# US 8,771,951 B2 · Methods for PCR and HLA typing using raw blood

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- Number: US 8,771,951 B2
- Document ID: US8771951B2
- Status: Granted
- Kind: B2
- Application number: 12/924,301
- Filed: 2010-09-24
- Published: 2011-05-19
- Granted: 2014-07-08
- Earliest claimed: 2009-11-16
- Assignees: Genomics USA, Inc.
- Applicants: Michael E. Hogan; Georgina Lopez Padilla; Melissa R. May; Andrew T. Abalos; Frederick H. Eggars; Kevin M. O'Brien
- Inventors: Michael E. Hogan; Georgina Lopez Padilla; Melissa R. May; Andrew T. Abalos; Frederick H. Eggars; Kevin M. O'Brien
- Web page: https://patentgrep.com/patent/US8771951B2

## CPC

- C12Q 1/6881: for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
- C12Q 1/689: for bacteria
- C12Q 2600/156: Polymorphic or mutational markers
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## 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 secondary 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 method of allelotyping a gene or set thereof by amplifying the gene(s) using tandem PCR on DNA or RNA comprising the sample, hybridizing the resulting amplicon or sets thereof to probes with sequences of gene-associated allele variations. A detectable signal indicating hybridization corresponds to an allelotype of the gene or a set of allelotypes for the set of genes.

## Claims

### Claim 1

A method for amplifying a DNA of interest, comprising:
obtaining a raw sample comprising DNA;
performing a first PCR on the raw sample to produce a first amplicon;
diluting the first amplicon; and
performing a second PCR thereon until all primers used in the second PCR reaction are consumed to produce a second amplicon, thereby amplifying the input sample DNA to a final amplified DNA product concentration that is limited by the primer concentration in the second PCR reaction, said second PCR reaction independent of the amount or purity of the DNA comprising the original sample, wherein primers for the second PCR reaction target DNA sequences that are contained within the amplified product of the first PCR reaction and wherein primers for the second PCR reaction are a set of multiple exon-specific primers having sequences 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.

### Claim 2 (depends on 1)

The method of claim 1, comprising:
performing the first PCR on a set of gene targets in parallel on the raw sample 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.

### Claim 3 (depends on 2)

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.

### Claim 4 (depends on 2)

The method of claim 2, wherein the gene targets are HLA-DRB1, DQ-A1 and DQB1; are DQ-A1 and DQ-B1 or are HLA-B and KIR.

### Claim 5 (depends on 2)

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.

### Claim 6 (depends on 2)

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.

### Claim 7 (depends on 1)

The method of claim 1, further comprising labeling the second PCR primers with one or more fluorophores.

### Claim 8 (depends on 7)

The method of claim 7, wherein the fluorophor is a cyanine dye.

### Claim 9 (depends on 8)

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.

### Claim 10 (depends on 9)

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-DQB1 gene.

### Claim 11 (depends on 1)

The method of claim 1, wherein primers for the first PCR are locus-specific primers.

### Claim 12 (depends on 11)

The method of claim 11, wherein the primers have sequences shown in SEQ ID NOS: 1-14.

### Claim 13 (depends on 1)

The method of claim 1, further comprising:
sequencing the second amplicon for an analysis thereof.

### Claim 14 (depends on 13)

The method of claim 13, 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.

### Claim 15 (depends on 1)

The method of claim 1, wherein the raw sample is fresh or rehydrated and comprises unprocessed fluid blood, dried unprocessed blood, a fresh buccal swab sample, a dried buccal swab sample, fecal material, a vaginal sample or a sample obtained by swabbing an animate or inanimate surface or object.

### Claim 16 (depends on 1)

The method of claim 1, wherein the DNA is mitochondrial DNA.

### Claim 17

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.

### Claim 18 (depends on 17)

The method of claim 17, further comprising labeling the second PCR primers with one or more fluorophores.

### Claim 19 (depends on 18)

The method of claim 18, wherein the fluorophor is a cyanine dye.

### Claim 20 (depends on 18)

The method of claim 18, 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.

### Claim 21 (depends on 20)

The method of claim 20, 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.

### Claim 22 (depends on 20)

The method of claim 20, further comprising sequencing the second amplicon(s) for an analysis thereof.

### Claim 23 (depends on 22)

The method of claim 22, 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.

### Claim 24 (depends on 17)

The method of claim 17, wherein the second PCR is linear PCR and the second amplicon(s) is cRNA(s).

### Claim 25 (depends on 17)

The method of claim 17, wherein the second PCR is real time PCR and the primers are exon specific to the first cDNA amplicon(s).

### Claim 26 (depends on 17)

The method of claim 17, wherein the raw sample is fresh or rehydrated and comprises unprocessed fluid blood, dried unprocessed blood, a fresh buccal swab sample, a dried buccal swab sample, fecal material, a vaginal sample or a sample obtained by swabbing an animate or inanimate surface or object.

### Claim 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.

### Claim 28 (depends on 27)

The method of claim 27, wherein the first amplicon or amplicon set is cDNA amplified from RNA comprising the sample and the second PCR is linear PCR or real time PCR performed thereon.

### Claim 29 (depends on 27)

The method of claim 27, wherein the signal is fluorescence, said second PCR primer pairs labeled with one or more fluorophores.

### Claim 30 (depends on 29)

The method of claim 29, wherein the fluorophor is a cyanine dye.

### Claim 31 (depends on 27)

The method of claim 27, wherein the gene of interest is 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.

### Claim 32 (depends on 27)

The method of claim 27, wherein the locus-specific primers have sequences shown in SEQ ID NOS: 1-14.

### Claim 33 (depends on 27)

The method of claim 27, wherein the raw biological sample is fresh or rehydrated and comprises blood, dried blood, a buccal sample, fecal matter or a vaginal sample or other sample obtained by swabbing an animate surface or object.

### Claim 34 (depends on 27)

The method of claim 27, wherein the individuals comprise a population in a field environment.