Cloning Methods 10r39

CLONING Components & Methods

CLONING: Components & Methods Linkers Adaptors T-cloning Topo-TA cloning pEntr-Topo cloning Gateway Technology

oining Of DNA Molecules Together By Ligation LIGATION OF STICKY ENDS IS DESIRABLE. What do we do when insert has blunt ends ? How do we increase the efficiency of ligation in such cases ? Strategies for converting blunt ends into cohesive ends: 9.Linkers 10.Adaptors 11.Homopolymer Tailing 12.TA cloning

LINKERS

tegy to add cohesive ends to a blunt-ended molecule

ially-synthesized short pieces of ds DNA of known nucleotide sequence

vantage: The restriction site er is also present in the fragment.

ADAPTORS Short synthetic oligonucleotides with a cohesive end.

• Problem: Cohesive ends could base pair with each other to form dimers. This results in a blunt-ended molecule which can still ligate to the blunt-ended DNA molecule. However we need to digest this to obtain a cohesive end molecule.

ADAPTORS (contd.)

ptors synthesized such that cohesive end is different.

P terminus is modified to 5’-OH nus.

e pairing occurs but is not stabilized on reaction.

ptors can not themselves ligate but e blunt ended molecule.

er ligation to the DNA fragment, the rmal 5’-OH terminus is treated with

Homopolymer Tailing Another method of producing cohesive ends on a blunt end DNA molecule. Deoxynucleotidyl transferase adds a series of nucleotides onto the 3’-OH termini of a ds DNA molecule.

Generally complementary homopolymer tails a added to the vector as well as insert.

Due to the differences in the length of the pol attached, a nick or discontinuities are produced This is repaired by Klenow polymerase.

In case the homopolymers are more than 20 n long, the base-paired associations are quite stro These molecules are introduced into bacterial ce and host cell polymerase and DNA ligase repair recombinant DNA.

TA-cloning

merases possesses terminal transferase activity that adds a single dA-o of PCR products.

ectors provide the corresponding T for base-pairing.

A PCR Product A

ges: Quick and efficient cloning method. No need to dephosphorylate the vector

ntage: Restriction digested fragments and PCR products of proof-readin u, Pwo or Tli DNA Pol. yield blunt-end products. can be employed.

-TA cloning: more efficient method of clo

ADVANTAGES REQUIREMENTS • Highly efficient PCR products • One-step cloning A linearized activated vector with dT-overhangs at 3’end • Saves time Activated vector: Topoisomerases covalently bound to the

TOPOISOMERASES Source: Vaccinia virus Action: The phospho-tyrosyl bond between the DNA and en attacked by the 5’-hydroxyl and releases the topoisomera

Different Ways of TA-cloning

Different Ways of TA-cloning

GATEWAY CLONING •

Recombination-based cloning technology

•

Replaces the use of restriction endonucleases and ligases with site specific recombinases

•

Universal system for cloning and subcloning DNA fragments

•

Rapid and efficient technology

Basis of Gateway Cloning Technology

Recombination-based technology Recombination of phage lambda in E. c

INTEGRATION att P: Phage attachment site (245bp) att B: Bacterial attachment site(25 bp) Proteins Involved: Int (integrase) & IHF(integration Host Factor) EXCISION att L: left attachment junction (100 bp) att R: right attachment junction(168 bp) Proteins Involved: Int IHF & Xis attL x attR attB x attP (excisionase)

Commercial Vectors have been modified for efficient cloning

ite (43 bases removed) modified to make excision reaction irreversible efficient ions introduced in core att sites to eliminate stop codons and ensure sp ions in 5 bp regions flanking the 15 bp core region of attB sites to minim ary structure formation.

Gateway Technology is Flexible

ccdB attL1

attL2

Entry Clone

attR1

+

ccdB attR2 LR clonase

Destination Vector

attB1

attB2

Expression Clone

attP1

+

attP2

Donor Vector

BP clonase KanR

AmpR

AmpR

KanR

Requirements for Gateway System REQUIREMENTS Entry clone Destination Vector LR clonase enzyme (int, IHF & xis)

TYPES OF SELECTION

Positive Selection: Antibiotic resistance gen

Negative Selection: ccdB gene (interferes w DNA gyrase gene) Vector has ccdB gene and thus propagated i E. coli strain DB3.1. gyrA462 No degradatio (DB3.1) ccdB (toxin) Chromosome is degraded ccdA Reing by (antidote) DNA Gyrase

Different ways to generate the entry clone P1

ccdB

P2

Donor Vector

L1

+

B1

Gene

B2

L2

+

TOPO-Activated Entry Vector

attB PCR Product

1. BP Cloning

Gene PCR Product

1. TOPO® Cloning BP Clonase™

TOPO®

L1

Gene

L2

Entry Clone Ligase

1. Restriction/Ligase Cloning L1

L2

digested Entry Vector

4. Pre-made entry clone 5. Custom-made entry clone L1

+

B1

Gene

digested DNA Fragment

ORF

L2

ORF Collection

1.BP Cloning gene a ttL 1 ccd B a ttP 1 a ttB 1

gen e

a ttB 2

+

E n tr y C lo n e

a ttP 2 a ttR 1

Donor V e c to r K an R

a ttL 2

ccdB

a ttR 2

+

K an R

BP Clonase™

1. BP Cloning - Primer Design for PCR

90-99% correct clones on Kan plates

•

GGGG and the attB1 sequence must be added to the 5’-primer (sense)

•

GGGG and the attB2 sequence must be added the 3’-primer Geneto Specific (antisense) Primer Sequence attB1

5’ – GGGGACAAGTTTGTACAAAAAAGCAGGCTNNN… attB2

2. Restriction/Ligase cloning Use when there are convenient sites to cut insert out of another plasmid Must cut out ccdB gene by using one of four RE sites flanking the ccdB Reading frame of insert must be considered, as well as downstream expression elements Various reading frames of pENTR vectors are available

3. TOPO Cloning

ectional and faster cloning by pEntr-D Topo reaction of the PCR product.

ward primer should contain CACC sequence e 5’ end.

ures directional cloning.

y efficient.

ry clone ready for LR reaction with the nation vectors.

ecombination Forms a Cointegrate Molec

mbination of Entry vector with a Destination vector

irements: ry clone with the GOI between and L2 site stination vector with attR1 and ites & ccdB gene clonase enzyme

mation of Cointegrate molecule ntegrate molecule resolves through a second tion into two daughter molecules. ardless of which pair of sites and att R1 or and att R2 to first from an integrate.

Types of Destination Vectors Destination vectors available for expression in

E. coli

Yeast Baculovirus Insect cells Mammals Plants

des the available vectors, any vector can be converte Gateway vector

Conversion of a vector into Gateway vect

available which can be vector by restriction n.

emember: ion vector ligation mix propagated in DB3.1 cells.

ing a protein fusion vector, ensure the reading frame of the protein w.r

assettes A-C are blunt-ended molecules they may be inserted in either d ck for the orientation of the inserted cassette.

confirm the type of cassette by restriction digestion.

l clones should be selected on chloramphenicol+ antibiotic resistance o

ateway cloning system is superior system

ecombination-based cloning.

xtremely rapid.

ery simple to perform.

ghly efficient cloning as 99% clones are ect.

aithful maintenance and orientation of GOI.

arallel cloning can be easily carried out.

ighly versatile system.

ny vector can be converted into GW system.

ectors available for many biological systems.

ultisite gateway systems are now available.

Suggested Reading Gene Cloning and DNA Analysis: T. A. Brown Gateway Manual: www.invitrogen.com