A Sequence Listing has been submitted as a ASCII text file, which is incorporated by reference herein. The ASCII text file is named “Sequence_Listing.txt”, was created on Sep. 25, 2009, and has a size of 1,547,033 bytes.
The present invention relates to a probe set and a method for identifying an allele of human HLA.
Human leukocyte antigen (HLA) is known to include multiple HLA types, such as HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, HLA-DR, and HLA-MICA. An HLA allele is designated with a four or more digit number by the WHO HLA Nomenclature Committee. The principle of the nomenclature is that the first two digits correspond to the serotypes; the third and fourth digits distinguish the alleles of different amino acid sequences (subtypes); and the fifth digit distinguishes the alleles of different base sequences but encoding the same amino acid sequence. Typing of these alleles has been conventionally conducted at the serological level. Although this serological method does not require special sample processing, and enables easy typing using antigen-antibody reaction, the serotypes are the roughest classification corresponding to the first two digits of the numbers according to the nomenclature described above.
Many of other commercially available kits of the type associated with genomic extraction do not have enough accuracy to identify each allele individually. It is the current state that such a kit distinguishes multiple alleles as a group. Moreover, even a kit based on the SBT (Sequencing Based Typing) method, which enables the most detailed polymorphic analysis, often fails to solve the problem of ambiguity by one analysis since most samples are heterozygotes requiring reexamination. Such problematic alleles are listed collectively in http://www.ihwg.org/protocols/sbt/ambiguities2.pdf by the International Histocompatibility Working Group (IHWG).
On the other hand, with the development of advanced medical treatment in recent years, detailed HLA typing is required in organ transplantation, etc. In addition, associations of HLA with diabetes, cancer, and other multifactorial diseases have been suggested. With such a background, a test method is desired that can identify each allele individually. Upon such demands it is an object of the present invention to provide a probe set that is useful for identifying each allele of HLA individually, and a method for identification of an HLA allele by the use thereof.
A probe set for identifying an allele of HLA according to the present invention is a probe set comprising multiple probes that can be used for identifying HLA allele contained in a specimen, characterized in that each of the multiple probes comprises a partial sequence containing a base represented by a capital letter in a sequence of each allele in an allele list in the description.
An embodiment of the present invention is a probe set comprising multiple probes that can be used for identification of an HLA-A allele in a specimen, characterized in that each of the multiple probes comprises a partial sequence containing a base represented by a capital letter in a sequence of each allele in an allele list in the description.
Another embodiment of the present invention is a method for identification of an HLA-A allele contained in a specimen using a probe set, characterized in that the probe set is the probe set described above.
Another embodiment of the present invention is a probe set comprising multiple probes that can be used for identification of an HLA-B allele in a specimen, characterized in that each of the multiple probes comprises a partial sequence containing a base represented by a capital letter in a sequence of each allele in an allele list in the description.
Another embodiment of the present invention is a method for identification of an HLA-B allele contained in a specimen using a probe set, characterized in that the probe set is the probe set described above.
Another embodiment of the present invention is a probe set comprising multiple probes that can be used for identification of an HLA-C allele in a specimen, characterized in that each of the multiple probes comprises a partial sequence containing a base represented by a capital letter in a sequence of each allele in an allele list in the description.
Another embodiment of the present invention is a method for identification of an HLA-C allele contained in a specimen using a probe set, characterized in that the probe set is the probe set described above.
Another embodiment of the present invention is a probe set comprising multiple probes that can be used for identification of an HLA-DP allele in a specimen, characterized in that each of the multiple probes comprises a partial sequence including a base represented by a capital letter in the sequence of each allele in the allele list in the description.
Another embodiment of the present invention is a method for identification of an HLA-DP allele contained in a specimen using a probe set, characterized in that the probe set is the probe set described above. Another embodiment according to the present invention is a probe set for identification of an HLA-DQ allele that is a probe set comprising multiple probes that can be used for identification of an HLA-DQ allele in a specimen, characterized in that each of the multiple probes comprises a partial sequence containing a base represented by a capital letter in a sequence of each allele in an allele list in the description.
Another embodiment of the present invention is a probe set comprising multiple probes that can be used for identification of an HLA-DQ allele in a specimen, characterized in that each of the multiple probes comprises a partial sequence containing a base represented by a capital letter in a sequence of each allele in an allele list in the description.
Another embodiment of the present invention is a method for identification of an HLA-DQ allele contained in a specimen using a probe set, characterized in that the probe set is the probe set described above.
Another embodiment of the present invention is a probe set comprising multiple probes that can be used for identification of an HLA-DR allele in a specimen, characterized in that each of the multiple probes comprises a partial sequence containing a base represented by a capital letter in a sequence of each allele in an allele list in the description.
Another embodiment of the present invention is a method for identification of an HLA-DR allele contained in a specimen using a probe set, characterized in that the probe set is the probe set described above.
Another embodiment of the present invention is a probe set comprising multiple probes that can be used for identification of an HLA-MICA allele in a specimen, characterized in that each of the multiple probes comprises a partial sequence containing a base represented by a capital letter in a sequence of each allele in an allele list in the description.
Another embodiment of the present invention is a method for identification of an HLA-MICA allele contained in a specimen using a probe set, characterized in that the probe set is the probe set described above.
The probe set according to the present invention, and identification of an allele of each HLA type by the use thereof can contribute to diathesis diagnoses and tailor-made medicines, which are required in organ transplantation, cancer, diabetes, and other multifactorial diseases.
Other features and advantages of the present invention will be apparent from the following description.
Preferred embodiments of the present invention will now be described in detail. Each probe that constitutes the probe set of the present invention has a partial sequence including a base represented by a capital letter in each allele sequence in the allele lists described later. Preferably, segments consisting of 10 to 30 bases including a base represented by a capital letter are selected from each allele sequence, and the probe set is composed of probes having the obtained partial base sequences respectively. As specific examples, the following compositions can be employed:
1) A probe set for HLA-A allele identification consisting of respective probes listed in one of the probe list A1 shown in Tables 1-1 to 1-7 and the probe list A2 shown in Tables 2-1 to 2-6 shown later;
2) A probe set for HLA-B allele identification consisting of probes listed in one of the probe list B1 shown in Tables 5-1 to 5-9 and the probe list B2 shown in Tables 6-1 to 6-8 shown later;
3) A probe set for HLA-C allele identification consisting of probes listed in one of the probe list C1 shown in Tables 9 and the probe list C2 shown in Table 10 shown later;
4) A probe set for HLA-DP allele identification consisting of probes listed in one of the probe lists DP1-DP4 shown in Tables 13-1 to 16-5 respectively as shown later;
5) A probe set for HLA-DQ allele identification consisting of probes listed in one of the probe lists DQ1 to DQ 4 shown in Tables 17A, 17B-1, 17B-2, 18A, 18B-1 and 18B-2 respectively as shown later;
6) A probe set for HLA-DR allele identification consisting of probes listed in one of the probe lists DR1 and DR2 shown in Tables 21-1 to 21-8 and Tables 22-1 to 22-7 respectively, as shown later; and
7) A probe set for HLA-MICA allele identification consisting of probes listed in one of the probe lists MICA1 and MICA2 shown in Tables 25-,1, 25-2 and Tables 26-1 to 26-2 respectively, as shown later.
