HK1217353B - Hla gene multiplex dna typing method and kit - Google Patents

Description

Multiplex DNA typing method and kit for HLA genes

Technical Field

The present invention relates to an ultra-high-resolution multiplex DNA typing method for HLA genes using a high-throughput DNA sequencer.

Background Art

Human leukocyte antigens (HLA), part of the human major histocompatibility complex (MHC), are closely involved in inducing immune responses by presenting peptides from foreign proteins such as pathogens, as well as peptides from self-proteins, to T cells. Six major HLA antigens are known: class I antigens (HLA-A, HLA-B, and HLA-C), which are expressed in nearly all cells, and class II antigens (HLA-DR, HLA-DQ, and HLA-DP), which are primarily expressed in cells of the immune system.

HLA class I antigens are composed of a highly polymorphic α chain and a nearly non-polymorphic β2-microglobulin. HLA class II antigens are composed of a highly polymorphic β chain and a less polymorphic α chain. The α chain of class I antigens is encoded by the HLA-A, HLA-B, and HLA-C genes, while the β chain of class II antigens is encoded by the HLA-DRB1, HLA-DRB3/4/5, HLA-DQB1, and HLA-DPB1 genes, and the α chain is encoded by the HLA-DRA1, HLA-DQA1, and HLA-DPA1 genes. At the genetic level, exons 2 and 3 of the gene encoding the α chain of HLA class I antigens are highly polymorphic, while exon 2 of the gene encoding the β chain of HLA class II antigens is highly polymorphic.

The HLA gene region is located on the short arm of human chromosome 6, at 6p21.3. From telomeric to centromeric, it is arranged in the order of class I (HLA-A, HLA-B, HLA-C, etc.), class III, and class II (HLA-DRA, HLA-DRB1, HLA-DRB3/4/5, HLA-DQA1, HLA-DQB1, HLA-DPA1, HLA-DPB1, etc.). Many genes are encoded at a very high density, and their association with transfusion, transplantation, and various diseases has been reported. HLA genes are absent from the class III region, but genes for complement components and tumor necrosis factor (TNF) are present.

Five structural polymorphisms have been identified in the HLA-DRB gene region, which encodes the β chain of the HLA-DR antigen. In DR1 and DR10 types, in addition to HLA-DRB1, pseudogenes such as HLA-DRB6 and HLA-DRB9 are also located on the same haplotype. In DR2 types, in addition to HLA-DRB1, pseudogenes such as HLA-DRB5 (DR51), HLA-DRB6, and HLA-DRB9 are also located on the same haplotype. In DR3, DR5, and DR6 types, in addition to HLA-DRB1, pseudogenes such as HLA-DRB3 (DR52), HLA-DRB2, and HLA-DRB9 are also located on the same haplotype. In DR4, DR7, and DR9, in addition to HLA-DRB1, pseudogenes such as HLA-DRB4 (DR53), HLA-DRB7, HLA-DRB8, and HLA-DRB9 are also located on the same haplotype. In contrast, in DR8, HLA-DRB genes other than HLA-DRB1 are not located on the same haplotype (Figure 1).

Each HLA gene exon contains numerous regions rich in polymorphism (polymorphic regions), and the base sequence (amino acid sequence) of a particular polymorphic region is often shared by multiple HLA alleles. In other words, each HLA allele is defined by the combination of multiple polymorphic regions. In HLA class I antigens, not only polymorphic regions within exons, but also exons 2 and 3, which have identical base sequences, are sometimes shared by multiple alleles.

Due to the high degree of polymorphism in HLA, a vast number of HLA alleles are known, and their representation has been established. Specifically, the first region (two-digit level) identifies serological HLA types; the second region (four-digit level) identifies alleles with amino acid substitutions within the same serological HLA type; the third region (six-digit level) identifies alleles with confirmed base substitutions without amino acid mutations; and the fourth region (eight-digit level) identifies alleles with base substitutions outside the gene region encoding the HLA molecule (introns) (Figure 2).

During bone marrow transplantation, it is said that if the HLA types (HLA-A, HLA-B, HLA-C, HLA-DRB1) of the recipient and the donor are completely matched at the four-digit level, the success rate of the transplant will be improved and the frequency of severe graft-versus-host disease (GVHD) will be reduced. Conversely, if the HLA types are inconsistent at the four-digit level, the risk of adverse reactions such as rejection increases. Therefore, accurate and precise HLA typing is extremely important in clinical practice.

As DNA typing methods for HLA genes, the SBT (sequence-based typing) method based on the polymerase chain reaction (PCR) and the SSO (sequence-specific oligonucleotide)-Luminex method are mainstream.

These previous DNA typing methods have the advantage of being able to quickly type a large number of samples. However, in the case of polymorphic regions and class I genes, the cis-trans positional relationship of exons on chromosomes cannot be accurately determined, resulting in phase ambiguity, which sometimes makes it difficult to perform high-precision HLA typing.

Furthermore, conventional methods employ PCR-based DNA typing centered on the exon regions of each HLA gene, thus ignoring base substitutions in intron and promoter regions. Consequently, detection of null alleles, which have the same gene structure as other expressed HLA genes but suppress their expression, may be overlooked.

For example, in the method described in Patent Document 1, since the PCR conditions for each HLA gene are different, PCR for each HLA gene must be performed independently, which has no effect on speeding up and simplifying the operation.

The present inventors have proposed a method for performing high-precision typing without phase ambiguity by using primer sets that specifically anneal to the upstream and downstream regions of each HLA locus (Patent Document 2 and Non-Patent Document 2). However, this method does not completely standardize the PCR conditions used for each gene, and a multiplex method for performing PCR simultaneously on all loci has not yet been achieved.

Furthermore, there has been no research on a high-speed PCR apparatus that significantly shortens the time required for temperature increase or decrease when performing PCR, for which speed-up of PCR is desired.

Prior art literature

Patent Literature

Patent Document 1: Japanese Patent Application Laid-Open No. 11-216000

Patent Document 2: International Publication No. WO2013/011734 Pamphlet

Non-patent literature

Non-patent document 1: Lind C. et al., Human Immunology, Vol. 71, pp. 1033-1042 (2010)

Non-patent document 2: Shiina T. et al., Tissue Antigens, Vol. 80, pp. 305-316 (2012).

