Methods for PCR and HLA typing using unpurified samples
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.
1. A method for amplifying a DNA of interest, comprising:
contacting fluidically isolated rings on a Guthrie card with samples of raw umbilical cord blood;
rehydrating the raw umbilical cord blood in each ring prior to a first PCR performed on each rehydrated raw sample;
performing the first PCR on said rehydrated raw samples without purifying DNA contained therein to produce first amplicons;
diluting the first amplicons; and
performing a second PCR thereon until all primers used in the second PCR reaction are consumed to produce second amplicons, thereby amplifying the DNA from the raw umbilical cord blood samples to final amplified DNA product concentrations that are limited by the primer concentration in the second PCR reactions, said second PCR reactions independent of the amount or purity of the DNA comprising the original raw umbilical cord blood, wherein the primers for the second PCR reaction are a set of multiple exon-specific primers with sequences shown SEQ ID NOS: 15-27 and target DNA sequences contained within the amplified product of the first PCR reaction.
2. The method of claim 1 , wherein said rehydrated raw umbilical cord samples comprise a set of gene targets, said method comprising:
performing the first PCR in parallel to produce a first set of amplicons;
diluting the first set of amplicons; and
performing a second PCR thereon, using the entire set of primary amplicon products as a set of templates for the second PCR reaction until all secondary PCR primers are consumed to produce a second amplicon set, thereby amplifying the DNA.
3. The method of claim 2 , wherein less than 5 gene targets, less than 10 gene targets or less than 20 gene targets are amplified in parallel.
4. The method of claim 2 , wherein the gene targets are HLA-DRB 1, DQ-A1 and DQB1, are DQ-A1 and DQ-B1 or are HLA-B and KIR.
5. The method of claim 2 , wherein the gene targets are two hypervariable regions near the mitochondrial origin of replication and one or more additional mitochondrial genes.
6. The method of claim 2 , wherein the gene targets are segments of microbe-specific microbial 16S DNA genes, said method detecting microbes in the raw samples.
7. The method of claim 1 , further comprising labeling the second PCR primers with one or more fluorophores.
8. The method of claim 7 , wherein the fluorophor is a cyanine dye.
9. The method of claim 8 , wherein the DNA comprises one or more genes of interest, further comprising:
hybridizing the second amplicon to probes having sequences of allele variations associated with the gene of interest;
detecting a fluorescence pattern from the hybridized amplicon; and
assigning an allelotype based on the fluorescence pattern.
10. The method of claim 9 , wherein the gene(s) of interest are an HLA-A gene, an HLA-B gene, an HLA-DRB1 gene, an HLA-DQA1 gene, or an HLA-DQB 1 gene.
11. The method of claim 9 , wherein hybridizing is performed on HLA-Chips containing microarrays.
12. The method of claim 1 , wherein primers for the first PCR are locus-specific primers.
13. The method of claim 12 , wherein the primers have sequences shown in SEQ ID NOS: 1-14.
14. The method of claim 1 , wherein the sample comprises DNA from a bacterium or a virus.
15. The method of claim 1 , further comprising:
sequencing the second amplicon for an analysis thereof; and
analyzing the sequencing data using the Ricimer allele calling algorithm.
16. The method of claim 15 , wherein analysis determines the type of viral or bacterial contamination.
17. The method of claim 15 , 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.
18. The method of claim 1 , wherein said rings are outlined with hydrophobic paint.
19. The method of claim 1 , wherein the DNA is mitochondrial DNA.