For example, the No. 0 probe in the probe list A1 has a 16-base sequence of “gccccgcttcatcgcC”, which is a segment containing the first capital lettered base C in A*010101, and the No. 0 probe in the probe list 2 has an 18-base sequence of “cttcatcgcCgtgggcta”, which is a segment also containing the first capital lettered base C in the same allele.
In the allele list, each allele is assigned with a unique number such as “A*xxxx” in accordance with “allele nomenclature” by Japanese Society for Histocompatibility and Immunogenetics, HLA Standardization Committee.
To identify an allele using a probe set according to the present invention, two methods are possible: one is detection by hybridization; and the other is direct detection by PCR without hybridization. In either method, each probe is an oligonucleotide of preferably more than 10 and less than 30 nucleotides in length and designed to include the base represented by a capital letter, i.e., a base specific for the allele to be identified.
Moreover, the probe arrays provided in the present invention present groups of varied bases for identification of each allele individually by positions chosen for the probes. As a method for detection of such a varied base, the detection method by hybridization, and the method of direct detection by PCR without hybridization can also be preferably used. Also in these cases, the probes are designed as oligonucleotides of preferably more than 10 and less than 30 nucleotides in length each containing a base represented by a capital letter.
When a variation is detected by hybridization, probes are preferably designed to have a variant base represented by a capital letter near the center of the probes, which makes Tm difference between full-matched and mismatched pairs larger, enabling easier separation of them by adjusting the reaction temperature of hybridization.
On the other hand, when the variation is directly detected by PCR, the variant base is rather placed near the 3′ end so that enzymatic recognition and elongation of annealed double strands will not occur. Also, some variation methods are possible, such as a method placing a variant base at the second from the 3′ end an artificial variant base at the third from the 3′ end as with Allele Specific Primer (Toyobo Co., Ltd.); a method circularizing probes by ligation with a mismatch placed near the 3′ end (Amersham Biosciences Co., Ltd.); TaqMan-MGB (ABI Co.); and 3′-end mismatch using LNA (Proligo Japan Co., Ltd.).
For example, a segment including the fourth capital letter of A*2302 is “ggagcagTggagagC”, and the corresponding segment of A*2303 of the same serotype is “ggagcagtTgagagc”, differing at the ninth base. By using a probe with a sequence of one of these segments, one can be distinguished from the other by mismatching.
The present invention will be described further by way of examples in the following.
Extraction of DNA from 1 ml of human blood was performed using GFX Genomic Blood DNA Purification Kit from Amersham Biosciences. The protocol is as follows:
Blood 1 ml→
Add RBC Lysis Solution [hemolysate]→
Mix gently at room temperature for 5 minutes→
Centrifuge at 12,000-16,000×g for 20 seconds→
Discard the supernatant leaving 20-50 μl→
Resuspend the precipitation→
Add Extraction Solution and vortex vigorously→
Stand at room temperature for 5 minutes [extraction of DNA]→
Heat the elusion buffer to 70° C.→
Add the sample→
Centrifuge at 5,000×g for 1 minute (binding of DNA)→
Add Extraction Solution (washing)→
Centrifuge at 5,000×g for 1 minute→
Add Washing Solution (washing)→
Centrifuge at 12,000×g for 3 minutes→
Set a GFX Column in a centrifugal tube→
Eluate with pure water→
Stand at room temperature for 1 minute→
Centrifuge at 5,000-8,000×g for 1 minute→
Concentrate to 230 μl . . . solution (1).
Next, quantitative PCR was carried out using QuantiTect SYBR Green PCR Kit from QIAGEN and GeneAmp5700 from ABI. The reaction composition and the protocol are shown below.
QuantiTect SYBR Green 2×premix: 10 μl
Solution (1): 1 μl
Solution of one of the probes in the probe list
A1 (10 μmol/μl): 1 μl
Mixed primers (10 pmol/1 μl)*: 3 μl
Ultra pure water: 5 μl
(Total: 20 μl)
*consisting of 1 μl each of the solutions respectively containing probes of the following sequences at 10 pmol/μl:
94° C.: 180 sec followed by 30 cycles of [94° C.: 10 sec→66° C.: 10 sec→72° C.: 20 sec.].
Referring to Amp Plot and Dissociation curves on a display of 5700 software, and to the allele-probe correspondence list A1 (Tables 3-1 to 3-9), it was identified as A*2402101.
Extraction of DNA from 1 ml of human blood was performed in the same manner as in Example 1. PCR of human HLA-A was then performed using ABI 9700 PCR Instrument and Ex Taq from Takara Bio Inc. The reaction composition and the protocol are as follows:
Ex Taq 2×premix: 20 μl
Solution (1): 3 μl
Cy-3 dUTP (1 mM): 2 μl
Mix primer (10 pmol/μl)*: 3 μl
Ultra pure water: 12 μl
(Total: 40 μl)
*consisting 1 μl each of the solutions respectively containing probes of the following sequences at 10 pmol/μl:
94° C.: 180 sec followed by 30 cycles of [94° C.: 10 sec→66° C.: 10 sec→72° C.: 20 sec]
After the completion of the reaction, unreacted dNTPs, etc., were removed using a purification column (QIAGEN QIAquick PCR Purification Kit) to obtain a sample.
At the same time, a DNA microarray was prepared to identify the allele in the specimen described above. The method for the preparation was in accordance with examples in Japanese Patent Application Laid-Open No. H11-187900. SH group was used as the functional group for immobilization. A glass substrate was treated by a silane-coupling agent to bind the SH group of the probes via a divalent reagent EMCS (N-(6-maleimidocaproyloxy) succinimide). Each probe in the probe list A2 was used for each dot.
The DNA microarray was blocked in advance with PBS supplemented with 1 wt % of BSA (bovine serum albumin) for two hours. The sample was adjusted to have a salt concentration equal to that of the PBS, and to contain 0.1 wt % of SDS (sodium dodecyl sulfate) and 25% of formamide.