Summary of the Invention

Technical problem to be solved by the invention

The present invention aims to develop a multiplex PCR method that allows for simultaneous PCR amplification of each HLA gene, including HLA-DRB3/DRB4/DRB5, in the same container using the same PCR conditions (annealing temperature) for each HLA gene, using newly designed gene-specific primers. Furthermore, the present invention aims to provide an ultra-high-resolution DNA typing method that eliminates ambiguity arising from phase ambiguity by setting the conditions (time required for the extension reaction) for the multiplex PCR method when the method is applied to a high-speed PCR apparatus, thereby achieving rapidity and simplification.

Methods used to solve technical problems

To solve the above-mentioned problems, the present inventors conducted intensive studies and, as a result, discovered that by redesigning primers for each HLA gene and selecting the DNA polymerase to be used, PCR could be performed under the same conditions regardless of the type of HLA gene, thereby completing the present invention.

That is, the present invention provides a method for DNA typing of HLA comprising the following steps.

(1) preparing a primer set that specifically hybridizes to the upstream region and the downstream region of at least two genes selected from genes belonging to HLA class I and class II in the human genome base sequence, and amplifies under the same PCR conditions;

(2) a step of simultaneously amplifying the at least two genes in the test sample (DNA) under the same PCR conditions in the same container using the primer set;

(3) a step of determining the base sequence of the PCR product; and

(4) Optionally, perform a homology search with a database.

Effects of the Invention

The present invention solves the problems of the prior art by designing PCR primers that can specifically amplify HLA genes (HLA-A, HLA-B, and HLA-C) and HLA class II antigens (HLA-DRB1, HLA-DRB3/4/5, HLA-DQA1, HLA-DQB1, HLA-DPA1, and HLA-DPB1) at the same annealing temperature, selecting a DNA polymerase suitable for the present DNA typing method, setting optimal PCR conditions that enable DNA typing of all HLA genes at once using a single PCR apparatus when using this enzyme, developing a multiplex PCR method for HLA-A, HLA-B, HLA-C, and HLA-DRB1 based on these conditions, further setting the time required for the extension reaction in the multiplex PCR method when using a high-speed PCR apparatus, and utilizing high-throughput sequencing technology.

The method of the present invention can obtain all the base sequences required for DNA typing of the HLA gene from a single molecule, and is therefore the ultimate DNA typing method that does not confirm phase ambiguity where the cis-trans positional relationship is unclear. For example, during transplantation, highly accurate HLA matching can be achieved between the desired transplant recipient and the donor candidate.

Since the entire base sequence of the HLA gene, including the promoter region, exon region, intron region and other surrounding regions, has been determined, null alleles and novel alleles whose expression is suppressed can be detected.

In the present invention, since suitable PCR conditions are set so that DNA typing of multiple to all HLA genes can be performed at once using a single PCR apparatus, speed and simplification of the operation are achieved.

The present invention provides a multiplex PCR method for a plurality of HLA genes, thereby realizing more accurate and rapid HLA typing between a transplant recipient and a donor candidate.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the HLA-DR gene region. Cited from "Transplantation and Blood Transfusion Examination," edited by Inoko Hidetoshi, Sasazuki Kenhiko, and Shizuo Takeo, Kodansha Science, 2004, p. 48.

Figure 2 shows the classification of HLA alleles. Used from the IMGT-HLA database (www.ebi.ac.uk/ipd/imgt/hla/).

[Figure 3] (a) is a diagram showing the relationship between HLA class I gene structure and molecular structure. (b) is a diagram showing the structure of the HLA class I gene promoter region. Cited from "Transplantation and Blood Transfusion Examination," edited by Inoko Hidetoshi, Sasazuki Kenhiko, and Shizuo Takeo, Kodansha Science, 2004, p. 35.

[ Fig. 4 ] is a schematic diagram showing the structure of each HLA gene.

Figure 5 (a) shows the relationship between the HLA class II gene structure and its molecular structure. (b) shows the structure of the HLA class II gene promoter region. Cited from "Transplantation and Blood Transfusion Examination," edited by Inoko Hidetoshi, Sasazuki Kenhiko, and Shizuo Takeo, Kodansha Science, 2004, pp. 46-47.

FIG6 is a diagram showing an agarose gel electrophoresis image showing the amplification status of the HLA-DRB1 gene amplified in Test Example 1. ...

[ Fig. 7 ] is a diagram showing an agarose gel electrophoresis image indicating the PCR status of the HLA gene amplified in Example 1.

[Figure 8] is a diagram showing an agarose gel electrophoresis image showing the PCR status of the HLA gene amplified in Example 2.

[ Figure 9] is a diagram showing an agarose gel electrophoresis image showing the PCR status of the HLA gene amplified in Example 3.

FIG. 10 is a diagram showing an agarose gel electrophoresis image showing the PCR status of the HLA gene amplified in Example 4.

FIG11 is a diagram showing an agarose gel electrophoresis image showing the PCR status of the HLA-A, HLA-B, HLA-C, and HLA-DRB1 genes amplified in Example 5.

FIG12 is a graph showing the results of measuring the length of the multiplex PCR products of the HLA-A, HLA-B, HLA-C, and HLA-DRB1 genes amplified in Example 5 using a bioanalyzer.

FIG. 13 is a diagram showing an agarose gel electrophoresis image showing the PCR status of the HLA-A, HLA-B, HLA-C, and HLA-DRB1 genes amplified in Comparative Example 1.

Figure 14 shows the results of measuring the length of the multiplex PCR products of the HLA-A, HLA-B, HLA-C, and HLA-DRB1 genes amplified in Comparative Example 1 using a bioanalyzer (upper panel) and an electrophoresis image thereof (lower panel).

DETAILED DESCRIPTION

Hereinafter, the DNA typing method of the present invention will be described in detail step by step.

