Exploiting the enzymatic recognition of an unnatural base pair to develop a universal genetic analysis system.
Clin Chem
; 49(3): 407-14, 2003 Mar.
Article
en En
| MEDLINE
| ID: mdl-12600952
BACKGROUND: With the invention of the DNA chip, genome-wide analysis is now a reality. Unfortunately, solid-phase detection systems such as the DNA chip suffer from a narrow range in quantification and sensitivity. Today the best methodology for sensitive, wide dynamic range quantification and genotyping of nucleic acids is real-time PCR. However, multiplexed real-time PCR technologies require complicated and costly design and manufacturing of separate detection probes for each new target. METHODS: We developed a novel real-time PCR technology that uses universal energy transfer probes constructed from An Expanded Genetic Information System (AEGIS) for both quantification and genotyping analyses. RESULTS: RNA quantification by reverse transcription-PCR was linear over four orders of magnitude for the simultaneous analysis of beta-actin messenger RNA and 18S ribosomal RNA. A single trial validation study of 176 previously genotyped clinical specimens was performed by endpoint analysis for factor V Leiden and prothrombin 20210A mutation detection. There was concordance for 173 samples between the genotyping results from Invader tests and the AEGIS universal energy transfer probe system for both factor V Leiden and prothrombin G20210A. Two prothrombin and one factor V sample gave indeterminate results (no calls). CONCLUSION: The AEGIS universal probe system allows for rapid development of PCR assays for nucleic acid quantification and genotyping.
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Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Ácidos Nucleicos
/
Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
Límite:
Humans
Idioma:
En
Revista:
Clin Chem
Asunto de la revista:
QUIMICA CLINICA
Año:
2003
Tipo del documento:
Article
País de afiliación:
Estados Unidos
Pais de publicación:
Reino Unido