cloning : dna preparation
TRANSCRIPT
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CLONING :
DNA PREPARATION
Phatareeya Laochinchat
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DNA Preparation
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DNA Preparation
Restriction enzyme
(RE) digestion
TA CloningSeamless Cloning
Ligation Independent
Cloning (LIC)
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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.
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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
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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.
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• 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)
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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
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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).
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5’-3’ exonuclease
3’overhang
DNA polymerase
ligation
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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
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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.
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Preparation of vector DNA
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Preparation of the insert
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Annealing of the insert and the LIC vector
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Advantages Disadvantages
Low cost Some types of sequence modifications not possible
Fast
Many different vector choices
Advantages/Disadvantages
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CLONING :
DNA END MODIFICATIONS
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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
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AP
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• 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
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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
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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.
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