JPH06303998A - Typing of hla - Google Patents

Abstract

PURPOSE:To provide an easy typing method for polymorphism of HLA genes on a base sequence level. CONSTITUTION:The typing method consists of (1) denaturation of the DNA fragment containing a HLA gene to make single-stranded DNA, (2) annealing of the single-stranded DNA with typing probes specifically combining with the specific regions of HLA gene to form bound bodies between the objective DNA fragments and the typing probes, (3) contact of the bound bodies with an insoluble supporting body carrying an immobilized ligand specifically combining with the objective DNA fragments and (4) subsequent elution of the typing probes from the supporting body by increasing the temperature gradually and analysis of the elution pattern.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for determining (typing) the HLA genotype of an individual, and more particularly to a typing method for directly examining the polymorphism of the HLA gene at the nucleotide sequence level. The HLA typing method of the present invention is useful in molecular biology, genetic engineering, and related industrial fields.

[0002]

2. Description of the Related Art Major Histocompatibility Complex (MHC)
HLA (Human Leukocyte), which is located on chromosome 17 in mouse and is located on the short arm of chromosome 6 in human.
e Antigen) gene complex corresponds to this.
Human histocompatibility is determined by the HLA gene, but the HLA gene and HLA antigen show a high degree of polymorphism.

The HLA antigen is deeply involved in biological defense mechanisms such as immune response and disease susceptibility. The detection of genetic mutations in the HLA region is carried out by selecting a donor (donor) and a recipient (recipient) pair in which the HLA gene is matched at the time of transplantation, and the tissue of Useful for typing. The closer the donor and recipient are genetically, the greater the success rate of the transplant. Also, HL
A typing provides important information in understanding genetic susceptibility to autoimmune diseases. Furthermore, since the HLA loci show a high degree of polymorphism, HLA typing is
It can be applied to personal appraisal, paternity appraisal, etc. in forensic medicine.

Conventionally, HLA typing has been carried out by a method of typing an HLA antigen by a microcytotoxic test for lymphocytes (serological method), lymphocyte mixed culture (cytological method), or the like. On the other hand, in recent years, H
DN for directly examining the polymorphism of LA gene at the nucleotide sequence level
Many proposals have been made regarding the A typing method.
These DNA typing methods are sensitive and more accurate than the immunological typing methods.

The HLA typing method at the DNA level is, for example, PCR-RFLP (Restricti).
on Fragment Length Polymo
rphism) method, PCR-SSO (Sequence)
Specific Oligonucleotide
e) method is known as a typical one (PCR
Experimental Manual, p. 227-235; Experimental Medicine, Vo
l. 8, p. 1094-1099).

The PCR-RFLP method is based on PCR (Poly
The polymorphic region of the HLA antigen gene is selectively amplified by merase chain reaction, and an allele (allele) is added to the amplified DNA.
le) A method of detecting whether or not it is cleaved by electrophoresis using a restriction enzyme that recognizes and cleaves a specific base sequence. However, this method has drawbacks such as the need to clarify the nucleotide sequence of the gene region to be typed and the need for various kinds of restriction enzymes.

The PCR-SSO method is a method for determining whether or not DNA amplified by PCR hybridizes with an alloantigen-specific oligonucleotide probe. In the dot blot method in the PCR-SSO method,
After the target HLA gene is amplified by PCR, it is immobilized on the membrane by dot blot, and each membrane is hybridized with a labeled probe,
The bound probe is detected by enzyme reaction or autoradiography. In the reverse dot blot method in the PCR-SSO method, an oligonucleotide probe is immobilized on a membrane, a labeled PCR product is hybridized, and a specifically bound PCR product is detected by a color reaction by an enzyme or the like ( Proc. Nat
l. Acad. Sci. USA, Vol. 86, p. 6
230-6234, 1989).

[0008] However, in the conventional PCR-SSO method, a signal is obtained only when the probe and the target DNA fragment are bound to each other, and therefore, the amount of information obtained from one probe is small. Therefore, as in HLA typing. To analyze the polymorphism of complex DNA, it is necessary to use a large number of probes.