Then, hybridization was performed using the above sample (PCR product) and the prepared DNA microarray. 50 μl of the sample was reacted with the blocked DNA microarray at 60° C. for 2 hours. Unreacted substances were washed off by washing three times with 2×SSC solution (NaCl 300 mM, Sodium Citrate (trisodium citrate dihydrate, C6H5Na3.2H2O) 30 mM, pH 7.0), followed by washing twice with 0.1×SSC solution. The DNA microarray was air-dried and the fluorometry measurement was conducted using GenePix4000B made by Axon. Referring to the allele-probe list A2 (Tables 4-1 to 4-9), the sample was identified as A*2402101.
The probe list A1 is shown in Tables 1-1 to 1-7 and the probe list A2 is shown in Tables 2-1 to 2-6. The allele-probe lists are shown in Tables 3-1 to 3-9 and Tables 4-1 to 4-9.
Extraction of DNA from 1 ml of human blood was performed using GFX Genomic Blood DNA Purification Kit from Amersham Biosciences in the same manner as in Example 1.
Next, quantitative PCR was carried out in the same manner as in Example 1 except that probes in the probe list B1 were used and 3 μl of the mixed primers consisting of 1 μl each of respective solutions of the following primers (10 pmol/μl):
After PCR amplification, the sample was identified being B*520101, referring to Amp Plot and Dissociation curves on a display of 5700 software and the allele-probe list B1 (described later).
Extraction of DNA from 1 ml of human blood was performed in the same way as in Example 1. PCR of human HLA-B was then performed in the same manner as in Example 2 except that 2 μl of the mixed primer consisting of 1 μl each of the respective solutions of the following primers at 10 pmol/μl and 13 μl of ultra pure water used:
At the same time, a DNA microarray was prepared to identify the allele in the specimen described above in the same manner as in Example 2, except that probes in the probe list B2 were to form the probe dots respectively.
Then, hybridization was performed using the above specimen and the prepared DNA microarray in the same manner as in Example 2. Fluorometry measurement was conducted with GenePix4000B (Axon). Referring to the allele-probe list B2 (described later), the sample was identified as B*520101.
The following Tables 5-1 to 5-9 show Probe list B1, and Tables 6-1 to 6-8 show Probe list B2. The Allele-probe list is shown in Tables 7 and 8.
Extraction of DNA from 1 ml of human blood was performed using GFX Genomic Blood DNA Purification Kit from Amersham Biosciences in the same manner as in Example 1.
Next, quantitative PCR was carried out in the same manner as in Example 1 except that probes in the probe list in Tables 9-1 to 9-4 were used respectively, and 3 μl of the mixed primers consisting of 1 μl each of the respective solutions of the following primers (10 pmol/μl) was used:
After PCR amplification, the sample was identified being Cw*120202, referring to Amp Plot and Dissociation curves on a display of 5700 software and the allele-probe list in Tables 11-1 to 11-4.
Extraction of DNA from 1 ml of human blood was performed in the same manner as in Example 1. PCR of human HLA-C was then performed in the same manner as in Example 2 except that 6 μl of the mixed primer consisting of 1 μl each of the solutions containing the following sequences at 10 pmol/μl respectively and 9 μl of ultra pure water was used.
At the same time, a DNA microarray was prepared to identify the allele in the specimen in the same manner as in Example 2. Probes in Tables 10-1 to 10-4 were used for the probe spots respectively.
Then, hybridization and fluorescence determination was performed using the above-prepared sample and the DNA microarray in the same manner as in Example 2 and the sample was identified as Cw*120202 referring to the probe-allele list in Tables 12-1 to 12-4.
In the following, Probe Lists C1 and C2 are shown In Tables 9-1 to 9-4 and Tables 10-1 to 10-4 respectively.
Extraction of DNA from 1 ml of human blood was performed using GFX Genomic Blood DNA Purification Kit from Amersham Biosciences in the same manner as in Example 1.
Next, quantitative PCR was carried out in the same manner as in Example 1 except that probes in the probe list 1 in Tables 13-1 to 13-3 or 14-1 to 14-3 were used and 3 μl of the mixed primers contains 1 μl of respective solutions of the following primers (10 pmol/μl):
After PCR amplification, referring to Amp Plot and Dissociation curves on a display of 5700 software, and to the list in Table 15-1 for the probes in Table 13-1, or to the list in Tables 15-2 to 15-5 for the probes in Tables 13-2 to 13-3, it was identified as DPA1*010301 and DPB1*0901.
Extraction of DNA from 1 ml of human blood was performed in the same way as in Example 1. PCR of human HLA-DP was then performed in the same manner as in Example 2 except that 6 μl of the mixed primer consisting of 1 μl each of the solutions containing the following sequences at 10 pmol/μl respectively and 9 μl of ultra pure water.
At the same time, a DNA microarray was prepared to identify the allele in the specimen described above in the same manner as in Example 2, except that probes in Tables 14-1 and 14-2 were used to form the probe spots respectively.
Then, hybridization was performed using the above specimen and the prepared DNA microarray in the same manner as in Example 2. Fluorometry measurement was conducted with GenePix4000B (Axon). Referring to the list in Table 16-1 when the probes in Table 14-1 were used, or to the list in Tables 16-2 to 16-5 when the probes in Table 14-2 were used, the sample was identified as DPA1*010301 and DPB1*0901.
