CLONING :

DNA PREPARATION

Phatareeya Laochinchat

DNA Preparation

DNA Preparation

Restriction enzyme

(RE) digestion

TA CloningSeamless Cloning

Ligation Independent

Cloning (LIC)

Restriction enzyme (RE) digestion

• RE is an enzyme that cuts DNA at or near specific

recognition nucleotide sequences known as restriction sites.

• RE function by cutting double stranded DNA at specific

4 to 8 base pair.

• The products of DNA cleavage

are either blunt ended or contain

5’ or 3’ overhangs.

Advantages/Disadvantages

Advantages Disadvantages

Low cost Possible sequence constraints due

to presence and/or translation of restriction site

Versatile

Many different vector choices

Directional cloning can be easily done

TA (PCR Cloning)

• TA cloning is a subcloning technique that avoids the use of

restriction enzymes and is easier and quicker than

traditional subcloning.

• The technique relies on the ability of adenine (A) and

thymine (T) on different DNA fragments to hybridize.

• PCR products are usually amplified using Taq DNA

polymerase which preferentially adds an A to the 3' end

of the product.

• PCR amplified inserts are cloned into linearized vectors that have complementary 3' T overhangs.

TA (PCR Cloning)

Advantages Disadvantages

High efficiency, with dedicated vectors

Limited vector choices

Amenable to high throughput Higher cost

Lack of sequence control at junction

Multi-fragment cloning is not straight forward

Advantages/Disadvantages

Seamless Cloning (Gene Assembly)

• Gibson Assembly enables multiple, overlapping DNA

fragments to be joined in a single-tube isothermal

reaction, with no additional sequence added (scar-less).

5’-3’ exonuclease

3’overhang

DNA polymerase

ligation

Advantages Disadvantages

Sequence-dependent Cost, relative to traditional methods

High cloning efficiency PCR primers for vector and

insert must be designed/orderedtogether

Efficiency assembly of multiple fragment

Exquisite control of higher-order gene assembly

Advantages/Disadvantages

LIC (Ligation Independent Cloning)

• Ligation Independent Cloning (LIC) is a technique

developed in the early 1990s as an alternative to restriction

enzyme/ligase cloning.

• Inserts are usually PCR amplified and vectors are made

linear either by restriction enzyme digestion or by PCR.

• This creative technique uses the 3’ → 5’ exonuclease

activity of T4 DNA Polymerase to create overhangs with complementarity between the vector and insert.

Preparation of vector DNA

Preparation of the insert

Annealing of the insert and the LIC vector

Advantages Disadvantages

Low cost Some types of sequence modifications not possible

Fast

Many different vector choices

Advantages/Disadvantages

CLONING :

DNA END MODIFICATIONS

Dephosphorylation Blunting

• Dephosphorylation is a common step in traditional cloning

to ensure the vector does not re-circularize during ligation.

• Fragments generated by restriction digestion have a 5'-

phosphate even after they have been treated with T4 DNA

Polymerase for blunting.

• Use Alkaline Phosphatase to remove the 5´ phosphate

AP

AP

• Phosphorylation of insert DNA that lacks terminal 5'

phosphates, such as PCR products and fragments with

synthetic linkers.

• T4 polynucleotide kinase can be used; this enzyme

catalyzes the transfer of phosphate from ATP to the 5'

terminus of dephosphorylated DNA or RNA

Phosphorylation of Insert DNA

Blunting/End-repair

• Blunt ends are always compatible with each other,

because there are no H-bonds being formed that would

define compatibility or incompatibility.

• Cohesive ends

Ligation

• Ligation is the process of joining two pieces of DNA from

different sources together through the formation of a

covalent bond.

• DNA ligase is the enzyme used to catalyze this reaction.

• DNA ligation requires ATP.