cloning : dna preparation
TRANSCRIPT
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.