(1) Primer set preparation steps

In the DNA typing method of the present invention, first, a primer set capable of annealing under the same conditions is prepared for comprehensively performing PCR on the upstream and downstream regions of each of the HLA-A, HLA-B, HLA-C, HLA-DQA1, HLA-DQB1, and HLA-DPA1 genes, the untranslated regions from the 5' untranslated region to exon 2 and from exon 2 to 3' of each of the HLA-DRB1 and HLA-DPB1 genes, and the untranslated regions from exon 2 to 3' of HLA-DRB3/4/5 in the human genomic base sequence.

The genomic base sequence of human chromosome 6 (6p21.3), which contains the region where HLA genes are present, has been elucidated, and the relationship between its gene structure and the structure of its expression product (HLA antigen) is also known (see Figures 3 and 5).

Specifically, the genes for HLA-A, HLA-B, and HLA-C, which regulate the classical HLA class I antigens, contain eight exons (Figure 3(a)), and two enhancer and promoter regions outside exon 1 regulate expression (Figure 3(b)).

Furthermore, it is known that a large number of polymorphic regions exist in exons 2, 3, and 4. Therefore, conventional DNA typing methods use primers prepared based on exons 2 and 3 in particular for PCR, which leads to the aforementioned problems ( FIG. 4 ).

In addition, HLA-DR, HLA-DQ, and HLA-DP, which define the classical HLA class II antigens, are composed of α and β chains, and each gene contains 5 or 6 exons (Figure 5(a)). A promoter region outside exon 1 regulates expression (Figure 5(b)).

Furthermore, it is known that a large number of polymorphic regions exist in exons 2 and 3. Therefore, conventional DNA typing methods use primers prepared based on exon 2 in particular for PCR, which leads to the aforementioned problems ( FIG. 4 ).

In the present invention, primer sets are prepared that are capable of performing PCR on the entire gene regions (including not only exons but also introns, 5' and 3' untranslated regions, and promoter regions) of classical class I antigens (HLA-A, HLA-B, HLA-C) and classical class II antigens (HLA-DRB1, HLA-DQA1, HLA-DQB1, HLA-DPA1, HLA-DPB1), and the untranslated regions from exons 2 to 3' of HLA-DRB3/4/5. The PCR products amplified using these primer sets are then subjected to high-throughput sequencing, as described below. This eliminates uncertainties such as phase ambiguity and allows for accurate detection of the presence of null alleles.

Specifically, primer sets corresponding to at least two genes among the PCR primer sets shown in Tables 1 to 3 below were prepared.

At least two genes means at least two genes selected from HLA-A, HLA-B, HLA-C, HLA-DQA1, HLA-DQB1, HLA-DPAl, HLA-DRB1, and HLA-DPB1, including all combinations of two, three, four, five, six, and seven genes selected from HLA-A, HLA-B, HLA-C, HLA-DQA1, HLA-DQB1, HLA-DPAl, HLA-DRB1, and HLA-DPB1, as well as all combinations of eight genes.

The primer sets corresponding to each gene are as follows.

Sequence numbers 1 to 3 in Table 1 represent PCR primer sets that specifically amplify the HLA-A gene, which is the MHC class I α chain. These primer sets contain base sequences located at positions that span the entire HLA-A gene region (including the promoter, exons, and introns) from both upstream and downstream sides in the human genomic sequence (reference sequence: hg19).

SEQ ID NOs. 1 and 2 have base sequences corresponding to bases 29,909,483 to 29,909,514 in the human genome base sequence (reference sequence: hg19).

SEQ ID NO: 3 has a base sequence complementary to the base sequence corresponding to bases 29,914,925 to 29,914,954 in the human genome base sequence (reference sequence: hg19).

The predicted length of the PCR product obtained using these primer sets is approximately 5,500 bases (bp).

Sequence numbers 4 and 5 in Table 1 are PCR primer sets that specifically amplify the HLA-B gene, which is the MHC class I α chain. These primer sets contain base sequences located at positions that span the entire HLA-B gene region (including the promoter, exons, and introns) from both upstream and downstream sides in the human genome sequence (reference sequence: hg19).

SEQ ID NO: 4 has a base sequence complementary to the base sequence corresponding to bases 31,325,796 to 31,325,824 in the human genome base sequence (reference sequence: hg19).

SEQ ID NO: 5 has a base sequence corresponding to bases 31,321,210 to 31,321,235 in the human genome base sequence (reference sequence: hg19).

The predicted length of the PCR product obtained using these primer sets is approximately 4,600 bases (bp).

Sequence numbers 6 to 8 in Table 1 are PCR primer sets for specifically amplifying the HLA-C gene, which is the MHC class I α chain. These primer sets contain base sequences located at positions that span the entire HLA-C gene region (including the promoter, exons, and introns) from both upstream and downstream sides in the human genomic sequence (reference sequence: hg19).

Sequence numbers 6 and 7 have base sequences complementary to the base sequence corresponding to bases 31,240,868 to 31,240,896 in the human genome base sequence (reference sequence: hg19).

SEQ ID NO: 8 has a base sequence corresponding to bases 31,236,075 to 31,236,114 in the human genome base sequence (reference sequence: hg19).

The predicted length of the PCR product obtained using these primer sets is approximately 4,800 bases (bp).

Sequence numbers 9 and 10 in Table 2 are PCR primer sets that specifically amplify the HLA-DR2 (DR15) and HLA-DR2 (DR16) subtypes of the HLA-DRB1 gene, which are MHC class II β chains. These primer sets contain base sequences located at positions flanking the untranslated region from exon 2 to 3' of the HLA-DRB1 gene in the human genomic sequence (reference sequence: hg19) from upstream and downstream sides.

SEQ ID NO: 10 has a base sequence corresponding to bases 32,546,609 to 32,546,629 in the human genome base sequence (reference sequence: hg19).

SEQ ID NO: 9 has a base sequence complementary to the base sequence corresponding to bases 32,552,130 to 32,552,153 in the human genome base sequence (reference sequence: hg19).

The predicted length of the PCR product obtained using these primer sets is approximately 5,600 bases (bp).

