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Cloning of PCR Fragment into T-Vector
Jung-Min Choi
Department of Biochemistry, College of Life Science and Biotechnology,
Mouse Genetics and Laboratory Animal Research Center, Yonsei University
Experimental Scheme
Mutagen
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TA Cloning
 TA Cloning uses the nontemplate-dependent terminal
transferase activity of some DNA polymerases.
 In TA Cloning Taq polymerase adds a 3'-A overhang to each end of
the PCR product.
 TA Cloning makes it possible to clone the PCR product into a
cloning vector with 3'-T overhangs.
 TA cloning is a popular method of cloning without the use of
restriction enzymes; instead, PCR products are amplified with only
Taq DNA polymerase and other polymerases. These polymerases
lack 5'-3' proofreading activity and add an adenosine triphosphate
residue to the 3' ends of the double-stranded PCR products. Such
PCR amplified products can thus be cloned in a linearized vector
that has complementary 3' thymidine triphosphate overhangs
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TA Cloning
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α-complementation
 The portion of the lacZ gene encoding the first 146 amino acids (the
α -fragment) are on the plasmid
 The remainder of the lacZ gene is found on the chromosome of the
host.
 If the α -fragment of the lacZ gene on the plasmid is intact (that is,
you have a non-recombinant plasmid), these two fragments of the
lacZ gene (one on the plasmid and the other on the chromosome)
complement each other and will produce a functional β galactosidase enzyme.
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TA Vector
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TOPO TA Cloning®
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Taq polymerase has a nontemplate-dependent
terminal transferase activity that adds a single
deoxyadenosine (A) to the 3´ ends of PCR
products.
The linearized vector supplied in this kit has
single, overhanging 3´ deoxythymidine (T)
residues. This allows PCR inserts to ligate
efficiently with the vector.
Topoisomerase I from Vaccinia virus binds to
duplex DNA at specific sites and cleaves the
phosphodiester backbone after 5′-CCCTT in
one strand (Shuman, 1991).
The energy from the broken phosphodiester
backbone is conserved by formation of a
covalent bond between the 3′ phosphate of the
cleaved strand and a tyrosyl residue (Tyr-274)
of topoisomerase I.
The phospho-tyrosyl bond between the DNA
and enzyme can subsequently be attacked by
the 5′ hydroxyl of the original cleaved strand,
reversing the reaction and releasing
topoisomerase (Shuman, 1994).
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Procedure
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ANALYSIS OF CLONED DNA
 Restriction mapping: determining the order of
restriction sites in a cloned fragment
 Gel electrophoresis: separates DNA fragments by
molecular weight
 Southern Blot analysis: DNA is transferred ("blotted")
to filter paper. Filter is exposed to a DNA probe. Binds
specifically to target DNA immobilized on filter
 DNA sequencing: provides complete order of bases in a
DNA fragment
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Exercise
5’
3’
3’
5’
The two oligonucleotides you used to make the fragment look like this:
18 mer
These oligonucleotides are written 5' to 3'. If you don't understand the oligo on
the right, then I suggest that you go back to review the lecture on PCR.
Suppose that you want to add an EcoRI site (GAATTC) to the end on the left,
and a BamHI site (GGATCC) to the end on the right.
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Answer
No problem! We just make our two oligonucleotides a bit longer, and
embed the extra sequence within.
The new oligo on the left will be
5’-NNGAATTCTCTATGGACCAGTACGAT-3’
The new oligo on the right will be
5‘-NNGGATCCCTCTATCCGTCTAGTCTA-3’
Please note that we added a "GC" base pair to each end to make the enzymes work
better - that is a subject for a future lecture, so don't worry about it just now. The
important thing is that we managed to change the ends of the DNA, just by adding a
bit of sequence to the 5' ends of each oligonucleotide.
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Exercise
The new PCR product would look like this:
Restriction Enzyme Digestion
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Exercises - Mutagenesis
Let’s make a change in the oligo on the left
Parental Oligo on the left
NOTE
To anneal correctly, it will
be better to extend the
primer length.
Mutant Oligo on the left
You see that we made a "G" at the third nucleotide instead of a "T". This will create a
transversion mutation in the product:
Both strands are affected, because the new version is simply copied into
its complementary nucleotides on the bottom strand. So you see, we can
make changes in the sequence that are internal.
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Creating a point mutation in the middle
of a DNA sequence
So you see, it is fairly straightforward to change a DNA sequence if it can be
covered by an oligonucleotide during polymerase chain reaction.
Suppose you want to do something a bit more challenging - creating a point mutation in
the middle of a DNA sequence, at the position marked with an "*" in the figure:
The ways of doing this in the old days were unspeakable, but now we can
simply get on the phone and order four oligonucleotides; two of which are
flanking and two of which cover and introduce the mutation into the amplified
material:
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We perform two PCR reactions to obtain the two halves of our final product, and
combine them in a third reaction, using the two "outside" oligonucleotides to
generate a chimeric product.
How does this happen? During the PCR process, the right side of the first
molecule can prime the synthesis from the left side of the second
Now we can simply cut the PCR product with EcoRI and BamHI, and drop it into
the vector, in place of the original version. Or, we can continue to manipulate the
DNA by PCR.
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