Disadvantages of using enzymes as labelling agents

By | 20.10.2017

In such applications the oligonucleotide must be labelled, so that its presence can be identified. P-ATP and T4 polynucleotide kinase. Radiolabelling has the advantages of very high sensitivity. P-disadvantages of using enzymes as labelling agents using kinase enzymes can only be carried out on a very small scale. Consequently colorimetric, chemiluminescent, and fluorescent labels have become increasingly popular and have taken over from radiolabels in most applications.

DNA molecules during PCR or other in vitro biochemical methods. Taq polymerase and is normally carried out on microgram scale. The label should be easy to attach under mild conditions using a simple, cheap and reproducible protocol. DNA to its target nucleic acid. The label should be stable to hybridization conditions e. C and be easily disposable.

This section focuses on oligonucleotide labelling, an active field in which huge progress has been made recently. The spacer separates the luminescent moiety from the DNA and can be used to change the hydrophobicity or hydrophilicity of the molecule and alter the flexibility and spacing of the label relative to the DNA. This is sometimes necessary for efficient hybridization. The reactive group provides a means of attaching the label to an oligonucleotide. Reaction of a phosphoramidite derivative of the labelling group with the oligonucleotide during solid phase synthesis.

This reaction produces a stable chemical bond such as an amide or thiourea linkage. Reaction of a thiol-modified oligonucleotide with an α,β-unsaturated ketone attached to a luminescent label or activated enzyme. DNA diagnostics and forensic screening. Some important methods of oligonucleotide labelling and applications of labelled oligonucleotides are described in more detail here. DNA1 and DNA2 to be brought together by simultaneous hybridization to a complementary template oligonucleotide. DNA 1 and DNA 2 can be any length, e.

100mer synthetic oligonucleotides or 500mer PCR products, whereas the template oligo need only be a short oligonucleotide of around 25 bases in length. Multiple ligation reactions can be carried out simultaneously using a mixture of DNA fragments and template oligonucleotides in order to assemble a long piece of DNA from several fragments. With reference to the phosphate group this is a β-elimination reaction. The purpose of the DMT group on the phosphate monomer is to act as a colorimetric monitor of coupling efficiency on the DNA synthesizer.

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