Sequence numbers 11 and 12 in Table 2 are PCR primer sets that specifically amplify HLA-DR3, HLA-DR5 (DR11), HLA-DR5 (DR12), HLA-DR6 (DR13), HLA-DR6 (DR14), and HLA-DR8 subtypes of the HLA-DRB1 gene, which are MHC class II β chains. These primer sets contain base sequences located at the following positions, which sandwich the untranslated region from exon 2 to 3' of the HLA-DRB1 gene from upstream and downstream in the human genomic sequence (reference sequence: hg19).

SEQ ID NO: 12 has a base sequence corresponding to bases 32,546,608 to 32,546,629 in the human genome base sequence (reference sequence: hg19).

SEQ ID NO: 11 has a base sequence complementary to the base sequence corresponding to bases 32,552,137 to 32,552,162 in the human genome base sequence (reference sequence: hg19).

The predicted length of the PCR product obtained using these primer sets is approximately 5,600 bases (bp).

Sequence numbers 13 to 15 in Table 2 represent PCR primer sets that specifically amplify the HLA-DRB3 gene, which is an MHC class II β chain. These primer sets contain base sequences located at positions flanking the untranslated region from exon 2 to the 3' region of the HLA-DRB3 gene in the human genomic sequence (reference sequence: 6_cox_hap2) from upstream and downstream.

SEQ ID NOs. 13 and 14 have base sequences complementary to the base sequence corresponding to bases 3,939,356 to 3,939,379 in the human genome base sequence (reference sequence: 6_cox_hap2).

SEQ ID NO: 15 has a base sequence corresponding to bases 3,934,187 to 3,934,207 in the human genome base sequence (reference sequence: 6_cox_hap2).

The predicted length of the PCR product obtained using these primer sets is approximately 5,200 bases (bp).

Sequence numbers 16 and 17 in Table 2 are PCR primer sets that specifically amplify the HLA-DRB4 gene, which is an MHC class II β chain. These primer sets contain base sequences located at positions flanking the untranslated region from exon 2 to the 3' region of the HLA-DRB4 gene in the human genomic sequence (reference sequence: chr6_mamn_hap4) from upstream and downstream.

SEQ ID NO: 16 has a base sequence complementary to the base sequence corresponding to bases 3,851,270 to 3,851,293 in the human genome base sequence (reference sequence: chr6_mann_hap4).

SEQ ID NO: 17 has a base sequence corresponding to bases 3,846,108 to 3,846,128 in the human genome base sequence (reference sequence: chr6_mann_hap4).

The predicted length of the PCR product obtained using these primer sets is approximately 5,200 bases (bp).

Sequence numbers 18 and 19 in Table 2 are PCR primer sets for specifically amplifying the HLA-DRB5 gene, which is an MHC class II β chain. These primer sets contain base sequences located at positions flanking the untranslated region from exon 2 to the 3' region of the HLA-DRB5 gene in the human genomic sequence (reference sequence: hg19) from upstream and downstream sides.

SEQ ID NO: 18 has a base sequence complementary to the base sequence corresponding to bases 32,489,933 to 32,489,956 in the human genome base sequence (reference sequence: hg19).

SEQ ID NO: 19 has a base sequence corresponding to bases 32,485,256 to 32,485,276 in the human genome base sequence (reference sequence: hg19).

The predicted length of the PCR product obtained using these primer sets is approximately 4,700 bases (bp).

Sequence numbers 20 and 21 in Table 3 are PCR primer sets for specifically amplifying the HLA-DQA1 gene, which is the MHC class II α chain. These primer sets contain base sequences located at positions that span the entire HLA-DQA1 gene region (including the promoter, exons, and introns) from both upstream and downstream sides in the human genomic sequence (reference sequence: hg19).

SEQ ID NO: 20 has a base sequence corresponding to bases 32,604,465 to 32,604,488 in the human genome base sequence (reference sequence: hg19).

SEQ ID NO: 21 has a base sequence complementary to the base sequence corresponding to bases 32,611,936 to 32,611,960 in the human genome base sequence (reference sequence: hg19).

The predicted length of the PCR product obtained using these primer sets is approximately 7,500 bases (bp).

Sequence numbers 22 to 26 in Table 3 represent PCR primer sets that specifically amplify the HLA-DQB1 gene, which is an MHC class II β chain. These primer sets contain base sequences located at positions that span the entire HLA-DQB1 gene region (including the promoter, exons, and introns) from both upstream and downstream sides in the human genomic sequence (reference sequence: hg19).

SEQ ID NO: 22 has a base sequence corresponding to bases 32,627,406 to 32,627,433 in the human genome base sequence (reference sequence: hg19).

Sequence numbers 23 and 25 have base sequences corresponding to bases 32,635,612 to 32,635,643 in the human genome base sequence (reference sequence: hg19).

SEQ ID NOs. 24 and 26 have base sequences complementary to the base sequence corresponding to bases 32,635,612 to 32,635,640 in the human genome base sequence (reference sequence: hg19).

The predicted length of the PCR product obtained using these primer sets is approximately 8,200 bases (bp).

Sequence numbers 27 to 29 in Table 3 represent PCR primer sets that specifically amplify the HLA-DPA1 gene, which is an MHC class II α chain. These primer sets contain base sequences located at positions that span the entire HLA-DPA1 gene region (including the promoter, exons, and introns) from both upstream and downstream sides in the human genomic sequence (reference sequence: hg19).

SEQ ID NO: 27 has a base sequence complementary to the base sequence corresponding to bases 33,041,573 to 33,041,600 in the human genome base sequence (reference sequence: hg19).

SEQ ID NO: 28 has a base sequence complementary to the base sequence corresponding to bases 33,041,573 to 33,041,598 in the human genome base sequence (reference sequence: hg19).

SEQ ID NO: 29 has a base sequence corresponding to bases 33,031,885 to 33,031,912 in the human genome base sequence (reference sequence: hg19).

The predicted length of the PCR product obtained using these primer sets is approximately 9,700 bases (bp).

Sequence numbers 30 and 31 in Table 3 are PCR primer sets for specifically amplifying the HLA-DPB1 gene, which is an MHC class II β chain. These primer sets contain base sequences located upstream and downstream of the untranslated region from exon 2 to 3' of the HLA-DPB1 gene in the human genomic sequence (reference sequence: hg19).

