CLONING Components & Methods

CLONING: Components & Methods

Linkers

Adaptors

T-cloning

Topo-TA cloning

pEntr-Topo cloning

Gateway Technology

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:

1.Linkers2.Adaptors3.Homopolymer Tailing4.TA cloning

Joining Of DNA Molecules Together By Ligation

LINKERS A strategy to add cohesive ends to a blunt-ended molecule

Artificially-synthesized short pieces of ds DNA of known nucleotide sequence.

Disadvantage: The restriction site in linker is also present in the cloning 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.) Adaptors synthesized such that cohesive end is different.

5’-P terminus is modified to 5’-OH terminus.

Base pairing occurs but is not stabilized ligation reaction.

Adaptors can not themselves ligate but to the blunt ended molecule.

After ligation to the DNA fragment, theabnormal 5’-OH terminus is treated withPNK.

Homopolymer Tailing

Deoxynucleotidyl transferase adds a series of nucleotides onto the 3’-OH termini of a ds DNAmolecule.

Generally complementary homopolymer tails areadded to the vector as well as insert.

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

In case the homopolymers are more than 20 nt long, the base-paired associations are quite strong.These molecules are introduced into bacterial cells and host cell polymerase and DNA ligase repair therecombinant DNA.

Another method of producing cohesive ends on a blunt end DNA molecule.

TA-cloning Taq Polymerases possesses terminal transferase activity that adds a single dA-overhangs to

the 3’ end of PCR products.

pGem-T vectors provide the corresponding T for base-pairing.

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

Disadvantage: Restriction digested fragments and PCR products of proof-reading enzymes

such as Pfu, Pwo or Tli DNA Pol. yield blunt-end products.A-TAILING can be employed.

A APCR Product

Topo-TA cloning: more efficient method of cloningADVANTAGES • Highly efficient• One-step cloning• Saves time

REQUIREMENTS PCR products A linearized activated vector with dT-overhangs at 3’ends

Activated vector: Topoisomerases covalently bound to the vector. TOPOISOMERASES

Source: Vaccinia virusAction: The phospho-tyrosyl bond between the DNA and enzyme attacked by the 5’-hydroxyl and releases the topoisomerase.

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

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

INTEGRATIONatt P: Phage attachment site (245bp)att B: Bacterial attachment site(25 bp)Proteins Involved: Int (integrase) &IHF(integration Host Factor)

EXCISIONatt L: left attachment junction (100 bp) att R: right attachment junction(168 bp)Proteins Involved: Int IHF & Xis (excisionase)

Basis of Gateway Cloning Technology

attB x attP attL x attR

Commercial Vectors have been modified for efficient cloning attR site (43 bases removed) modified to make excision reaction irreversible and

more efficient mutations introduced in core att sites to eliminate stop codons and ensure specificity mutations in 5 bp regions flanking the 15 bp core region of attB sites to minimize

Secondary structure formation.

LR clonase

++BP clonase

attL1 attL2

EntryClone

KanR

ExpressionClone

attB1 attB2

AmpR

attR1 attR2

ccdB

DestinationVector

AmpR

attP1 attP2

ccdB

DonorVector

KanR

Gateway Technology is Flexible

Requirements for Gateway SystemREQUIREMENTS

Entry clone Destination Vector LR clonase enzyme (int, IHF & xis)

TYPES OF SELECTIONPositive Selection: Antibiotic resistance gene

Negative Selection: ccdB gene (interferes with DNA gyrase gene)Vector has ccdB gene and thus propagated inE. coli strain DB3.1.

ccdB (toxin)

Rejoining by DNA Gyrase

Chromosome is degraded

ccdA(antidote)

gyrA462(DB3.1)

No degradation

Different ways to generate the entry clone

2. TOPO® Cloning

TOPO®BP Clonase™

1. BP Cloning

PCR Product

+TOPO-ActivatedEntry Vector

L1 L2

Gene+attB PCR Product

B2GeneB1Donor Vector

P2ccdBP1

Entry Clone

L2GeneL1

+digested DNA Fragment

GeneB1digested Entry Vector

L2L1

4. Pre-made entry clone5. Custom-made entry clone

Ligase

3. Restriction/Ligase Cloning

ORF Collection

L2ORFL1

1.BP Cloning

90-99% correct cloneson Kan plates

geneattB1 attB2

+attP1 attP2

ccdB

DonorVector

KanR

+

gene

attL1 attL2

EntryC lone

KanRccdBattR1 attR2

BP Clonase™

1. BP Cloning - Primer Design for PCR

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

• GGGG and the attB2 sequence must be added to the 3’-primer (antisense)

attB1

5’ – GGGGACAAGTTTGTACAAAAAAGCAGGCTNNN…

attB2

5’ – GGGGACCACTTTGTACAAGAAAGCTGGGTNNN…

Gene SpecificPrimer Sequence

2. Restriction/Ligase cloningUse 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

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

Forward primer should contain CACC sequence at the 5’ end.

Ensures directional cloning.

Very efficient.

Entry clone ready for LR reaction with the destination vectors.

Recombination Forms a Cointegrate MoleculeRecombination of Entry vector with a Destination vector

Requirements: • Entry clone with the GOI between attL1 and L2 site• Destination vector with attR1 and R2 sites & ccdB gene• LR clonase enzyme

•Formation of Cointegrate molecule•Cointegrate molecule resolves through a second reaction into two daughter molecules.• Regardless of which pair of sites attL1 and att R1 orattL2 and att R2react to first from an integrate.

Types of Destination Vectors

Destination vectors available for expression in E. coli

Yeast

Baculovirus

Insect cells

Mammals

Plants

Besides the available vectors, any vector can be converted to Gateway vector

Conversion of a vector into Gateway vector

Cassettes available which can be cloned in a vector by restrictionand ligation.

Things to remember:1. Destination vector ligation mixshould be propagated in DB3.1 cells.

2. If designing a protein fusion vector, ensure the reading frame of the protein w.r.t. the tag.

3. Since cassettes A-C are blunt-ended molecules they may be inserted in either directions.Always check for the orientation of the inserted cassette.

4. Always confirm the type of cassette by restriction digestion.

5. The final clones should be selected on chloramphenicol+ antibiotic resistance of vectorbackbone.

Gateway cloning system is superior system1. Recombination-based cloning.

2. Extremely rapid.

3. Very simple to perform.

4. Highly efficient cloning as 99% clones are correct.

5. Faithful maintenance and orientation of the GOI.

6. Parallel cloning can be easily carried out.

7. Highly versatile system.

8. Any vector can be converted into GW system.

9. Vectors available for many biological systems.

10.Multisite gateway systems are now available.

Suggested Reading

Gene Cloning and DNA Analysis: T. A. Brown

Gateway Manual: www.invitrogen.com