JPH0288967A - Novel oligonucleotide probe - Google Patents
Novel oligonucleotide probeInfo
- Publication number
- JPH0288967A JPH0288967A JP63239823A JP23982388A JPH0288967A JP H0288967 A JPH0288967 A JP H0288967A JP 63239823 A JP63239823 A JP 63239823A JP 23982388 A JP23982388 A JP 23982388A JP H0288967 A JPH0288967 A JP H0288967A
- Authority
- JP
- Japan
- Prior art keywords
- type discrimination
- oligonucleotide probe
- specific
- oligonucleotide
- executed
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 108020005187 Oligonucleotide Probes Proteins 0.000 title claims abstract description 8
- 239000002751 oligonucleotide probe Substances 0.000 title claims abstract description 8
- 108091007433 antigens Proteins 0.000 claims abstract description 6
- 239000000427 antigen Substances 0.000 claims abstract description 5
- 102000036639 antigens Human genes 0.000 claims abstract description 5
- 101710181478 Envelope glycoprotein GP350 Proteins 0.000 claims description 3
- 210000000265 leukocyte Anatomy 0.000 claims description 2
- 108090000623 proteins and genes Proteins 0.000 claims description 2
- 150000007523 nucleic acids Chemical group 0.000 claims 2
- 108091028043 Nucleic acid sequence Proteins 0.000 claims 1
- 108020004707 nucleic acids Proteins 0.000 claims 1
- 102000039446 nucleic acids Human genes 0.000 claims 1
- 239000002773 nucleotide Substances 0.000 claims 1
- 125000003729 nucleotide group Chemical group 0.000 claims 1
- 238000009396 hybridization Methods 0.000 abstract description 8
- 108091034117 Oligonucleotide Proteins 0.000 abstract description 7
- 108091008146 restriction endonucleases Proteins 0.000 abstract description 6
- 239000000523 sample Substances 0.000 abstract description 5
- 238000007796 conventional method Methods 0.000 abstract description 3
- 238000004458 analytical method Methods 0.000 abstract description 2
- 102000015789 HLA-DP Antigens Human genes 0.000 abstract 1
- 108010010378 HLA-DP Antigens Proteins 0.000 abstract 1
- 238000012850 discrimination method Methods 0.000 abstract 1
- 230000002194 synthesizing effect Effects 0.000 abstract 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 20
- 239000000243 solution Substances 0.000 description 13
- 239000012528 membrane Substances 0.000 description 11
- 238000000034 method Methods 0.000 description 10
- 239000011780 sodium chloride Substances 0.000 description 10
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 8
- 239000004677 Nylon Substances 0.000 description 7
- 229920001778 nylon Polymers 0.000 description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 description 4
- 239000001509 sodium citrate Substances 0.000 description 4
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 4
- 239000001488 sodium phosphate Substances 0.000 description 4
- 229910000162 sodium phosphate Inorganic materials 0.000 description 4
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 4
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 210000000601 blood cell Anatomy 0.000 description 3
- 210000005259 peripheral blood Anatomy 0.000 description 3
- 239000011886 peripheral blood Substances 0.000 description 3
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 2
- 241000972773 Aulopiformes Species 0.000 description 2
- 229920001917 Ficoll Polymers 0.000 description 2
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 2
- 238000000376 autoradiography Methods 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 230000002380 cytological effect Effects 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 210000004698 lymphocyte Anatomy 0.000 description 2
- 239000004033 plastic Substances 0.000 description 2
- 229920003023 plastic Polymers 0.000 description 2
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 2
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 2
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 2
