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Enzymes for Molecular Biology

Cloning & DNA Recombination

Cloning and DNA recombination technologies have contributed many insights into the workings of genes and cells in health and disease. Now cloning techniques and applications have advanced further with innovative applications such as high-throughput assembly of combinatorial libraries and mapping of epigenetic modifications.

End terminal modification may be required before template DNA can be cloned into a suitable plasmid vector. DNA identified for cloning is generally isolated from the source by restriction enzyme digestion or PCR amplification. Specialized enzymes, such as Klenow Fragment, T4 DNA polymerase and T4 polynucleotide kinase, modify 3’ or 5’ ends to improve covalent joining between DNA fragments and vector.

The next cloning step is ligation with an enzyme suitable for annealing the ends of vector and insert.

Tailing is typically done to prepare a T-vector for use in TA cloning or to A-tail a PCR product produced by a high-fidelity polymerase (not Taq) for use in TA cloning. Products of amplification with Taq DNA polymerase are already A-tailed.

Enzyme Terminal modification

End fill 5’→3’ End fill 3’→5’  A-tailing for cloning End 5’ phosphorylation

P7060L Klenow Fragment

✔ Yes ✔ Yes ✘ No ✘ No

P7080L T4 DNA Polymerase*

✔ Yes ✔ Yes ✘ No ✘ No

P7010 Klenow (3’→5’ exo-)

✘ No ✘ No ✔ Yes ✘ No

Y9040L T4 Polynucleotide Kinase

✘ No ✘ No ✘ No ✔ Yes

Y9140† End Repair Mix

✔ Yes ✔ Yes ✘ No ✔ Yes
Enzyme Ligation

Ligation of sticky cohesive ends Ligation of blunt ends Nick sealing

L6090L E. coli DNA Ligase

✔ Yes ✘ No ✔ Yes

L6010L T3 DNA Ligase

✔ Yes A/T > G/C [1.0 M NaCl] ✔ Yes ✔ Yes

L6030 T4 DNA Ligase

✔ Yes ✔ Yes ✔ Yes
EN11 T4 DNA Ligase
✔ Yes ✔ Yes ✔ Yes

L6020L T7 DNA Ligase

✔ Yes ✘ No ✔ Yes
EN13 Tth DNA Ligase* ✔ Yes ✘ No ✔ Yes

L6060L Taq DNA Ligase
Coming soon

✘ No ✘ No ✔ Yes

Sequence and ligation independent cloning (SLIC) exploits exonuclease enzyme activity:

A library intended for next-generation sequencing starts with fragmented DNA. Enzyme action completes the cloning steps.

All the necessary enzyme components for DNA library construction can be assembled with adapters into a “home-brew” library preparation kit.

Gene synthesis is based on joining oligodeoxynucleotides that have long regions of complementary overlap.

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