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Enzymes for Molecular Biology

Cloning and DNA Recombination

Sticky or blunt: modify, anneal and ligate

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.

Enzymes for cloning

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.

Terminal modification of DNA

*The 3’→5’ exonuclease activity of T4 DNA Polymerase is essential to the protocol for sequence and ligation independent cloning (SLIC).

† The conversion to blunt-ended DNA is accomplished by exploiting the 5’→3’ polymerase and 3’→5’ exonuclease activities of T4 DNA Polymerase (P7080). T4 Polynucleotide Kinase (Y9040) ensures that the ends of the blunt-ended DNA fragments are 5’-phosphorylated for subsequent ligation by T4 DNA Ligase.

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DNA ligation

* Thermostable; for applications that require high temperature, high-stringency ligation of duplex DNA structures.
† Thermostable; for accurate ligation of nicks without gaps or mismatched base pairs.

It’s a SLIC way to clone

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

Cloning for next-generation sequencing

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

  • icon-genomicsBlunt fragments

    Ends are blunted and 5’ phosphorylated with the enzymes T4 DNA polymerase, Klenow Fragment and T4 PNK.

  • icon-bio-informaticsA-tail the ends

    Next, the 3’ ends are A-tailed using either of these enzymes: Taq DNA polymerase or Klenow Fragment (3’→5’ exo-).

  • icon-barcodeAdd adapters

    Then, complementary adapters or barcodes are added by ligation with the final enzyme, T4 DNA ligase.

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

Gene synthesis

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

  • icon-temperatureThermostable Taq DNA ligase

    Improve synthesis by using thermostable Taq DNA ligase rather than T4 DNA ligase.

    Higher ligation temperatures increase the probability of forming correct ligation products.

  • icon-targetNick-selective ligase

     

    Use a nick-selective ligase to avoid deletions caused by ligation across gaps.

    Long synthesized DNAs are not joined out of order.

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FAQs about enzymes for cloning and recombination

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