tcaggccaccaac;
tcaggccaccaac;
caggccaccaac;
tcaggccaccaac;
tcaggccgccaaT;
tcaggccgccaaT;
tcaggccgccaaT;
tcaggccgccaaT;
tcaggccgccaaT;
caaT;
tcaggccgccaaT;
aggccgccaaT;
tcaggccgccaaT;
tcaggccaccaac;
acgagctggacgaggccgtgaccctgcagcgccgagtcc;
gagctggacgaggccgtgaccctgcagcgccga;
acgagctggGcgggcccatgaccctgcagcgccgagtcc;
acgagctgggcgggcccatgaccctgcagcgccgag;
acgagctgggcgggcccatgaccctgcagcgccga;
acgagctgggcgggcccatgaccctgcagcgccgag;
acgagctgggcgggcccatgaccctgcagcgccgag;
acgagctgggcgggcccAtgaccctgcagcgccgag;
acgagctggacgaggccgtgaccctgcagcgccgagtcc;
acgagctggacgaggccgtgaccctAcagcgccgag;
acgagctggGcgggcccatgaccctgcagcgccgagtcc;
acgagctggGcgggcccatgaccctgcagcgccgagtcc;
acgagctggacgaggccgtgaccctgcagcgccgag;
acgagctggacgaggccgtgaccctgcag;
gacgaggccgtgaccctgcag;
acgagctggacgaggccgtgaccctgcagcgccgag;
acgagctggacgaggccgtgaccctgcagcgccgag;
gacgaggccgtgaccctgcag;
acgagctggacgaggccgtgaccctgcag;
acgagctggacgaggccgtgaccctgcag;
acgagctggacgaggccgtgaccctgcag;
acgagctggtcgggcccAtgaccctgcagcgccgag;
acgagctggacgaggccgtgaccctgcagcgccgag;
acgagctggacgaggccgtgaccctgcagcgccgag;
gTcgggcccatgaccctgcag;
acgagctggacgaggccgtgaccctgcagcgccgag;
acgagctggacgaggccgtgaccctgcagcgccgag;
acgagctggacgaggccgtgaccctgcagcgTcga;
acgagctggacgaggccgtgaccctgcagcgccgag;
acgagctggacgaggccgtgaccctgcagcgccgag;
acgagctggGcgggcccatgaccctgcagcgccgag;
acgagctgggcgggcccAtgaccctgcagcgccgag;
acgagctggacgaggccgtgaccctgcagcgccgag;
gacgaggccgtgaccctgcagcgccgag;
gacgaggccgtgaccctgcagcgccga;
acgagctggacgaggccgtgaccctgcagcgccgag;
acgagctggTcgggcccatgaccctgcagcgccgag;
acgagctggacgaggccgtgaccctgcagcgccgag;
acgagctggacgaggccgtgaccctgcag;
acgagctggacgaggccgtgaccctgcagcgccgag;
acgagctgggcgggcccatgaccctgcagcgccgag;
acgagctggGcgggcccatgaccctgcag;
acgagctggtcgggcccAtgaccctgcag;
acgagctggacgaggccgtgaccctgcag;
acgagctggacgaggccgtgaccctgcagcgccgag;
gacgaggccgtgaccctgcagcgccgag;
gacgaggccgtgaccctgcag;
acgagctggGcgggcccatgaccctgcagcgccga;
acgagctggTcgggcccatgaccctgcagcgccga;
gagctgggcgggcccatgaccctgcagcgccga;
acgagctggacgaggccgtgaccctgcagcgccgag;
gacgaggccgtgaccctgcag;
acgagctgggcgggcccAtgaccctgcagcgccgag;
acgagctgggcgggcccAtgaccctgcagcgccgag;
gagctggGcgggcccAtgaccctgcag;
gagctggGcgggcccatgaccctgcag;
gagctggacgaggccgtgaccctgcag;
acgagctggGcgggcccatgaccctgcagcgccgag;
acgagctggacgaggccgtgaccctgcag;
acgagctggTcgggcccatgaccctgcag;
acgagctggacgaggccgtgaccctgcag;
acgagctggacgaggccgtgaccctgcag;
gacgaggccgtgaccctgcag;
gacgaggccg;
gagctggacgaggccgtgaccctgcag;
acgagctggGcgggcccatgaccctgcag;
acgagctgggcgggcccatgaccctgcag;
acgagctggacgaggccgtgaccctgcagcgc;
acgagctggTcgggcccatgaccctgcag;
gagctggacgaggccgtgaccctgcag;
gagctggacgaggccgtgaccctgcag;
acgagctggacgaggccgtgaccctgcagcgccgag;
acgagctggGcgggcccatgaccctgcagcgccgag;
acgagctggacgaggccgtgaccctgcagcgccgag;
acgagctggacgaggccgtgaccctgcagcgccga;
acgagctggacgaggccgtgacc;
gagctggacgaggccgtgaccctgcag;
gagctggGcgggcccatgaccctgcag;
gagctggGcgggcccatgaccctgcag;
gagctggGcgggcccatgaccctgcag;
gtcgggcccAtgaccctgcag;
gGcgggcccatgaccctgcag;
acgagctggacgaggccgtgaccctgcag;
acgagctgggcgggcccatgaccctgcagcgccgag;
acgagctggacgaggccgtgaccctgcagcgccgag;
acgagctggacgaggccgtgaccctgcagcgccgag;
acgagctgggcgggcccAtgacc;
acgagctggGcgggcccatgaccctgcagcgccgag;
acgagctgggcgggcccAtgaccctgcagcAccgag;
acgagctgggcgggcccatgaccctgcagcgccgag;
acgagctggacgaggccgtgaccctgcagcgccga;
acgagctggacgaggccgtgaccctgcagcAccgag;
cgagctgggcgggcccAtgaccctgcagcgccga;
acgagctggacgaggccgtgaccctgcagcgccgag;
acgagctggacgaggccgtgaccctgcagcgccgag;
acgagctggacgaggccgtgaccctgcagcgccgag;
acgagctggacgaggccgtgaccctgcagcgccgag;
acgagctggacgaggccgtgaccctgcagcgccgag;
acgagctggacgaggccgtgaccctgcagcgccgag;
acgagctggacgaggccgtgaccctgcagcgccgag;
acgagctgggcgggcccatgaccctgcagcgccgag;
In the following, Probe lists DP1-DP4 are shown in Tables 13-1 to 13-3 and Tables 14-1 to 14-3 respectively. Probe-Allele Lists DP1-4 are shown in Tables 15-1 to 15-5 and Tables 16-1 to 16-5.
Extraction of DNA from 1 ml of human blood was performed using GFX Genomic Blood DNA Purification Kit from Amersham Biosciences in the same manner as in Example 1.
Next, quantitative PCR was carried out in the same manner as in Example 1 except that probes in the probe lists DQ1A and DQ1B were used and 2 μl of the mixed primers consisting of 1 μl each of respective solutions of the following primers (10 pmol/μl) and 6 μl of ultra pure water were used:
After PCR amplification, referring to Amp Plot and Dissociation curves on a display of 5700 software, and to the allele-probe list (Table 19A, 19B-1 and 19B-2), it was identified as DQA1*0103 and DQB1*060101.
Extraction of DNA from 1 ml of human blood was performed in the same way as in Example 3. PCR of human HLA-DQ was then performed in the same manner as in Example 2 except that 3 μl of the mixed primer consisting of 1 μl each of the solutions containing the following sequences at 10 pmol/μl respectively, and 12 μl of ultra pure water were used:
At the same time, a DNA microarray was prepared to identify the allele in the specimen described above in the same manner as in Example 2, except that probes in the probe list of Tables 18A, 18B-1 and 18B-2 were used to form the probe spots respectively.
Then, hybridization was performed using the above specimen and the prepared DNA microarray in the same manner as in Example 2. Fluorometry measurement was conducted with GenePix4000B (Axon). Referring to the allele-probe list (Tables 20A, 20B-1 and 20B-2), it was identified as DQA1*0103 and DQB1*060101.