- 235000019515 salmon Nutrition 0.000 description 2
- 230000000405 serological effect Effects 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- LWIHDJKSTIGBAC-UHFFFAOYSA-K tripotassium phosphate Chemical compound [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 230000007018 DNA scission Effects 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 102000018713 Histocompatibility Antigens Class II Human genes 0.000 description 1
- 108010027412 Histocompatibility Antigens Class II Proteins 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 108700018351 Major Histocompatibility Complex Proteins 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 108010021757 Polynucleotide 5'-Hydroxyl-Kinase Proteins 0.000 description 1
- 102000008422 Polynucleotide 5'-hydroxyl-kinase Human genes 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 238000007605 air drying Methods 0.000 description 1
- 239000012670 alkaline solution Substances 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 229910052792 caesium Inorganic materials 0.000 description 1
- TVFDJXOCXUVLDH-UHFFFAOYSA-N caesium atom Chemical compound [Cs] TVFDJXOCXUVLDH-UHFFFAOYSA-N 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- YTRQFSDWAXHJCC-UHFFFAOYSA-N chloroform;phenol Chemical compound ClC(Cl)Cl.OC1=CC=CC=C1 YTRQFSDWAXHJCC-UHFFFAOYSA-N 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 229960000633 dextran sulfate Drugs 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- YQOKLYTXVFAUCW-UHFFFAOYSA-N guanidine;isothiocyanic acid Chemical compound N=C=S.NC(N)=N YQOKLYTXVFAUCW-UHFFFAOYSA-N 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 239000001103 potassium chloride Substances 0.000 description 1
- 235000011164 potassium chloride Nutrition 0.000 description 1
- 229910000160 potassium phosphate Inorganic materials 0.000 description 1
- 235000011009 potassium phosphates Nutrition 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000007894 restriction fragment length polymorphism technique Methods 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000020382 suppression by virus of host antigen processing and presentation of peptide antigen via MHC class I Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000002054 transplantation Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Abstract
Description
【発明の詳細な説明】
〈産業上の利用分野〉
ヒトの主要組織適合性複合体である白血球膜抗原（ＩＩ
Ｌ＾）は、臓器等の移植に重要な関わりをもつ。[Detailed Description of the Invention] <Industrial Application Field> Leukocyte membrane antigen (II), which is a major histocompatibility complex in humans.
L^) has an important relationship with organ transplantation.
本発明は、このＩ（Ｌ＾の判別法に関するものである。The present invention relates to a method for determining this I(L^).
〈従来の技術〉
１１Ｌ＾の型判別法は、従来血清学的または細胞学的方
法により行われている。即ち、ＩＩＬＡクラス１抗原で
あるＡ、Ｂ、Ｃおよびクラス■抗原のうちＤｌ、ＤＱの
各抗原は血清学的方法、換言すれば特異抗体と補体を用
いたリンパ球傷害テストにより型判別が行なわれている
。またクラス■抗原のうちＤ抗原は、Ｍ Ｌ Ｃ（ｍｉ
ｘｅｄ ｌｙｍｐｈｏｃｙｔｅ ｃｕｌｔｕｒｅ）法で
、ＤＰ膜抗原、Ｐ Ｌ Ｔ （ｐｒｉｍｅｄ ｌｙｍｐ
ｈｏｃｙｔｅｔｙｐｉｎｇ）法でそれぞれ判定されてい
る。<Prior Art> Type determination of 11L^ has conventionally been carried out by serological or cytological methods. In other words, the types of IILA class 1 antigens A, B, C and class II antigens Dl and DQ can be determined by serological methods, in other words, lymphocyte damage tests using specific antibodies and complement. It is being done. Furthermore, among class ■ antigens, D antigen is MLC (mi
DP membrane antigen, PLT (primed lymphocyte culture) method
They were determined using the hocytetyping method.
また、最近の分子生物学の進歩によりＨＬＡに関しても
遺伝子をプローブとして用い、検体ＤＮＡの制限酵素に
よる切断断片の長さの違い（ＲＦＬ Ｐ ”Ｒｅ５ｔｒ
ｉｃｔｉｏｎ Ｆｒａｇｍｅｎｔ Ｌｅｎｇｔｈ Ｐｏ
ｌｙｒａｏｒｐｈｉｓｍ）から型判別を行なうことが、
試みられている（Ｍａｅｄａ等 ｔｌｕｉａｎ Ｉｍ
ｍｕｎｏｌｏｇＹ ２１巻ｐ２３９ 、１９８８）。In addition, with recent advances in molecular biology, HLA genes can also be used as probes to detect differences in length of fragments cut by restriction enzymes (RFLP ``Re5tr'') of sample DNA.
iction Fragment Length Po
lyraorphism).
