Methods for PCR and HLA typing using unpurified samples

US9416419B2 · kind B2 · utility

Inventors

• Michael E. Hogan · Princeton, US
• Georgina Lopez Padilla · Tucson, US
• Melissa Rose May · Tucson, US
• Andrew T. Abalos · Tucson, US
• Frederick H. Eggars · Sahuarita, US
• Kevin M. O'Brien · San Diego, US

Key dates

• Filing date: Oct 12, 2011
• Grant date: Aug 16, 2016
• Expiry date: Aug 7, 2032

Classification

• Technology area (CPC C): Chemistry; Metallurgy
• CPC primary: C12Q2600/16
• WIPO field: Biotechnology
• WIPO sector: Chemistry

Abstract

Provided are methods for amplifying a gene or RNA or sets thereof of interest using a tandem PCR process. The primers in the first PCR or set of PCR reactions are locus-specific. The primers in the second PCR or set of PCR reactions are specific for a sub-sequence of the locus-specific primers and completely consumed during the second PCR amplification. For RNA amplification, the first PCR is reverse transcription and the resulting cDNA(s) provide a template for cRNA synthesis, endpoint PCR or real time PCR. Also provided is a tandem PCR method which accepts raw, completely unpurified mouthwash, cheek swabs and ORAGENE™-stabilized saliva as the sample input, the resulting amplicons serving as the substrate for complex, microarray-based genetic testing. Also provided is a method of allelotyping a gene or set thereof by amplifying the gene(s) using tandem PCR on DNA or RNA comprising the sample.