1. 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

2. Experimental Scheme 2 Mutagen

3. 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 3

4. TA Cloning 4

5. α-complementation 5  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.

6. TA Vector 6

7. TOPO TA Cloning®  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). 7

8. Procedure 8

9. ANALYSIS OF CLONED DNA 9  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

10. Exercise 10 The two oligonucleotides you used to make the fragment look like this: 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. 18 mer 5’ 3’5’ 3’

11. Answer 11 The new oligo on the right will be 5‘-NNGGATCCCTCTATCCGTCTAGTCTA-3’ The new oligo on the left will be 5’-NNGAATTCTCTATGGACCAGTACGAT-3’ No problem! We just make our two oligonucleotides a bit longer, and embed the extra sequence within. 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.

12. Exercise 12 The new PCR product would look like this: Restriction Enzyme Digestion

13. Exercises - Mutagenesis 13 Let’s make a change in the oligo on the left Parental Oligo on the left 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. To anneal correctly, it will be better to extend the primer length. NOTE

14. Creating a point mutation in the middle of a DNA sequence 14 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:

15. 15 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 15 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.