Nucleic Acid Amplification Technologies

Saldanha J, Lelie N, Heath A. Establishment of the first international standard for nucleic acid amplification technology (NAT) assays for HCV RNA. WHO Collaborative Study Group. Vox Sang 1999 76 149-58. 

Nucleic Acid Amplification Technologies (NAATs) such as PCR, are generally followed (often in real-time) by some means to detect the accumulation of nucleic acid amplicon. However, as pyrophosphate is a by-product of nucleic acid biosynthesis, the generation of pyrophosphate can also be used to follow NAATs. 

Vaneechoutte, M., Van Eldere, J. 1997. The possibilities and limitations of nucleic acid amplification technology in diagnostic microbiology. J. Med. Microbiol. 46 188-194. 

Abramson RD and Myers TW (1993) Nucleic acid amplification technologies. Current Opinion in Biotechnology 4 41-47. 

Nucleic acid probe methods which rely on the specificity of nucleic acid hybridization reaction, combined with the powerful methods of nucleic acid amplification, are expected to spawn many new diagnostic tests which may be useful in the detection and treatment of infectious diseases, cancer, and inherited disorders. The impact of development and applications of diagnostic tests based on nucleic acid probes and on Mab technology wiU be felt increasingly as deeper understanding of diseases at a molecular level is gained. 

Nucleic acid-based technologies Nucleic acid probe Polymerase chain reaction-DNA amplification 6S rRNA sequencing techniques Automated riboprinting... 

Wilding P. Nucleic Acid Amplification in Microchips, In Cheng JKL, ed. Biochip technology. Philadelphia Harwood Academic Publishers, 2001 173-84. 

In the sample preparation step, the nucleic acid content from the sample is extracted, concentrated, and purified to meet the quality required by the downstream amplification process. Sample preparation of nucleic acid targeting technologies involves several processes. In most cases, lysis is an initially required process to break open intact structures of targeted species (e.g., cell wall, membrane, and capsules) and to release nucleic acids. Additional purification or extraction of the released nucleic acids from the lysate may be conducted to minimize inhibitory effects on downstream amplification. Following the lysis step, the chemicals introduced (e.g., membrane-solubilizing ionic detergents or proteinases), cell debris, and other interferents that come with samples are removed. For diluted specimens (i.e., urine), nucleic acid concentration may be required in order to reduce the limit of detection of amplification and detection assays. 

In summary, bDNA has a number of distinct theoretical and practical advantages over target amplification systems for direct quantitation of specific nucleic acid sequences. The following sections review the specific clinical and research applications of this technology. 

To properly accession and purify nucleic acids for analysis, the receiving laboratory must know the sample type. Both heparin and urine have been reported to inhibit PCR, to the detriment of blood samples containing the former and nearly all samples of the latter. In recent years, extraction technology and amplification chemistry have improved so that each sample type is susceptible to analysis. Proper identification of sample type provides the receiving laboratory with an opportunity to apply appropriate techniques to its extraction and analysis. 

Nygaard V, Hovig E. Options available for profiling small samples a review of sample amplification technology when combined with microarray profiling. Nucleic Acids Res 2006 34 996-1014. 

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