It has been attempted (Maeda et al.
munologY Vol. 21, p. 239, 1988).
〈発明が解決しようとしている問題点〉従来の方法のう
ちで、特に細胞学的な方法であるＭＬＣ及びＰＬＴ法は
、判定に要する時間及び手間が多く、判定の精度も必ず
しも満足できるものではなかった。また、ＤＮＡの制限
酵素による切断パターンの違いにより型判別を行なう方
法も得られたパターンの解析に手間取り、簡便さに欠け
ていた。<Problems to be solved by the invention> Among conventional methods, especially the cytological methods MLC and PLT, the time and effort required for determination are large, and the accuracy of determination is not always satisfactory. Ta. Furthermore, a method of type discrimination based on differences in DNA cleavage patterns by restriction enzymes requires time and effort to analyze the resulting patterns, and lacks simplicity.
〈問題点を解決するための手段〉
本発明は、）ｆＬＡ−ＤＰ膜抗の型判別を配列特異的オ
リゴヌクレオチドをプローブとして行うことを特徴とす
る。その結果、従来の制限酵素の切断パターンの違いに
より型判別を行う方法に比べ、パターン解析の必要性が
なくなり、簡便に、高い精度で型判別ができる。<Means for Solving the Problems> The present invention is characterized in that type discrimination of fLA-DP membrane antibodies is carried out using a sequence-specific oligonucleotide as a probe. As a result, compared to conventional methods in which type discrimination is performed based on differences in the cleavage patterns of restriction enzymes, there is no need for pattern analysis, and type discrimination can be performed simply and with high accuracy.
配列特異的オリゴヌクレオチドは、ＤＰ膜抗の塩基配列
（Ｉｎｏｋｏ等 ｉｎ Ｐｒｅｓｅｎｔ ａｎｄ ｆｕ
ｔｕｒｅ ｄｅ−ｖｅｌｏｐｍｅｎｔ ｏｒ ｔｒａｎ
ｓｐｌａｎｔａｔｉｏｎ、東海大学出版会１９８７）よ
り各ＤＰ型に特異的な部分を選択し、ＤＮＡ合成装置３
８１Ａ（アプライド・バイオシステムズ社）により合成
した。Sequence-specific oligonucleotides are based on the base sequence of DP membrane antibodies (Inoko et al. in Present and fu
ture de-development or tran
Select a portion specific to each DP type from ``Splantation, Tokai University Press, 1987'', and add it to the DNA synthesizer 3.
81A (Applied Biosystems).
試料は、通常判定すべき個人から採取した血液を用いる
。血液から血球を集め高分子ＤＮＡ及び全ＲＮＡを抽出
し、直接膜に固定するかゲル電気泳動後、膜に移行させ
てから固定する。The sample used is usually blood collected from the individual to be evaluated. Blood cells are collected from blood, high-molecular DNA and total RNA are extracted, and either directly fixed on a membrane or transferred to a membrane after gel electrophoresis and then fixed.
固定したＤＮＡ又はＲＮＡと３１ｐ等にて標識したオリ
ゴヌクレオチドプローブとのハイブリダイゼーションを
実施し、ハイブリダイズするかしないかにより型判別を
行う。以下実施例により本発明を更に詳細に説明するが
、これは本発明の範囲を同等制限するものではない。Hybridization is performed between the fixed DNA or RNA and an oligonucleotide probe labeled with 31p, etc., and type discrimination is performed based on whether hybridization occurs or not. The present invention will be explained in more detail with reference to Examples below, but these are not intended to limit the scope of the present invention.