[0009]

SUMMARY OF THE INVENTION The object of the present invention is to obtain HL
It is intended to provide a method for easily typing the polymorphism of the A gene at the nucleotide sequence level. The present inventor has focused on the strength of interaction in complementary DNA-DNA hybridization. The melting temperature (Tm) of a DNA-DNA hybrid depends on stability, and when the base sequences of DNAs are completely complementary, they form a hybrid up to a high temperature, but at one or more sites. When there is a mismatch (inappropriate base pair), the stability is remarkably reduced and dissociation occurs at a low temperature. The melting temperature also differs depending on the type of mismatch.

Therefore, an oligonucleotide probe having a sequence complementary to the DNA region to be analyzed (probe DN
A) is prepared, and the target DNA and probe D in the sample DNA are prepared.
After forming a hybrid with NA, the probe DNA
When the melting temperature of the target DNA is analyzed, information on the region of the target DNA contained in the sample DNA is obtained, and the base sequence in the region can be estimated from this.

In order to capture the target DNA fragment, D having a base sequence complementary to the target DNA fragment is used.
By using an insoluble support on which NA is immobilized as a ligand, a hybrid product of the probe DNA and the target DNA is immobilized on the support, and then the probe D
The melting of NA and the target DNA can be detected by dissociation of the probe DNA from this support.

By dissociating the probe DNA by columnizing the insoluble support and gradually increasing the temperature while precisely controlling the column temperature, the dissociation temperature of the probe DNA can be accurately measured and analyzed. With HLA typing is possible. The present invention has been completed based on these findings.

[0013]

Thus, according to the present invention, (1) a DNA fragment containing an HLA gene is denatured into a single strand, and (2) a specific region of the HLA gene is specifically An insoluble support on which a ligand that specifically binds to the target DNA fragment is immobilized by annealing with a binding probe that binds to form a bond between the target DNA fragment and the typing probe (3) The binding probe of the target DNA fragment and the typing probe is bound to the support, and the temperature is gradually raised to elute the typing probe from the support, and the elution is performed. An HLA typing method is provided which is characterized by analyzing patterns.

The present invention will be described in detail below. The insoluble support used in the present invention is usually in the form of a film or a particle, for example, an inorganic polymer such as silica gel, porous glass or graphite; an organic polymer such as nylon, nitrocellulose or polytetrafluoroethylene; aluminum,
Examples thereof include metal particles such as apatite; ceramic particles such as alumina. In addition, these surfaces may be chemically or physically modified. When the support is in the form of particles, the size of the particles is usually 0.1 to 5
00 μm, preferably 1 to 10 μm, and the pore size of the particles is usually 100 to 4000 Å, preferably 1000.
It is about 4000Å.

The ligand immobilized on the insoluble support is a synthetic oligonucleotide (synthetic DNA). The length of the ligand is preferably longer than that of the typing probe, usually 20 to 100 bases (base), preferably 3
It is 0 to 60 base. The base sequence of the synthetic DNA serving as the ligand is made to be complementary to the base sequence in the target DNA fragment.

The method of binding the ligand and the support is carried out by using various functional groups (carboxyl group, amino group,
(Hydroxyl group, etc.) as it is, or after being treated with a condensing agent or an activator (tresyl chloride, water-soluble carbodiimide, etc.), it is reacted with a synthetic DNA having a terminal functional group such as an amino group or a thiol group.

The typing probe is a synthetic oligonucleotide (synthetic DNA) and its length is usually 15
˜50 bases, preferably 20˜30 bases. One type of probe may be used, but usually several types of regions within the HLA gene are selected and multiple types of probes are selected so that typing can be performed efficiently according to the polymorphism of the target HLA gene. design.

The typing probe may be bound with a labeling substance in order to increase the detection sensitivity. As the substance to be labeled, a fluorescent dye, a hapten such as biotin, an enzyme such as alkaline phosphatase and the like are used. Fluorescent dyes include fluorescein, rhodamine, and Europi
um or the like is used. At this time, if several dyes having different fluorescence wavelengths such as fluorescein and rhodamine are used, two or more types of typing probes can be detected at the same time, and rapid analysis is possible.

As a method for binding the labeling substance to the synthetic DNA, the functional groups (isothiocyanate group, carboxyl group, amino group, etc.) in the labeling substance can be used as they are, or after activation, functional groups such as amino group, thiol group, etc. A method of reacting with a synthetic DNA having a group is used. The labeling substance is usually
It is attached to the end of synthetic DNA.