SEQ ID NO: 30 has a base sequence corresponding to bases 33,048,182 to 33,048,207 in the human genome base sequence (reference sequence: hg19).

SEQ ID NO: 31 has a base sequence complementary to the base sequence corresponding to bases 33,055,428 to 33,055,453 in the human genome base sequence (reference sequence: hg19).

The predicted length of the PCR product obtained using these primer sets is approximately 7,300 bases (bp).

These primers can be prepared using methods commonly used in the art. The primer sets listed in Tables 1 to 3 represent the most preferred examples. In the methods of the present invention, any set of sense and antisense primers that can anneal to positions sandwiching the entire HLA gene region from both upstream and downstream can be used similarly.

It should be noted that in this specification, even if the base sequence corresponds to the same position in the reference sequence (or the base sequence complementary thereto), primers that differ by one to several bases are each given a unique sequence number. These differences of one to several bases lead to polymorphisms.

(2) PCR steps

In the method of the present invention, PCR is performed on the test sample (DNA) using the primer set prepared in the above step (1).

PCR was performed according to a conventional protocol, as described below.

1. Depending on the form of the test sample, DNA is extracted from the sample.

2. Quantify the extracted DNA and set the appropriate primer concentration to prepare the reaction solution.

3. Set reaction conditions and perform PCR.

Example: Heat denaturation step (usually 92-98°C)

Annealing step (usually 55-72°C)

Extension step (usually 65-80°C)

In the method of the present invention, the temperature of the annealing and extension steps is preferably set to approximately 65°C to 70°C, more preferably 65°C to 68°C. By performing annealing and extension at approximately 65°C to 70°C, HLA alleles can be generated at equal ratios (uniformly).

4. The obtained PCR product is purified and used for the subsequent base sequence determination step.

By using a newly defined primer set in the present invention, multiple different genes can be amplified simultaneously under the same conditions in a reaction in one test tube.

The enzyme (DNA polymerase) used in the present invention is not particularly limited, and commercially available products may be used, such as PrimeSTAR GXL DNA Polymerase, Tks Gflex DNA Polymerase, TaKaRa LA Taq, and Long PCR Enzyme Mix from Thermo Scientific.

(3) Base sequence determination step.

Next, the base sequence of the PCR product (amplified DNA) generated in step (2) is determined. This step is preferably performed using a technique known as high-throughput sequencing (or ultra-high-speed sequencing, or massive parallel sequencing). For information on high-throughput sequencing, see, for example, "Ji Ji Medicine," Vol. 27, No. 1, 2009 (Yodosha).

This specification describes a method for determining sequences using the Roche 454 GS system. However, base sequences can also be determined using the Life Technologies Ion Torrent PGM system and the Illumina MiSeq system.

1. The PCR product obtained in step (2) above was fragmented into approximately 500 bases using a nebulizer.

2. Add DNA adapters to the ends of the fragmented DNA fragments.

3. After converting the DNA fragments to single-stranded DNA fragments, the DNA fragments attached with DNA adapters are bound to beads via the adapters. The resulting beads are then embedded in a water-in-oil emulsion (creating a microreactor environment in which one DNA fragment is bound to each bead).

4. Emulsion PCR is performed to form copies of each DNA fragment on each bead. (Each DNA fragment is clonally amplified within each microreactor, allowing for the simultaneous amplification of many fragments without competing with other sequences.) Next, the emulsion is disrupted and the beads bearing the amplified DNA fragments are recovered.

5. Concentrate the beads and load them into a Pico Titer plate (size sufficient to fit one bead into one well).

6. For each bead, the pyrophosphate produced during the polymerase reaction is detected using the luciferase fluorescence reaction. The DNA base sequence is determined based on the intensity and pattern of the luminescence. Four nucleic acids (A, C, G, and T) are added in a specific order, and the chemiluminescence pattern corresponding to the added nucleic acids is recorded. The base sequence is determined based on the combination of signal intensity and positional information.

(4) DNA typing steps

Next, the sff file obtained in the above step (3) is classified according to the MID tag and compared with the data of the base sequence database of known HLA alleles, thereby determining the HLA alleles at the fourth region level (8-digit level) of the DNA contained in the test sample.

The method of the present invention is exemplified by the primer sets listed in Tables 1 to 3 above. The present invention has the characteristic of determining the sequence of DNA amplified across nearly the entire region by setting primers across the entire region of each HLA class I and HLA class II gene. This eliminates ambiguity and provides information on null alleles.

In the present invention, by newly setting a primer set having the same annealing temperature during PCR for each HLA gene, PCR for multiple genes can be performed simultaneously using a single PCR apparatus.

Furthermore, in the present invention, by setting the above-mentioned novel primer set, multiplex PCR can be performed simultaneously on multiple HLA genes in one test tube.

Furthermore, it was confirmed that the combination of primer sets and enzymes used in the present invention is also applicable to high-speed PCR devices, thereby enabling PCR to be performed more quickly and with higher precision than before.

Example

The present invention will be described in more detail below with reference to specific examples, but the present invention is not limited to these examples.

(Test Example 1)

[Purpose]

A PCR enzyme suitable for the method of the present invention is selected.

[method]

PCR was performed using two samples of extracted genomic DNA heterozygous for DR9 and DR15 as templates using a primer set that specifically amplifies exon 2 from the promoter region of HLA-DRB1 (see Non-Patent Document 2).

It should be noted that the genomic DNA was selected for the following reasons: since the length of the DR9 PCR product is 11.2 kb and the length of the DR15 PCR product is 6.1 kb, it is easy to confirm that the two HLA alleles are amplified in equal amounts. The DR9 PCR product is the longest in the present invention, and if a DR9 PCR product is obtained, it is believed that other HLA genes can also be amplified in the same manner.

(1) PCR was performed using the 23 commercially available long-range PCR enzymes listed in Table 4. Specifically, 1.5 to 3 μL (4 pmol/μL) of PCR primers, PCR buffer according to the protocol for each enzyme, dNTPs, and long-range PCR enzyme were added to 50 ng of genomic DNA solution, and the total volume of the reaction solution was adjusted to 20 μL with sterile water.