〈実施例〉
実施例１
ヒト末梢血より高分子ＤＮＡを抽出して、制限酵素処理
、電気泳動の後、サザントランスファー法によりＤＮＡ
をナイロン膜に移行させ、オリゴヌクレオチドプローブ
によりＤＰタイピングを行う方法を下に示す。<Example> Example 1 High-molecular DNA was extracted from human peripheral blood, treated with restriction enzymes, electrophoresed, and then transformed into DNA using the Southern transfer method.
The method for transferring DP to a nylon membrane and performing DP typing using an oligonucleotide probe is shown below.
ヒトの末梢血より１０＠個の血球を集め、リン酸緩衝液
：Ｐ Ｂ Ｓ （１３７ｍＭ塩化ナトリウム、 ２．７
ｍＭ塩化カリウム、 ８ａＭリン酸ナトリウム、 １．
’５＋ａＭリン酸カリウム）に分散させる。３倍量の緩
衝液（５０ｍＭトリス−塩酸、ｐＨ８，０，２０ｄ Ｅ
ＤＴ＾、 ＩＱＯｓＭ塩化ナトリウム、１％５ＤＳ）を
加え細胞を溶解させる。プロテアーゼ処理、フェノール
抽出により、除タンパク後、ＲＮＡ分解酵素（ＲＮａｓ
ｅＡ、ＲＮａｓｅＴｌ）によりＲＮＡを除く。これに２
倍量の冷エタノールを加え、高分子ＤＮＡを得る。高分
子ＤＮＡを種々の制限酵素で切断、０．７％アガロース
ゲル電気泳動を行う。Collect 10 blood cells from human peripheral blood and add them to phosphate buffer: PBS (137mM sodium chloride, 2.7
mM Potassium Chloride, 8aM Sodium Phosphate, 1.
'5+aM potassium phosphate). 3 volumes of buffer (50mM Tris-HCl, pH 8,0, 20dE
DT^, IQOsM sodium chloride, 1% 5DS) to lyse the cells. After protein removal by protease treatment and phenol extraction, RNA degrading enzyme (RNas)
RNA is removed using eA, RNaseTl). 2 to this
Add twice the amount of cold ethanol to obtain high-molecular DNA. High-molecular DNA is cut with various restriction enzymes and subjected to 0.7% agarose gel electrophoresis.
電気泳動終了後、ゲルをアルカリ変性液（０，４Ｍ水酸
化ナトリウム、 ０．６Ｍ塩化ナトリウム）にて３０分
振盪後同じアルカリ液を使いサザン法によりナイロン膜
にＤＮＡを移行させる。γ−”Ｐ−ＡＴＰとポリヌクレ
オチドキナーゼによりオリゴヌクレオチドの５末端を標
識。ナイロン膜をプラスチック袋に入れ、プレハイブリ
ダイゼーション溶液（０，９Ｍ塩化ナトリウム、０．０
９Ｍクエン酸ナトリウム、０，１％牛血清アルブミン、
０．１％フィコール、０．１％ポリビニルピロリドン、
０．５％ＳＤＳ、 １００μ９／ｒｅρ変性サケ精子Ｄ
ＮＡ）を加え、６５℃で３時間プレハイブリダイゼーシ
ョンを行う。次にプレハイブリダイゼーション溶液を捨
て、ハイブリダイゼーション溶、液（プレハイブリダイ
ゼーション溶液と同じ）及び標識したオリゴヌクレオチ
ドをカウント２×１０’ｅｐｍで加える。６５℃で１晩
ハイブリダイゼーシヨンを行う。ナイロン膜を袋からと
り出し、２ＸＳＳＣ（０，０１５Ｍ塩化ナトリウム、０
．００１５Ｍクエン酸ナトリウム）、 ０．１％ＳＯ８
中で５５℃、１０分間インキュベートする。風乾後ナイ
ロン膜をプラスチックラップで包み、オートラジオグラ
フィーを行う。After electrophoresis, the gel is shaken for 30 minutes in an alkaline denaturing solution (0.4 M sodium hydroxide, 0.6 M sodium chloride), and then the DNA is transferred to a nylon membrane by the Southern method using the same alkaline solution. Label the 5-end of the oligonucleotide with γ-”P-ATP and polynucleotide kinase. Place the nylon membrane in a plastic bag and add prehybridization solution (0.9 M sodium chloride, 0.0
