Methods for pcr and hla typing using unpurified samples

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.

What is claimed is: 
     
         1 . A method for amplifying one or more RNAs of interest, comprising:
 obtaining a sample from an individual;   performing a first reverse transcription reaction on said sample to produce a first cDNA products(s);   diluting the first cDNA product(s) as template into a first PCR reaction; and   performing PCR thereon until all primers are consumed to produce amplicon(s), thereby amplifying the RNA(s) of interest.   
     
     
         2 . The method of  claim 1 , wherein said sample is a raw umbilical cord blood sample from an individual, a sample of mouthwash expelled from said individual, cheek swabs from said individual, a saliva sample from said individual, a sample from a bacterium or a virus. 
     
     
         3 . The method of  claim 2 , further comprising labeling the PCR primers with one or more fluorophores. 
     
     
         4 . The method of  claim 3 , wherein the fluorophor is a cyanine dye. 
     
     
         5 . The method of  claim 4 , further comprising:
 hybridizing the second amplicon or set of amplicons to probes having sequences complementary to an area of interest in a gene sequence;   detecting a fluorescence pattern from the hybridized amplicon(s); and   identifying one or more genes or allelotypes thereof based on the fluorescence pattern.   
     
     
         6 . The method of  claim 5 , wherein the gene(s) are one or more of an HLA-A gene, an HLA-B gene or an HLA-DRB1 gene, an HLA-DQA1 gene, or an HLA-DQB1 gene or combinations thereof. 
     
     
         7 . The method of  claim 5 , wherein hybridizing is performed on microarrays competent to measure allele variation within the HLA genes. 
     
     
         8 . The method of  claim 7 , wherein said microarrays are fluidically isolated by removable gaskets or by functionally-equivalent hydrophobic barriers. 
     
     
         9 . The method of  claim 7 , further comprising analyzing the hybridization data using the Ricimer allele calling algorithm. 
     
     
         10 . The method of  claim 9 , wherein analysis determines the type of viral or bacterial contamination. 
     
     
         11 . The method of  claim 9 , wherein analysis determines one or more of identity, paternity of an individual, forensic information, tissue matching, risk factors for the development of disease, or response to medication. 
     
     
         12 . The method of  claim 1 , wherein the second PCR is linear PCR and the second amplicon(s) is cRNA(s). 
     
     
         13 . The method of  claim 1 , wherein the second PCR is real time PCR and the primers are exon specific to the first cDNA amplicon(s). 
     
     
         14 . The method of  claim 1 , wherein the first amplicon(s) are one or more of an HLA-A, an HLA-B or an HLA-DBR1, an HLA-DQA1, or an HLA-DQ-B1 cDNA(s) and the exon-specific primers have a sequence shown in SEQ ID NOS: 15-27. 
     
     
         15 . The method of  claim 2 , wherein obtaining said umbilical cord blood sample comprises the step of:
 contacting said sample on Guthrie cards, and   rehydrating said sample.   
     
     
         16 . The method of  claim 15 , wherein said Guthrie cards contain fluidically isolated rings. 
     
     
         17 . The method of  claim 16 , wherein said rings are outlined with hydrophobic paint. 
     
     
         18 . The method of  claim 2 , wherein said saliva sample is a sample stabilized by a sample collection kit.