(2) After incubation at 94°C for 2 minutes, a two-step process of incubation at 98°C for 10 seconds and at 68°C for 5 minutes was repeated 30 times. The PCR was performed using the GeneAmp PCR System 9700 (Life Technologies). After PCR, the amplification of the PCR product was confirmed by agarose gel electrophoresis. The electrophoresis image is shown in Figure 6.

[Results and Investigation]

By performing PCR according to a standard protocol and electrophoresis of these PCR products on agarose gels, PCR products derived from both HLA alleles were obtained at the target molecular weight positions for both samples using four enzymes: PrimeSTAR GXL DNA Polymerase (TaKaRa), TaKaRa LA Taq (TaKaRa), Tks Gflex DNA Polymerase (TaKaRa), and Long PCR Enzyme Mix (Thermo SCIENTIFIC) ( Figure 6 ).

However, the results of Test Example 1 shown below revealed that, of the four enzymes described above, three—PrimeSTAR GXL DNA Polymerase (TaKaRa), TaKaRa LA Taq (TaKaRa), and Tks Gflex DNA Polymerase (TaKaRa)—were able to amplify PCR products derived from each HLA gene allele approximately equally, confirming that these three enzymes are particularly suitable for the method of the present invention.

(Example 1)

[Purpose]

PrimeSTAR GXL DNA Polymerase (TaKaRa) was used as an enzyme to confirm the amplification status of each HLA gene.

[method]

PCR was performed using PrimeSTAR GXL DNA Polymerase (TaKaRa) with the extracted genomic DNA as a template and primer sets specific for each HLA class I and HLA class II gene (see Tables 1 to 3: Sequence Numbers 1 to 31). The specific steps are as follows.

(1) To 50 ng of genomic DNA solution, add 4 μL of 5x PrimeSTAR GXL buffer, 1.6 μL of dNTP solution, 1 to 3 μL (4 pmol/μL) of PCR primers, and 0.4 μL of Prime STAR GXL polymerase. Adjust the total volume of the reaction solution to 20 μL with sterile water.

(2) After incubation at 94°C for 2 minutes, the reaction was repeated 30 times with two steps of incubation at 98°C for 10 seconds and at 70°C for 5 minutes. The PCR was performed using the GeneAmp PCR System 9700 (Life Technologies). After PCR, the amplification of the PCR product was confirmed by agarose gel electrophoresis. The electrophoresis image is shown in Figure 7.

[Results and Investigation]

Examination of PCR conditions using PrimeSTAR GXL DNA Polymerase (TaKaRa) and agarose gel electrophoresis of these PCR products revealed that a single PCR product at the target molecular weight was obtained for each of the HLA class I and HLA class II genes (Figure 7).

(Example 2)

[Purpose]

The purpose was to confirm the amplification status of each HLA gene using Tks Gflex DNA Polymerase (TaKaRa) as an enzyme.

[method]

PCR was performed using Tks Gflex DNA Polymerase (TaKaRa) with the extracted genomic DNA as a template and primer sets specific for each HLA class I and HLA class II gene (see Tables 1 to 3: SEQ ID NOs. 1 to 31). The specific steps are as follows.

(1) Add 10 μL of 2x Gflx PCR Buffer, 1-3 μL (4 pmol/μL) of PCR primers, and 0.2 μL of Tks Gflex DNA Polymerase to 50 ng of genomic DNA solution. Adjust the total volume of the reaction solution to 20 μL with sterile water.

(2) After incubating at 94°C for 2 minutes, a two-step process of 98°C for 10 seconds and 68°C for 5 minutes was repeated 10 times. Subsequently, a two-step process of 98°C for 10 seconds and 65°C for 5 minutes was repeated 20 times. The GeneAmp PCR System 9700 (Life Technologies) was used for this PCR. After PCR, the amplification status of the PCR product was confirmed by agarose gel electrophoresis. The electrophoresis image is shown in Figure 8.

[Results and Investigation]

Examination of PCR conditions using Tks Gflex DNA Polymerase (TaKaRa) and agarose gel electrophoresis of these PCR products revealed that a single PCR product at the target molecular weight was obtained for each of the HLA class I and HLA class II genes ( Figure 8 ).

Compared with the results of Comparative Example 1, when Tks Gflex DNA Polymerase (TaKaRa) was used, each HLA gene was amplified equally and uniformly.

(Example 3)

[Purpose]

The differences in PCR performance between the GeneAmp PCR System 9700 (Life Technologies), a general-purpose PCR instrument, and the PCR Thermal Cycler Fast (TaKaRa), a high-speed amplification instrument specifically designed for PCR, were examined, and the time required for PCR was studied.

[method]

PCR was performed using Tks Gflex DNA Polymerase (TaKaRa) with the extracted genomic DNA as a template and primer sets specific for each HLA class I and HLA class II gene (see Tables 1 to 3: SEQ ID NOs. 1 to 31). The specific steps are as follows.

(1) Add 10 μL of 2x Gflex PCR Buffer, 1-3 μL (4 pmol/μL) of PCR primers, and 0.2 μL of Tks Gflx DNA Polymerase to 50 ng of genomic DNA solution, and adjust the total volume of the reaction solution to 20 μL with sterile water.

(2) After incubating at 94°C for 2 minutes, a two-step process of 98°C for 10 seconds and 68°C for 5 minutes was repeated 10 times. Subsequently, a two-step process of 98°C for 10 seconds and 65°C for 5 minutes was repeated 20 times. This PCR was performed using the GeneAmp PCR System 9700 (Life Technologies) and a PCR Thermal Cycler Fast (TaKaRa). After PCR, the amplification status of the PCR product was confirmed by agarose gel electrophoresis. The electrophoresis image is shown in Figure 9.

[Results and Investigation]

Agarose gel electrophoresis of PCR products using two PCR device models revealed no significant difference in band appearance between the two models, and a single PCR product was obtained at the target molecular weight for each of the HLA class I and HLA class II genes (Figure 9).

