4. 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.
10. 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.
11. • 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)
12.
13. 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
14. 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).
17. Advantages Disadvantages
Sequence-dependent Cost, relative to traditional
methods
High cloning efficiency PCR primers for vector and
insert must be designed/ordered
together
Efficiency assembly of multiple
fragment
Exquisite control of higher-
order gene assembly
Advantages/Disadvantages
18. 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.
25. 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
27. • 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
28.
29. 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
30.
31. 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.
