained and/or

multiplied copies -crucial

Host cells

E. Coli• Bacteria is the

commonest host cells

especially for initial

cloning

20-30min

12 hours

6-8 Billion

bacteria

Advantage of using of bacteria

* Grow faster

More product, less time

*produce high copy of

plasmids

*simple and inexpensive

culture

• Other eukaryotic cells are used for

further complicated application

Yeast cells

Plant cells

Mammalian cells

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 - Transformation methods

Gene Transfer -Transfection methods

More problematic, lower efficiency

Transiently or permanently existing in

the cells

5.Selection

Plate selection for yeasts

antibiotics or others for

mammalian cells

In E.coli:

--to select cells containing recombinant DNA

In Eukaryotes:

Plate selection

Detecting protein product

Hybridization to screen library

PCR screening

RE digestion

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

LacZ+/+ : blue colony

LacZ -/- : while colony

2nd selective marker is LacZ gene

a-complementation

if lacZ gene is interrupted by being inserted

with foreign gene, the colony will be white

Plate Screening

2. Blue-white screening

*plate contains appropriate antibiotics, IPTG and x-gal

*Selecting cells containing recombinant DNA

substrat

e

Lac I Lac Z Lac Y Lac A

Plac Olac

- galactosidase (-gal)

IPTG

asubunit

subunit

Background: Lac operon

P lacI

Blue colony

X-GAL

Plasmid: a subunit

Host cell: subunit

Subunits

recombine

in the cells

Separated in

gene

a-complementation

substrate

Colony and plaque hybridization

insert 0.8 kb

0.5 kb

1.0 kb

1.6 kb2.0 kb3.0 kb4.0 kb3.5 kb

•Size of the plasmids

•RE digestion product

recreated recombinant

Directly analyze vector DNA isolated from cells

RE digestion

Electrophoresis:

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

Recombinant DNA

Cloned recombinant DNA

Host Cell

Cloning

Target DNAVector

Selection

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.

3’ 5’

3’5’

PCR

3’ 5’

5’ 3’

5’5’

target sequence

Produces copies of selected DNA sequence that

occurs between two primer-binding sites.

PCR reaction system

√template: --dsDNA denatured into ssDNA by heating

--any source of DNA with some sequence

information known

√DNA polymerase

√reaction buffer

√dNTPs

--thermostable e.g Taq DNA pol.

√a pair of primers

--18 to 30 nt long (ssDNA) synthesized in vitro

--designed to anneal on opposite of the target

sequence

PCR is to amplify specific DNA

DNA template

Two primers

dNTPs

Taq polymerase

Buffer solution

What happened in

thermal cycler?

thermal cycler

Procedures：

1. 模板的变性

2. 引物的退火

3. 新链的延伸

Parameters:

Temperature, Time

Cycle

①

②

③

Three different steps proceed in each PCR cycle

DNA template

Repeated rounds of DNA duplication

pre-denaturation 95℃ for 5min;

Denaturation: 95℃ for 30~60s

Annealing: 37~68℃ for 30~60s

extension: 72℃ for 30s~2min

72℃ extension for 10 min

30 cycles

Round 1

Round 2

Round 3

Round 4

Round 5

Round 6

Number of DNA fragments

Accumulation of PCR product

Rate of PCR

2n

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

PCR product

1. Agarose Gel Electrophoresis

Identification of PCR-amplified DNA fragments

DNA

Marker

PCR

Product

Several

minutes

size separation

2. DNA Sequencing

RT-PCR

RT+PCRAAA(A)n

5‘-CapmRNA

(dT)12~18primer anneal

5‘-Cap

AAA(A)n

3‘ 5‘

Reverse transcriptasedNTP

5‘-Cap

AAA(A)n

5‘

cDNA:mRNA hybrid

RegularPCR

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

(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.

General rules for PCR Primer design

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

Two software widely used for PCR primer design

1. Primer Premier --------To design

Thank you!