In the case of GeneAmp PCR System 9700 (Life Technologies), the time required for PCR was 3 hours and 10 minutes, while in the case of PCR Thermal Cycler Fast (TaKaRa), it was 2 hours and 45 minutes.

Therefore, it was confirmed that the method of the present invention can be applied to high-speed PCR devices such as PCR Thermal Cycler Fast (TaKaRa), and contributes to the speeding up of PCR.

(Example 4)

[Purpose]

To confirm the reproducibility of PCR products using Tks Gflex DNA Polymerase (TaKaRa) and GeneAmp PCR System 9700 (Life Technologies) and to demonstrate that the PCR conditions set in the present invention are suitable for ultra-high-resolution DNA typing of the HLA gene, the base sequences of the PCR products were determined using multiple DNA samples.

[method]

1. Using Tks Gflex DNA Polymerase (TaKaRa), PCR was performed using genomic DNA from four samples (Samples 1 to 4) that had been extracted and HLA types confirmed, including allele combinations observed to be phase-ambiguous using conventional DNA typing methods, as templates using primer sets specific for each of the HLA class I and HLA class II genes (see Tables 1 to 3: SEQ ID NOs. 1 to 31). The specific steps are as follows.

(1) Add 10 μL of 2xGflxPCR Buffer, 1-3 μL (4 pmol/μL) of PCR primers, and 0.2 μL of Tks Gflex DNA Polymerase to 50 ng of genomic DNA solution. Adjust the total volume of the reaction solution to 20 μL with sterile water.

(2) After incubating at 94°C for 2 minutes, a two-step process of 98°C for 10 seconds and 68°C for 5 minutes was repeated 10 times. Subsequently, a two-step process of 98°C for 10 seconds and 65°C for 5 minutes was repeated 20 times. The GeneAmp PCR System 9700 (Life Technologies) was used for this PCR. After PCR, the amplification status of the PCR product was confirmed by agarose gel electrophoresis. The electrophoresis image is shown in Figure 10.

2. The base sequence of the PCR product was determined as follows.

(1) Purify the PCR product according to the standard protocol of QIAquick PCR Purification Kit (QIAGEN).

(2) The concentration was determined according to the standard protocol of PicoGreen dsDNA Quantitation Kit (Invitrogen).

(3) The purified PCR product was adjusted to a concentration of 500 ng/100 μL, and a rapid library was prepared according to the standard protocol of Genome Sequencer (GS) Junior (Roche). Emulsion PCR and sequencing were performed to obtain a base sequence of 20,000 reads per sample.

(4) These sequences were compared with all HLA allele data registered in the IMGT HLA database for homology analysis across all reads to select candidate HLA allele sequences.

(5) Using the candidate HLA allele sequence as a reference, GS Reference Mapper (Roche) was used to perform gene mapping under conditions that ensured 100% alignment between the reference and the reads. The gene mapping status was visually confirmed to identify the HLA allele.

[Results and Investigation]

1. Agarose gel electrophoresis of the PCR products revealed single amplified products at the target molecular weight for each of the HLA class I and HLA class II genes (Figure 10). Furthermore, sequencing of the PCR products by the Sanger method revealed HLA alleles consistent with existing reports, confirming the suitability of this PCR system for HLA typing.

2. Using four samples containing allele combinations for which phase ambiguity has been observed using existing DNA typing methods, HLA typing was performed using the Genome Sequencer (GS) Junior (Roche) using PCR products from the 5' untranslated region to exon 2 of HLA-A, HLA-B, HLA-C, HLA-DRB1, exon 2 to 3' untranslated region of HLA-DRB1, HLA-DRB3/4/5, HLA-DQA1, HLA-DQB1, HLA-DPA1, and HLA-DPB1, as well as exon 2 to 3' untranslated region of HLA-DPB1. The results showed that typing across all gene regions was possible for HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DRB3/4/5, and HLA-DQB1. For HLA-DQA1, HLA-DPA1, and HLA-DPB1, typing is only possible for the exon portion. Furthermore, novel alleles were detected in each of the HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 genes (Table 5). Therefore, this method is believed to be capable of performing 8-digit HLA typing without phase ambiguity and is an excellent tool for efficiently detecting base substitutions or insertions/deletions within promoters and introns that can cause null alleles.

(Example 5)

[Purpose]

The feasibility of multiplex PCR for HLA-A, HLA-B, HLA-C, and HLA-DRB1 was verified using Tks Gflex DNA Polymerase (TaKaRa) and GeneAmp PCR System 9700 (Life Technologies).

[method]

1. Using Tks Gflex DNA Polymerase (TaKaRa), genomic DNA from four samples (Samples 1 to 4) with previously extracted, clearly HLA-typed alleles containing allele combinations observed to be phase-ambiguous using conventional DNA typing methods was used as template. This DNA was mixed with gene-specific primer sets for HLA-A, HLA-B, HLA-C, and HLA-DRB1 (see Patent Document 3, Tables 1 and 2: SEQ ID NOs. 1 to 12) in a single tube, and PCR was performed using the mixture. The specific steps are as follows.

(1) Add 10 μL of 2xGflxPCR Buffer, 1-3 μL (4 pmol/μL) of PCR primers, and 0.2 μL of Tks Gflex DNA Polymerase to 50 ng of genomic DNA solution. Adjust the total volume of the reaction solution to 20 μL with sterile water.

(2) After incubating at 94°C for 2 minutes, the reaction was repeated 10 times with two steps of 98°C for 10 seconds and 68°C for 5 minutes. Furthermore, the reaction was repeated 20 times with two steps of 98°C for 10 seconds and 65°C for 5 minutes. A GeneAmp PCR System 9700 (Life Technologies) was used for this PCR. After PCR, the amplification status of the PCR product was confirmed by agarose gel electrophoresis. The electrophoresis image is shown in Figure 11. Furthermore, the length of the PCR product was accurately measured using a bioanalyzer (DNA12000 Chips). The electrophoresis image is shown in Figure 12.

2. The base sequence of the PCR product was determined as follows.

(1) Purify the PCR product according to the standard protocol of QIAquick PCR Purification Kit (QIAGEN).

(2) The concentration was determined according to the standard protocol of PicoGreen dsDNA Quantitation Kit (Invitrogen).