In the following, Probe Lists DQ1 and DQ2 are shown in Tables 17A, 17B-1 and 17B-2 and tables 18A, 18B-1 and 18B-2 respectively. Tables 19A, 19B-1 and 19B-2 and Tables 20A, 20B-1 and 20B-2 show Allele-Prove Lists.
Extraction of DNA from 1 ml of human blood was performed using GFX Genomic Blood DNA Purification Kit from Amersham Biosciences in the same manner as in Example 1.
Next, quantitative PCR was carried out in the same manner as in Example 1 except that probes in the probe list 1 in Tables 21-1 and 21-2 were used and 4 μl of the mixed primers consisting of 1 μl each of respective solutions of the following primers (10 pmol/μl) and 4 μl of ultra pure water were used:
Referring to Amp Plot and Dissociation curves on a display of 5700 software, it was found that probes 62, 12, and 152 were amplified. Therefore, it was identified as DRB1*040502 and DRB1*130202 referring to the allele-probe list 1 (Tables 23-1 to 23-13).
Extraction of DNA from 1 ml of human blood was performed in the same way as in Example 3. PCR of human HLA-DRB exon 2 was then performed in the same manner as in Example 2 except that 6 μl of the mixed primer consisting of 1 μl each of the solutions containing the following sequences at 10 pmol/μl respectively, and 9 μl of ultra pure water were used:
At the same time, a DNA microarray was prepared to identify the allele in the specimen described above in the same manner as in Example 2, except that probes in the probe list of Tables 22-1 to 22-7 were used to form the probe spots respectively.
Then, hybridization was performed using the above specimen and the prepared DNA microarray in the same manner as in Example 2. The fluorometry measurement was conducted with GenePix4000B (Axon).
As a result it was found that probes 59, 133, and 134 were amplified. Therefore, it was identified as DRB1*040502 and DRB1*130202 referring to the allele-probe list 1 (Tables 24-1 to 24-13).
acaactacggggttggtgagagcttcacagtgcagcggcgag;
acctaTtgcagacacaactacggggCtgtggagagcttcacagtgcagcggcgag;
acaactacggggctgtgg;
aaTtactgcagacacaactacggggttgtGgagagcttcacagtgcagcggcgag;
acaactacggggttggtgagagcttcacagtgcagcggcgag;
acaactacggggttgtGgagagcttcacagtgcagcggcgag;
acaactacggggttggtgagagcttcacagtgcagcggcgag;
acaactacggggttggtgagagcttcacagtgcagcggcgag;
acaactacggggttggtgagagcttcacagtgcagcggcgag;
acaactacggggttggtgagagcttcacagtgcagcggcgag;
aActactgcagacacaactacggggttgtGgagagcttcacagtgcagcggcgag;
acaactacggggttgtGgagagcttcacagtgcagcg;
aActactgcagacacaactacggggttggtgagagcttcacagtgcagcggcgag;
aaTtactgcagacacaactacggggttggtgagagcttcacagtgcagcggcgag;
gtGgagagcttcacagtgcagcggcga;
acaactacggggttgtGgagagcttcacagtgcagcggcgag;
acaactacggggttggtgagagcttcacagtgcagcggcgag;
acaactacggggttggtgagagcttcacGgtgcagcggcgag;
tacggggttgtGgagagcttcacagtgcag;
aActactgcagacacaactacggggttgtGgagagcttcacagtgcagcggcgag;
aActactgcagacacaactacggggttgtGgagagcttcacagtgcagcggcgag;
ggggttggtgagagcttcacagtgcagcgg;
aActactgcagacacaactacggggttgtGgagagcttcacagtgcagcggcgag;
acaactacggggttgtGgagagcttcacagtgcagcggcga;
ggggttgtGgag;
acaactacggggttgtGgagagcttcacagtgcagcggcgag;
acaactacggggttggtgagagcttcacagtgcagcggcgag;
acaactacggggttgtGgagagcttcacagtgcagcggcga;
acaactacggggttgtg;
acaactacggggttggtgagagcttcacagtgcagcggcgag;
acaactacggggttgtGgagagcttcacagtgcagcggcgag;
acaactacggggttgtGgagagcttcacagtgcagcggcgag;
acaactacggggCtgtggagagcttcacagtgcagcgg;
ctacggggttgtggagagcttcacagtgcagcggcgag;
acaactacggggttgtGgagagcttcacagtgcagcggcgag;
acaactacggggttgtGgagagcttcacagtgcagcggcgag;
acaactacggggttgtGgagagcttcacagtgcagcggcgag;
acaactacggggttggtgagagcttcacagtgcagcggcgag;
acctactgcagacacaactacggggttgtGgagagcttcacagtgcagcggcgag;
acaactacggggttgtGgagagcttcacagtgcagcggcgag;
acctactgcagacacaactacggggttggtgagagcttcacagtgcagcggcga;
acaactacggggttggtgagagcttcacagtgcagcggcgag;
acaactacggggttggtgagagcttcacagtgcagcgAcgag;
acaactacggggttggtgagagcttcacGgtgcagcggcgag;
acctactgcagacacaactacggggttgtGgagagcttcacagtgcagcggcgag;
acctactgcagacacaactacggggttggtgagagcttcacagtgcagcggcga;
acaactacggggttggtgagagcttcacagtgcagcgg;
ctacggggttggtgagagcttcacagtgcagcggcgag;
ctacggggttgtGgagagcttcacagtgcagcggcgag;
tacggggttgtGgagagcttcacagtgcagcgg;
ttcaca;
acaactacggggttgtGgagag;
tcacagtgca;
ctacggggttggtgagagcttcacagtgcagcggcgag;
gttggtgagagcttcacagtg;
gttgtGgagagcttcaca;
acaactacggggttgtggagagAttcacagtgcagcggcgag;
acaactacggggttggtgagagcttcacagtgcagcggcgag;
ggggttgtGgagag;
acaactacggggttggtg;
acaactacggggCtgtggagagcttcacagtg;
acaactacggggttggtgagagcttcacagtgcagcggcgag;
acaactacggggttggtgagagcttcacagtgcagcggcgag;
acaactacggggttggtgagagcttcacagtgcagcggcgag;
acaactacggggttggtgagagcttcacagtgcagcggcgag;
ggggttgtggag;
acaactacggggttggtgagagcttcacagtgcagcggcgag;