9M sodium citrate, 0.1% bovine serum albumin,
0.1% Ficoll, 0.1% polyvinylpyrrolidone,
0.5% SDS, 100 μ9/reρ denatured salmon sperm D
NA) and perform prehybridization at 65°C for 3 hours. The prehybridization solution is then discarded and the hybridization solution (same as the prehybridization solution) and labeled oligonucleotide are added at a count of 2 x 10' epm. Hybridization is carried out overnight at 65°C. Take out the nylon membrane from the bag and add 2XSSC (0,015M sodium chloride, 0
．． 0015M sodium citrate), 0.1% SO8
Incubate for 10 minutes at 55°C. After air drying, wrap the nylon membrane in plastic wrap and perform autoradiography.
実施例２
ヒト末梢血より血球を集め、ＧＴＣ液（４Ｍグアニジン
イソチオシアネート、０．５％ サルコシルナトリウ
ム、 ２５ｍＭクエン酸ナトリウム）を１０１加える。Example 2 Blood cells were collected from human peripheral blood, and 10 ml of GTC solution (4M guanidine isothiocyanate, 0.5% sodium sarcosyl, 25mM sodium citrate) was added.
混合液を２２ゲージの注射針の中で圧を十分にかけて２
回通しＤＮＡを切断する。超遠心機のローター５Ｗ２８
用のポリアロマ−の遠心管へ１／３容のセシウムＴＦ＾
（１，５１１Ｆ／＋Ｉ２）液を入れ、その上に上記の試
料液を上層後、５ｌｌ１２８で２８．ＯＯＯｒｐｍ ２
４時間遠心し上清を捨てる。ＲＮＡベレットをＲＮＡ溶
解液（１０ｍ輩トリスー塩酸、 ｐＨ７，５，５ｍＭ
ＥＤＴＡ、 １％５ＤＳ）に溶かす。塩化ナトリウムを
０．１Ｍ加えた後、フェノールクロロホルム（１： １
、Ｖ／Ｖ）を等量加え、よく撹拌後、遠心により２層
に分ける。水層を取り、再度同様の操作を繰り返す。水
層に２倍容のエタノールを加え、得られた沈澱を７０％
エタノールをで洗浄後回収する。全ＲＮＡは、＋５Ｘ
５ＳＣ（０，１１２５Ｍ塩化ナトリウム、Ｏ，０１１２
５Ｍクエン酸ナトリウム）、６％ホルムアミド液にて変
性し、６５℃１０分間保温後、水中にて冷却し、ナイロ
ン膜上にスポットする。Apply sufficient pressure to the mixture in a 22 gauge syringe needle.
Cut the passed DNA. Ultracentrifuge rotor 5W28
Add 1/3 volume of cesium TF to a polyaromer centrifuge tube.
Pour the (1,511F/+I2) solution, layer the above sample solution on top, and add 5 liters of 128 to 28. OOOrpm 2
Centrifuge for 4 hours and discard the supernatant. Transfer the RNA pellet to RNA lysis solution (10mA Tris-HCl, pH 7, 5, 5mM).
Dissolve in EDTA, 1% 5DS). After adding 0.1M sodium chloride, phenolchloroform (1:1
, V/V), stir well, and separate into two layers by centrifugation. Remove the water layer and repeat the same operation again. Add 2 times the volume of ethanol to the aqueous layer and dilute the resulting precipitate to 70%.
