Structural Biochemistry/Enzymes Used for Cloning

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Enzymes that modify nucleic acids are used to synthesize, degrade, join, and/or remove portions of nucleic acids. Cloning enzymes are enzymes that are important in nucleic acid cloning procedures. The activities of the cloning enzymes consist of ligases, kinases, phosphatases, and RecA Protein.

DNA ligase, for example is used in joining DNA molecules together. Within the cell, the double ends of the strands are attached covalently. While RNA primers replace DNA during DNA replication, DNA ligase seals in the gaps.

1. Ligases are used to join nucleic acid segments, especially when one is cloning a DNA fragment into the vector DNA.

2. Phosphatases remove the 5′-phosphate from nucleic acid strands. This prevents vector downgrading, which would reduce the number of background colonies as well as producing substrate to which a kinase can attach a new radio-labeled phosphate.

3. Kinases add new phosphate groups to nucleic acids. This is usually done in order to label the nucleic acid fragments or the synthetically made oligonucleotide.

4. RecA Protein and AgarACE® Enzyme are used primarily to protect in certain cloning procedures or facilitate the nucleic acid purification. The E. coli RecA Protein is able to facilitate the pairing of homologous DNA sequences. AgarACE® Enzyme is a patented agarose-lysing enzyme produced for the harvest of DNA from agarose gels.