Methods for PCR and HLA typing using raw blood

A method for amplifying one or more RNAs of interest, comprising:

• obtaining a raw biological sample from an individual;
• performing a first reverse transcription PCR on the raw biological sample to produce a first cDNA amplicon(s);
• diluting the first amplicon(s) and performing a second PCR thereon until all primers are consumed to produce a second amplicon(s), thereby amplifying the RNA(s) of interest;
• 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 shown in at least SEQ ID NO:15 and 16 and additional primer pairs selected from the group consisting of SEQ ID NOs:17-27.

A method for allelotyping a gene of interest, comprising:

• obtaining a raw biological sample from one or more individuals;
• performing a first PCR on the raw biological sample using primers specific to the gene locus or a defined set of gene loci to produce a first amplicon or first set of amplicons;
• diluting the first amplicon or first set of amplicons and performing a second PCR with the amplicon(s) serving as the template for the second PCR reaction using primers specific to an exon or a set of exons within the gene locus until all primers are consumed to produce an amplicon set from the second PCR reaction, wherein said primers specific to the exon shown in at least SEQ ID NO:15 and 16 and additional primer pairs selected from the group consisting of SEQ ID NOs:17-27;
• hybridizing the second amplicon or amplicon set to probes having sequences of allele variations associated with the gene or gene set of interest;
• detecting a signal from the hybridized amplicon or amplicon set;
• and
• assigning an allelotype based on the detected hybridization signal.