In the method of the present invention, it is preferable to use the technique of affinity chromatography in which an insoluble support on which a ligand that specifically binds to a target DNA fragment is immobilized is used as a stationary phase. It is also preferable to use high performance liquid chromatography (HPLC) for rapid processing.

The chromatographic tube of the column is usually 100 kg.
There is no particular limitation as long as it can withstand a hydrostatic pressure of / cm ² and is inert to the solution. For example, metals such as stainless steel, plastics such as high-density polyethylene and polystyrene, and inorganic substances such as glass can be used. The inner diameter of the chromatographic tube is usually 1 to 20 mm, preferably 1 to 5 mm. The length of the chromatographic tube is usually 1-3.
It is 0 cm, preferably 1 to 10 cm.

The flow rate at the time of liquid transfer to the column is usually 0.0
It is 1 to 1 ml / min, preferably 0.05 to 0.5 ml / min. The solution sent to the column is not particularly limited as long as it does not attack the support or the chromatographic tube. For example, N
An aqueous solution containing a solute such as aCl or EDTA, which may be mixed with an organic solvent such as methanol, ethanol, or acetonitrile.

The length of the target DNA fragment is usually 50.
base to 10 kb, preferably 100 to 2 kb
Asase, those prepared by PCR, those digested with a restriction enzyme or by a physical method, those incorporated into a vector, etc. are used. The DNA fragment may be single-stranded or double-stranded.

The temperature of the column is controlled by using a constant temperature bath, a column heater or the like, but a constant temperature water bath is particularly preferable for accurately controlling the temperature in the column. If the temperature rise rate is too fast, it will be difficult to identify the presence or absence of mutation and the type, but if it is too slow, there will be a problem that the analysis time will be long. Than this,
It is usually 0.1 ° C./minute to 3 ° C./minute, preferably about 0.5 ° C./minute to 1.0 ° C./minute.

The binding between the typing probe and the target DNA fragment is carried out by denaturing the target DNA fragment by heat or alkali treatment and then mixing the typing probe.

The typing probe and the purpose D
When the bound NA fragment is placed in a column kept at a constant temperature, it is bound to the insoluble support in the column.

In the method of the present invention, (1) a DNA fragment containing the HLA gene is denatured to form a single strand, and then (2) H
It is annealed with a typing probe that specifically binds to a specific region of the LA gene to form a conjugate of the target DNA fragment and the typing probe. If you select multiple specific regions of the HLA gene and create multiple types of typing probes that specifically bind to these regions, you can accurately type many types of HLA genes with several types of typing probes by the following operations. can do.

Thereafter, the sample DNA subjected to the above treatment
(3) is contacted with an insoluble support on which a ligand that specifically binds to the target DNA fragment is immobilized,
A conjugate of the NA fragment and the typing probe is bound to the support. The insoluble support on which the ligand is immobilized is
It is preferred to pack in a column and operate with a liquid chromatography device. Then, (4) the temperature is gradually raised to elute the typing probe from the support. In this case, if the specific region of the target DNA fragment and the typing probe are completely complementary, the elution temperature will be high, and if there is a mismatch, the elution temperature will be low, and the temperature will differ depending on the number and type of mismatches. To do. Therefore, by analyzing the elution pattern such as the elution temperature of the typing probe, the HLA gene exhibiting a high degree of polymorphism can be easily typed. Objective DN
As the sample DNA fragment containing the A fragment, it is desirable to use the one amplified by the PCR method.

[0029]

EXAMPLES The present invention will be described more specifically with reference to the following examples.

[Example 1] (1) Design of probe for HLA-DQA typing The HLA-DQA gene is divided into 1 to 4 types as shown in Table 1, and type 1 is 1.1 or 1. .2,
And 1.3 subtypes.

[0031]

[Table 1] Three types of probes were designed as shown in Table 2 so that all the combinations of the six types of DQA-type homozygotes and heterozygotes (21 patterns in total) can be distinguished.

[0032]

[Table 2]

DNAs of these sequences (PR-1, PR-
2 and PR-3) were synthesized by a DNA synthesizer, and FITC (fluorescein isothiocyanate) was labeled by a conventional method. These typing probes are designed to specifically bind to specific regions of the HLA genes (subtype 1.1 and type 3) shown in Table 1 (Table 1).
(Regions corresponding to the symbols PR-1, PR-2, and PR-3 shown in the figure).