ctacggggttggtga;
acaactacggggttggtgagagcttcacagtg;
acaactacggggttgtGgagagcttcacagtgcagcggcgag;
acaactacggggttgtGgagagcttcacagtgcagcggcgag;
acaactacggggttggtgagagcttcacagtgcagcggcgag;
acaactacggggttgtggagagcttcacagtgcagcgg;
acaactacggggttgtggagagcttcacagtgcagcgg;
acaactacggggttgtGgagagcttcacagtgcagcgg;
acaactacggggttgtGgagagcttcacagtgcagcggcgag;
acaactacggggttggtgagagcttcacagtgcagcgg;
acaactacggggttgtGgagagcttcacagtgcagc;
acaactacggggttggtg;
acaactacggggttggtg;
ctacggggttggtgagagc;
acaactacggggttggtgagagcttcacag;
acaactacggggttggtgagagcttcacagtgcagcggcgag;
acaactacggggttggtgagagcttcacagtg;
acctactgcagacacaactacggggttggtgagagcttcacGgtgcagcggcgag;
acaactacggggttggtgagagcttcacggtgcagcggcgag;
acaactacggggttggtgagagcttcacagtgcagcggcgag;
aactacggggttggtgagagcttcacggtg;
acctactgcagacacaactacggggttggtgagagcttcacagtgcagcggcgag;
acctactgcagacacaactacggggttgtGgagagcttcacagtgcagcggcgag;
acaactacggggttgTtgagagcttcacGgtgcagcggcga;
acaactacggggttgtGgagagcttcacGgtgcagcggcgag;
acaactacggggttggt;
cacaactacggggttgtGgagagcttcacagtgcagcggcgag;
acaactacggggttggtgagagcttcacGgtgcagcggcgag;
ggggttggtgag;
acaactacggggttggtgagagcttcacGgtgcagcggcgag;
acaactacggggttgtGgagagcttcacagtgcagcggcgag;
acaactacggggttggtgagagcttcacGgtg;
acaactacggggCtgtggagagcttcacagtgcagcggcgag;
acggggttggtgagagcttcacGgtg;
acaactacggggttggtgagagcttcacagtg;
ctacggggttggtg;
acaactacggggttggtgagagcttcacGgtgcagcggcgag;
acaactacggggttggtg;
acaactacggggttggtgagagcttcacagtgcagcggcgag;
ctacggggttggtgagagcttcacagtgc;
acaactacggggttgtGgagagcttcacagtgcagcggcga;
acaactacggggttggtgagagcttcacggtgcagcggcga;
acaactacggggCtgtGgagagcttcacGgtgcagcggcgag;
acaactacggggttggtgagagcttcacagtgcagcggcgag;
acaactacggggttggtgagagcttcacagtgcagcgg;
accgtgtgcagacacaactacggggttggtgagagcttcacagtgcagAggcgag;
acaactacggggttggtgagagcttcacagtgcagAggcgag;
acaactacggggttggtgagagcttcacagtgcagcggcgag;
acctactgcagacacaactacggggttggtgagagcttcacGgtgcagcggcgag;
acaactacggggttggtgagagcttcacagtgcagcggcgag;
aactacggggttggtgagagcttcacagtgcagcggcgag;
acctactgcagacacaactacggggttgtGgagagcttcacagtgcagcggcgag;
acctactgcagacacaactacggggttgtGgagagcttcacGgtgcagcggcgag;
cacaactacggggttggtgagagcttcacagtgcagcggcga;
aactacggggCtgtggagagcttcacagtgcagcggcgag;
ctacggggCtgtggagagcttcacagtgcagcggcgag;
ggggttgtGgagagcttcacagtgcagcggcgag;
cttcacagtg;
cttcacGgtg;
cag;
ctacggggttggtgagagcttcacagtgcagcggcgag;
acaactacggggttggtgagagcttcacagtgcagcggcgag;
acctaTtgcagacacaactacggggttgtGgagagcttcacagtgcagcggcga;
acctactgcagacacaactacggggttggtgagagcttcacagtgcagcggcgag;
acctactgcagacacaactacggggttggtgagagcttcacagtgcagcggcgag;
acaactacggggttgtGgagagcttcacagtgcagcggcgag;
acctaTtgcagacacaactacggggttgtGgagagcttcacagtgcagcggcgag;
ctacggggttgtGgagagcttcacagtgcagcggcgag;
ctacggggttggtgagagcttcacagtgcagcggcgag;
ggggttggtgagagcttcacagtgcagc;
ggggCtgtggaga;
acaactacggggttggtgagag;
cacaactacggggttggtg;
tacggggttggtgagagcttcac;
acaactacggggttgtGgagagcttcacagtgcagcggcgag;
ggggttggtgag;
ctacggggttggtgagag;
acaactacggggttggtgagagcttcacagtgcagcggcgag;
acaactacggggttggtgagagcttcacagtgcagcggcgag;
acaactacggggttggtgagagcttcacagtgcagcggcgag;
acaactacggggttggtgagagcttcacagtgcagcggcga;
acctactgcagacacaactacggggttggtgagagcttcacagtgcagcggcgag;
acaactacggggttggtgagagcttcacagtgcagcggcgag;
ggggttggtgagagcttcacagtgcagcggc;
acaactacggggttgtGgagagcttcacagtgcagcggcga;
ggggttgtGgagagcttcacagtgcagcggc;
aactacggggttgtGgagagcttcacagtgcagcggcga;
acaactacggggttggtgagagcttcacagtgcagcggcgag;
acaactacggggttgtggagagcttcacagtgcagcggcgag;
acaactacggggttggtgagagcttcacagtgcagcggcgag;
acaactacggggttgtGg;
ctacggggttgtGgagagcttcacagtgcagcggcgag;
acaactacggggttgtGgagagcttcacagtgcagcggcgag;
acaactacggggCtgtggagagcttcacagtgcagcggcgag;
tacggggCtgtggag;
acaactacggggttgtggagagcttcacagtgcagcgg;
gCtgtgg;
acaactacggggCtgtggagagcttcacagtgcagcggcgag;
acctaTtgcagacacaactacggggCtgtggagagcttcacagtgcagcggcgag;
acaactacggggctgtggagagcttcacagtgcagcggcgag;
acaactacggggCtgtggagagcttcacagtgcagcggcgag;
acctactgcagacacaactacggggttgtGgagagcttcacagtgcagcggcgag;
acaactacggggttgtggagagcttcacagtgcagcggcgag;
acaactacggggttgtGgagagcttcacagtgcagcggcgag;
acctactgcagacacaactacggggttggtgagagcttcacagtgcagcggcgag;
acaactacggggttggtgagagcttcacagtgcagcgg;
acctactgcagacacaactacggggttggtgagagcttcacGgtgcagcggcgag;
ggggttggtgagagcttcacagtgcagcggcgag;
acctactgcagacacaactacggggttgtGgagagcttcacagtgcagcggcgag;
aactacggggttggtgagagcttcacagtgcagcggcgag;
acaactacggggttggtgagagcttcacagtgcagcggcgag;
acaactacggggttggtgagagcttcacagtgcagcggcg;
tacggggttgtGgagagcttcacagtg;
ctacggggttgtGgagagcttcacagtg;
acaactacggggttgtGgagagcttcacagtgcagcggcgag;
acaactacggggttgtGgagagcttcacagtgcagcggcgag;
acaactacggggttggtgagagcttcacagtgcagcggcgag;
acaactacggggttggtgagagcttcacagtgca;