(3) The purified PCR product was adjusted to a concentration of 500 ng/100 μL, and a rapid library was prepared according to the standard protocol of Genome Sequencer (GS) Junior (Roche). Emulsion PCR and sequencing were performed to obtain a base sequence of 20,000 reads per sample.

(4) These sequences were compared with all HLA allele data registered in the IMGT HLA database for homology analysis across all reads to select candidate HLA allele sequences.

(5) Using the candidate HLA allele sequence as a reference, GS Reference Mapper (Roche) was used to perform gene mapping under conditions that ensured 100% alignment between the reference and the reads. The gene mapping status was visually confirmed to identify the HLA allele.

[Results and Investigation]

When PCR was performed independently on each of the HLA-A, HLA-B, HLA-C, and HLA-DRB1 genes, agarose gel electrophoresis of the PCR products revealed a single amplified product at the target molecular weight (lanes 1-4 in Figure 11 ). On the other hand, when these primers were mixed and PCR was performed, multiple bands were observed, presumably representing PCR products from each gene (lane 5 in Figure 11 ). Detailed measurement of the PCR product lengths using a bioanalyzer (Agilent DNA 12000 kit) confirmed amplification of PCR products from each gene (Figure 12 ). Furthermore, HLA typing of the PCR products from each gene using the Genome Sequencer (GS) Junior (Roche) revealed that all samples were consistently assigned to the HLA alleles identified in Example 4. Therefore, the multiplex method of the present invention, using a high-speed PCR device, is considered an excellent tool for simplifying and expediting the often complex PCR procedures used in clinical settings.

(Comparative Example 1)

[Purpose]

It was confirmed that the primer set of the present invention is suitable for multiplex amplification.

[method]

The following experiment was conducted: PCR was performed under the same conditions as in Example 5 using primer sets for each of the HLA-A, HLA-B, HLA-C, and HLA-DRB1 genes disclosed in Patent Document 2.

[Results and Investigation]

After PCR, the amplification status of the PCR product was confirmed by agarose gel electrophoresis. The electrophoresis image is shown in Figure 13 (left). Furthermore, the length of the PCR product was accurately measured using a bioanalyzer (Agilent DNA 12000 kit). The electrophoresis image is shown in Figure 14 (left).

For comparison, the results when the primer set newly set in the present invention was used are shown on the right side of the same figure.

As shown in FIG14 , when the primer set described in the prior art (Patent Document 2) (previously developed primer set) was used, specific HLA-B and HLA-C were not sufficiently amplified, particularly when the HLA-A, HLA-B, HLA-C, and HLA-DRB1 genes were simultaneously amplified in the same vessel. In contrast, when the novel primer set of the present invention (newly developed primer set) was used, all genes were uniformly amplified.

Sequence Listing

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Claims (4)

1. A non-diagnostic method for HLA DNA typing, characterized in that it comprises: (1) The steps of preparing primer sets that specifically hybridize with the upstream and downstream regions of four genes selected from the human genome base sequence HLA-A, HLA-B, HLA-C and HLA-DRB1, and amplify them under the same PCR conditions. The HLA-A gene is amplified using primer sets consisting of oligonucleotides with base sequences shown in sequence number 1 or 2 and oligonucleotide sequences with base sequences shown in sequence number 3. The HLA-B gene is amplified using a primer set consisting of oligonucleotides with the base sequence shown in sequence number 4 and oligonucleotides with the base sequence shown in sequence number 5. The HLA-C gene is amplified using primer sets consisting of oligonucleotides with base sequences shown in sequence number 6 or 7 and oligonucleotide sequences shown in sequence number 8. The HLA-DRB1 gene is amplified using primer sets consisting of oligonucleotides with the base sequence shown in sequence number 9 and oligonucleotides with the base sequence shown in sequence number 10, and/or primer sets consisting of oligonucleotides with the base sequence shown in sequence number 11 and oligonucleotides with the base sequence shown in sequence number 12. (2) The step of simultaneously amplifying the four genes in the genomic DNA of the test sample under the same PCR conditions in the same container using the primer set; (3) Steps for determining the base sequence of PCR products; and (4) Optionally perform the steps of retrieval of the same source as the database. 2. The method of claim 1, wherein the temperature conditions for PCR are as follows: after incubation at 94°C for 2 minutes, the two steps of incubation at 98°C for 10 seconds and at 68°C for 5 minutes are repeated 10 times as one step, and then the two steps of incubation at 98°C for 10 seconds and at 65°C for 5 minutes are repeated 20 times as one step. 3. Primer set for HLA gene DNA typing of genomic DNA, containing the following primer sets for amplifying four HLA genes: The primer set used to amplify the HLA-A gene consists of the following primers: primers consisting of oligonucleotides with the base sequence shown in sequence number 1 or 2, and primers consisting of oligonucleotides with the base sequence shown in sequence number 3. The primer set used to amplify the HLA-B gene consists of the following primers: primers consisting of oligonucleotides with the base sequence shown in sequence number 4, and primers consisting of oligonucleotides with the base sequence shown in sequence number 5. The primer set used to amplify the HLA-C gene consists of the following primers: primers consisting of oligonucleotides based on the base sequence shown in sequence number 6 or 7, and primers consisting of oligonucleotides based on the base sequence shown in sequence number 8, and... The primer set used for amplifying the HLA-DRB1 gene consists of primers comprising the oligonucleotide sequence shown in sequence number 9 and primers comprising the oligonucleotide sequence shown in sequence number 10; and/or the primer set used for amplifying the HLA-DRB1 gene consists of primers comprising the oligonucleotide sequence shown in sequence number 11 and primers comprising the oligonucleotide sequence shown in sequence number 12. 4. A DNA typing kit for HLA genes, comprising the primer set for amplifying four HLA genes as described in claim 3 and one or more DNA polymerases selected from the following: PrimeSTAR GXL DNA Polymerase manufactured by TaKaRa, TaKaRa LA Taq manufactured by TaKaRa, Tks Gflex DNA Polymerase manufactured by TaKaRa, and Long PCR Enzyme Mix manufactured by Thermo Scientific.