(2) Preparation of ligand-immobilized column From the HLA-DQA gene, 45ba common to each type
se was selected and a DNA synthesizer was used to synthesize a DNA having the nucleotide sequence shown in Table 3. This synthetic DNA is the target D
It is a ligand that specifically binds to the NA fragment.

[0035]

[Table 3]

6 mg of this DNA is used as an equal amount of complementary strand DNA
After annealing with (protecting DNA) in 300 μl of 1 M NaCl, 0.04 M NaHCO ₃ (pH 7.
5) Add 300 μl and add 300 mg of tresyl chloride activated silica gel (Nucleosil 1000-
OH, particle size 7μm, pore size 1000Å, made by Nagel)
And reacted for 24 hours. After the reaction is completed, excess DNA is removed, and then washed in 2.4M TEACl at 65 ° C to dissociate the DNA double strands into single strands.
An immobilized support was prepared. (Fixed amount: 2.0 mg / g
dry gel) This support is used as a packing buffer (0.5M N
aCl, 10 mM phosphoric acid, 1 mM EDTA, pH 7.
0) suspended in a packer, buff for packing
er was sent to fill the column (φ2.1 m × 10 m) with the support.

(3) Preparation of target DNA fragment by PCR HLA- was prepared by PCR using the primers shown in Table 4.
242 bp of the DQA region was amplified.

[0038]

[Table 4]

(4) Analysis of DNA fragment of interest by column The ligand-immobilized column prepared in (2) above was connected to a high performance liquid chromatography device (manufactured by Waters). (Solution: 40%, EtOH, 10 mM phosphoric acid b
buffer, 1 mM EDTA, pH 6.7, flow rate 0.
1 ml / min) 10 μl of the PCR product obtained in (3) above was heat-denatured, and then a typing probe was mixed and annealed.
Injection was performed on a column kept at 30 ° C. 10
After washing the inside of the column by flowing a solution for a minute, the column temperature is raised (30 ° C. to 60 ° C., heating rate 0.5 ° C./min)
The eluted typing probe was used as a fluorescence detector (FS-80
10, manufactured by Tosoh).

(5) HLA typing by analysis of analysis results Each typing probe and DN of each type of HLA-DQA
The A sequences correspond as shown in Tables 5-7.

[0041]

[Table 5]

[0042]

[Table 6]

[0043]

[Table 7] The elution patterns A, B, and C in these tables show that the elution temperatures are relatively high (A), medium (B), and low (C).
It corresponds to each.

As shown in Table 8, by analyzing this elution pattern, 21 types of HLA-DQA can be distinguished by using these three probes.

[0045]

[Table 8]

Table 9 shows the measurement results of the elution time and elution temperature of each sample. Experimental conditions Eluent: 40% ethanol, 1 mM EDTA, 10 m
M Phosphate buffer (pH 7.0) Flow rate: 0.1 ml / min Column temperature: 30 ° C.-60 ° C. (0.5 ° C./min) 10 minutes after the sample was added, the temperature increased.

[0047]

[Table 9] (Footnote) Elution temperature = (elution time−10) / 2 + 30 ° C. Table 10 shows the elution pattern and average elution time for each probe.

[0048]

[Table 10]

[0049]

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to rapidly perform HLA typing by using a relatively small number of typing probes, and to examine compatibility in organ transplantation and bone marrow transplantation, and in forensic medicine. It can be used in fields such as personal assessment, paternity assessment, diagnosis of disease susceptibility, and anthropology.

Claims (1)

[Claims] 1. A DNA fragment of interest comprising: (1) denaturing a DNA fragment containing an HLA gene to form a single strand, and (2) annealing it with a typing probe that specifically binds to a specific region of the HLA gene. Form a conjugate between the fragment and the typing probe, (3) and then target DN
A ligand that specifically binds to the A fragment is brought into contact with an immobilized insoluble support to allow the conjugate of the target DNA fragment and the typing probe to bind to the support, (4) and then the temperature is gradually increased. By so doing, the typing probe is eluted from the support, and the elution pattern is analyzed, which is an HLA typing method.