acaactacggggttggtgagagcttcacagtgcagcggcgag;
ctacggggttgtGgagagcttcacagtgcagcggcgag;
ggttgAtgagagcttcaca;
acctactgcagacacaactacggggttgtGgagagcttcacagtgcagcggcgag;
acaactacggggttgtGgagagcttcacagtgcagcggcgag;
acctactgcagacacaactacggggttgtGgagagcttcacagtgcagcggcgag;
acaactacggggttgtGgagagcttcacagtgcagcgg;
acctactgcagacacaactacggggttggtgagagcttcacagtgcagcggcgag;
gacacaactacggggttgtGgagagcttcacagtgcagcggcgag;
aactacggggttggtgagagcttcacGgtgcagcggc;
aactacggggttgtGgagagcttcacagtgcagcggc;
acaactacggggttggtgaga;
acaactacggggttggtgagagcttcacagtgcagcggcgag;
acaactacggggttgtGgagagcttcacagtgcagcggcgag;
Cgggttgtggagagcttcac;
acaactacggggttggtgagagcttcacagtgcagcggcgag;
ctacggggttggtgagagcttcaca;
acaactacggggttggtgagagcttcacagtgcagcggcga;
acaactacggggttgtGgagagcttcacagtgcagcggcgag;
acaactacggggttggtg;
acaactacggggttggtgagagcttcacagtgcagcggcgag;
acaactacggggttgtggagagcttcacagtgcagcgg;
acaactacggggttggtgagagcttcacagtgcagcggcgag;
acaactacggggttggtgagagcttcacGgtgcagcggcga;
acaactacggggttggt;
acaactacggggttggtgagagcttcacagtgcagcggcgag;
ggggttgtGgagagcttcacagtgcagcggcg;
acaactacggggttggtgagagcttcacagtgcagcggcgag;
acaactacggggttgtGgagagcttcacagtgcagcggcgag;
acaactacggggttgtGgagagcttcacagtgcagcggcga;
acaactacggggttgtGgagagcttcacagtgcagcggcgag;
ggggttggtgagag;
acaactacggggttggtgagagcttcacagtgcagcggcgag;
acaactacggggttggtgagagcttcacGgtgcagcggcgag;
acaactacggggttgtGgagagcttcacagtgcagcggcgag;
acaactacggggttggtgagagcttcacagtgcagcgg;
acaactacggggttggtgagagcttcacagtgcagcggcgag;
acaactacggggttgtggagagcttcacagtgcagcggcgag;
acaactacggggttgtGgagagcttcacagtg;
acaactacggggttgtGgagagcttcacagtgcagcggcga;
acaactacggggttgtGgagagcttcacagtgcagcggcgag;
ctacggggttggtgagagcttcacGgtgcagcggcgag;
acaactacggggttgtGg;
acaactacggggttgtGgagagcttcacagtgcagcggcgag;
acaactacggggttgtGgagagcttcacagtgcagcggcgag;
acaactacggggttgtGgagagcttcacagtgcagcggcgag;
acaactacggggttggtgagagcttcacagtgcagcggcga;
acaactacggggttggtgagagcttcacGgtgcagcggcgag;
acaactacggggttgtGgagagcttcacagtgcagcggcga;
ctacggggttggtgagagcttcacagtgcagcggcgag;
tacggggttgtGgagagcttcacagtgcagcgg;
gttgtGg;
gttggtg;
tacggggttggtgagagcttcacagtg;
Ggagagcttcacagtgcag;
ggggttgtGgag;
acaactacggggttgtGgagagcttcacagtgcagcgg;
gttgtGgagagcttcacagtgcagcggcga;
acctactgcagacacaactacggggttggtgagagcttcaca;
ggggttgtGgaga;
ggggttgtGgaga;
acaactacggggttggtgagagcttcacagtgcagcggcgag;
acaactacggggttgtGgagagcttcacagtgcagcggcgag;
tacggggttggtgagagcttcacagtgcagcggcgag;
ctacggggttggtgagagcttcacagtgcagcggcgag;
acaactacggggttgtGgagagcttcacagtgcagcggcgag;
acaactacggggttggtgagagcttcacagtgcagcggcgag;
acaactacggggCtgtGgagagcttcaca;
acaactacggggCtgtggagagcttcacagtgcagcggcgag;
ctacggggttggtgagagcttcaca;
ctacggggttgtGgagagcttcaca;
acaactacggggttgtGgagagcttcacagtgcagcggcgag;
ggggttgtGgagagcttcacagtgcagcggc;
acaactacggggttgtGgagagcttcacagtgcagcggcgag;
acaactacggggttgtGgagagcttcacagtgcagcgg;
acaactacggggttggtgagagcttcacagtgcagcggcgag;
acaactacggggttgtGgagagcttcacagtgcagcggcgag;
acaactacggggttgtGgagagcttcacagtgcagcggcgag;
acaactacggggttgtGgagagcttcacagtgcagcggcgag;
acaactacggggttggtgagagcttcacagtgcagcggcgag;
acaactacggggttggtgagagcttcacagtgcagcggcga;
acaactacggggttggtgagagcttcacagtgcagcggcgag;
acaactacggggttgtggagagcttcacagtgcagcggcgag;
acaactacggggttggtgagagcttcacagtgcagcggcgag;
acaactacggggttgtGgagagcttcacagtgcagcggcgag;
acaactacggAgttgtGgagagcttcacagtgcagcgg;
acaactacggggttgtGgagagcttcacagtgcagcgg;
acaactacggggttgtGgagagcttcacagtgcagcggcgag;
acctactgcagacacaactacggggttggtgagagcttcacagtgcagcggcgag;
acaactacggggttggtg;
acctactgcagacacaactacggggttgtGgagagcttcacagtgcagcggcgag;
tacggggttgtGgagagcttcacagtg;
tacggggttgtGgagagcttcacagtgcagcgg;
ggggttgtggagagcttcacagtgcagcggcgag;
ctacggggttgtGgagagc;
acaactacggggttggtgagagcttcacagtgcagcggcgag;
acaactacggggttgtggagagcttcacagtgcagcggcgag;
actacggggttgtGgagagc;
acaactacggggttggtgagagcttcacagtgcagcggcgag;
cacaactacggggttgtGgagagcttcacagtgcagcggcgag;
acaactacggggttgtGgagagcttcacagtgcagcgg;
aactacggggttggtgagagcttcaca;
ctacggggttggtg;
gttggtg;
ggggttggtgag;
acaactacggggttggtgagagcttcacagtgca;
acaactacggggttggtgagagcttcacagtgcagcggcgag;
aaTtactgcagacacaactacggggttggtgagagcttcacagtgcagcggcgag;
aaTtactgcagacacaactacggggttggtgagagc;
acaactacggAgttggtg;
aaTtactgcagacacaactacggggttggtgagagc;
acaactacggggttggtgagagc;
acaactacggggttggtgagagcttcaca;
acaactacggggttggtgagagcttcacagtgcagcggcg;
acaactacggggttggtg;
acaactacggggttggtg;
acaactacggggttggtgagagcttcacagtgcagcgg;
acaactacggggttggtgagagcttcacagtgcagcgg;
acaactacggggttggtg;
aaTtactgcagacacaactacggggttggtgagagcttcacagtgcagcggcgag;
acaactacggggttggtg;
acaactacggggttggtgagagcttcacagtgcagcggcgag;
acaactacggggttggtgagagcttcacagtgcagcggcgag;
ggggttggtgaga;
acaactacggggttgtGgagagcttcacagtgcagcggcgag;
