One-step nucleic acid amplification

From Ganfyd

A name given to biochemical kits designed to simplify the detection of specific nucleic acid sequences, usually by reverse-transcriptase polymerase chain reaction of mRNA to confirm active transcription of genes specific to a particular cell-type. One application is the detection of metastatic breast cancer cells in the sentinel axillary lymph node by using CK19 and mammoglobin as breast-cancer specific gene transcripts.

The technique has been used in colorectal cancer,^[1] but is less established.

A typical kit will contain:

• Homogenisation reagent - to aid in breakdown of tissues and cells to release nucleic acid
• Reverse transcriptase - converts mRNA to more stable DNA
• Reagents for polymerase chain reaction amplification of the resulting DNA

Normally, PCR products are detected by ethidium bromide staining and gel electrophoresis to identify an appropriately-sized DNA product. However, many commercial kits are designed to give results quickly enough - often less than an hour - to allow intra-operative decision-making. To achieve this, fluorescent reporter probes may be used. These are oligonucleotides that bind to the intended DNA product. The attached fluorescent probe which is normally 'quenched', but as the PCR's DNA polymerase incorporates the nucleotides, its exonuclease activity cleaves off the quenching part of the probe allowing fluorescence, which can be measured.

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