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Nucleic Acid Sequence-Based Amplification - NASBA |
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| NASBA (nucleic acid sequence-based amplification) is a continuous, isothermal, enzyme-based method for the amplification of nucleic acid. The technique employs a mixture of reverse transcriptase, ribonuclease-H, RNA polymerase and two transcript-specific DNA primers. The forward primer has a 5' extension containing the promoter sequence for bacteriophage T7 DNA-dependent RNA polymerase, whereas the reverse primer has a 5' extension containing a complementary binding sequence for an electrochemiluminescent (ECL) tag. During the amplification process, the 5' primer extensions are fully incorporated into the amplified sequence allowing highly efficient production of specific RNA template (directed by the RNA polymerase; see Figure). The technique is particularly suited for the amplification of single stranded RNA and under optimum conditions a 1012-fold level of amplification is possible. |
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| After amplification, detection can be performed using either the ECL or MP system. In the ECL detection system, specificity is enhanced by an additional capture probe, which confirms the presence of RNA amplicon of interest. An aliquot of the amplification reaction is added to a hybridization solution containing both the capture probe and the ECL probe (Figure). The capture probe is specific for the RNA amplicon of interest, whilst the ECL probe is generic and has complementary region to the RNA amplicon. After incubation, the magnetic beads carrying the hybridized amplicon/ECL probe complexes are captured on the surface of an electrode by means of a magnet. Voltage applied to this electrode triggers the ECL reaction. The light emitted by the hybridized ruthenium-labelled probe is proportional to the amount of amplicon generated in the corresponding amplification reaction. |
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| In the microplate detection method, an aliquot of the amplification reaction is immobilized by a capture probe attached to a microtitre plate (Figure). A detection probe is added and binds to the immobilized amplicon. A detection molecule is added and the colorimetric signal generated is measured in a standard 96-well microtitre plate spectrophotometer at 405nm." |
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