aactacggggttggtgagagcttcacagtgcag;
acaactacggggttggtg;
ggggttggtgagag;
acaactacggggttggtg;
acaactacggggttggtgagagcttcaca;
aaTtactgcagacacaactacggggttggtgagagcttcacagtgcagcggcgag;
aaTtactgcagacacaactacggggttggtgagagcttcacagtgcagcggcgag;
acaactacggggttggtgagagcttcacagtgcagcggcgag;
acaactacggggttggtgagagcttcacagtgcagcggcgag;
acaactacggggttgCtgagagcttcacagtgcagcggcgag;
acaactacggggttggtgagagcttcacagtgcagcggcgag;
acaactacggggttggtgagagcttcacagtgcagcggcgag;
acaactacggggttggtgagagcttcacagtgcagcggcgag;
aaTtactgcagacacaactacggggttgtGgagagcttcacagtgcagcggcgag;
acaactacggCgttgtggagagcttcacagtgcagcggcgag;
acaactacggggttgtGg;
ctacggggttggtgagagcttcaca;
acctactgcagaTacaactacggggttgtggagagcttcacagtgcagcggcgag;
acaactacggggttgtggagagcttcacagtgcagcggcgag;
acaactacggggttgtggagagcttcacagtgcagcggcgag;
ggggttgtggagag;
acaactacggggttgtggagagcttcacagtgcagcggcgag;
acaactacggggttggtgagagcttcaca;
acctactgcagacacaactacggggttggtgagagcttcacagtgcagcggcgag;
ggggttggtgagagcttcacag;
acctactgcagacacaactacggggttggtgagagcttcacagtgcagcggcgag;
cacaactacggggttggtgagagcttcacagtgcagcgg;
acaactacggggctgtGgagagcttcacagtgcagcggcga;
acaactacggggttggtg;
acaactacggggttggtg;
acaactacggggttggtgagagcttcacagtgcagcggcgag;
acaactacggggttggtgagagcttcacagtgcagcggcgag;
acaactacggggttggtgagagcttcacagtgcagcggcgag;
ctacggggttggtgagagcttcacagtgcagcgg;
acaactacggggctgtGgagagcttcaca;
In the following, Probe List DR1 and 2 are shown in Tables 21-1 to 21-8 and Tables 22-1 to 22-7 respectively. Allele-Probe Lists 1 and 2 are shown in Tables 23-1 to 23-13 and Tables 24-1 to 24-13 respectively.
Extraction of DNA from 1 ml of human blood was performed using GFX Genomic Blood DNA Purification Kit from Amersham Biosciences in the same manner as in Example 1.
Next, quantitative PCR was carried out in the same manner as in Example 1 except that probes in Tables 25-1 and 25-2 were used and 2 μl of the mixed primers consisting of 1 μl each of respective solutions of the following primers (10 pmol/μl) and 6 μl of ultra pure water:
After PCR amplification, referring to Amp Plot and Dissociation curves on a display of 5700 software, and to the allele-probe list 1 (Tables 27-1 and 27-2), it was identified as MICA*00201.
Extraction of DNA from 1 ml of human blood was performed in the same way as in Example 3. PCR of human HLA-MICA was then performed in the same manner as in Example 2 except that 3 μl of the mixed primer consisting of 1 μl each of the solutions containing the following sequences at 10 pmol/μl respectively, and 12 μl of ultra pure water were used:
At the same time, a DNA microarray was prepared to identify the allele in the specimen described above in the same manner as in Example 2, except that probes in the probe list of Table 26-1 were used to form the probe spots respectively.
Then, hybridization was performed using the above specimen and the prepared DNA microarray in the same manner as in Example 2. The DNA microarray was air-dried and the fluorometry measurement was conducted with GenePix4000B (Axon). Referring to the allele-probe correspondence list 2 (Tables 28-1 and 28-2), it was identified as MICA*00201.
In the following, Probe List M1 and M2 are shown in Tables 25-1 and 25-2 and Tables 26-1 and 26-2 and Tables 27-1 and 27-2 and Tables 28-1 and 28-2 respectively.
The present invention is not limited to the above embodiments and various changes and modifications can be made within the spirit and scope of the present invention. Therefore, to apprise the public of the scope of the present invention, the following claims are made.
This application claims priority from Japanese Patent Application Nos. 2003-430553 filed on Dec. 25, 2003, 2003-430554 filed on Dec. 25, 2003, 2003-430555 filed on Dec. 25, 2003, 2003-430556 filed on Dec. 25, 2003, 2003-430557 filed on Dec. 25, 2003, 2003-430558 filed on Dec. 25, 2003 and 2003-430559 filed on Dec. 25, 2003, which are hereby incorporated by reference herein.
Number | Date | Country | Kind |
---|---|---|---|
2003-430553 | Dec 2003 | JP | national |
2003-430554 | Dec 2003 | JP | national |
2003-430555 | Dec 2003 | JP | national |
2003-430556 | Dec 2003 | JP | national |
2003-430557 | Dec 2003 | JP | national |
2003-430558 | Dec 2003 | JP | national |
2003-430559 | Dec 2003 | JP | national |
This application is a divisional of application Ser. No. 10/582,327, which is the National Stage of International Application No. PCT/JP2004/019763, filed Dec. 24, 2004. The contents of each of the foregoing applications are incorporated by reference herein.
Number | Date | Country | |
---|---|---|---|
Parent | 10582327 | Jun 2006 | US |
Child | 13467972 | US |