Collect after washing with ethanol. Total RNA is +5X
5SC (0,1125M sodium chloride, O,0112
5M sodium citrate) and 6% formamide solution, kept at 65°C for 10 minutes, cooled in water, and spotted on a nylon membrane.
次にプレハイブリダイゼーション溶液（０，９Ｍ塩化ナ
トリウム、０．０５Ｍリン酸ナトリウム、ｐＨ７，７，
０，０００５Ｍ ＥＤＴＡ、０．０１％フィコール、０
．０１％ポリビニルピロリドン、５０％ホルムアミド、
１０％デキストラン硫酸、■００μｇｈＱ変性サケ精子
ＤＮＡ）中にて４２℃６時間プレハイブリダイゼーショ
ンを行う。６時間後に液を捨てて、ハイブリダイゼーシ
ョン溶液（プレハイブリダイゼーション溶液と同じ）及
び３ｆｐで５末端標識したオリゴヌクレオチドを加え、
４２℃で１晩ハイブリダイゼーノヨンを行う。ナイロン
膜を袋よりとり出し、５ｘ ５ＳＰＥ（０，９Ｍ塩化ナ
トリウム。Next, prehybridization solution (0.9M sodium chloride, 0.05M sodium phosphate, pH 7.7,
0,0005M EDTA, 0.01% Ficoll, 0
．． 01% polyvinylpyrrolidone, 50% formamide,
Prehybridization is performed at 42° C. for 6 hours in 10% dextran sulfate, 00 μghQ denatured salmon sperm DNA). After 6 hours, the solution was discarded, and a hybridization solution (same as the prehybridization solution) and an oligonucleotide labeled at the 5-end with 3fp were added.
Perform hybridization overnight at 42°C. Remove the nylon membrane from the bag and add 5x 5SPE (0.9M sodium chloride).
０．３Ｍリン酸ナトリウム、ｐｆＴ７．７，０．ＯＨＩ
Ｅｌ）ＴＡ）、０．１％ＳＤＳ中で室温５分間インキ
ュベートする。この操作を繰り返す。０． ＩＸ ５Ｓ
ＰＥ （０，０層８Ｍ塩化ナトリウム。0.3M sodium phosphate, pfT7.7,0. OHI
El) TA), incubate in 0.1% SDS for 5 minutes at room temperature. Repeat this operation. 0. IX5S
PE (0,0 layer 8M sodium chloride.
０．００６１ｉ１リン酸ナトリウム、 ｐ１１７．７，
０．０００６Ｍ ＥＤＴＡ）−〇、１％ＳＤＳ中で５０
℃ＩＯ分間インキュベートする。乾燥後オートラジオグ
ラフィーを行う。0.0061i1 Sodium Phosphate, p117.7,
0.0006M EDTA) - 50 in 1% SDS
Incubate for IO minutes. Autoradiography is performed after drying.
Claims (1)
配列中の少なくとも１個のヌクレオチドの変化の存在又
は、不存在を検出するオリゴヌクレオチドプローブであ
り、ＤＰｗ２、ＤＰｗ３、ＤＰｗ４、Ｃｐ６３の型判別
を行う時に使用する上記オリゴヌクレオチドプローブ。 ２、ＤＰ抗原のβ鎖遺伝子をコードする１９塩基の核酸
で下記の塩基配列の一部または、全部を含むことを特徴
とする特許請求範囲第１項記載のオリゴヌクレオチドプ
ローブ。 ＧＧＡＡＣＣＡＣＴＡＣＴＴＡＡＣＡＡＡ ＤＰ１ＡＧ
ＴＡＴＣＣＡＡＴＴＴＧＧＡＡＧＴＴ ＤＰ２ＴＧＧＡ
ＡＣＣＡＧＧＴＧＣＴＡＡＣＡＡ ＤＰ３ＡＧＴＡＴＣ
ＣＴＴＴＴＴＧＧＡＡＧＴＴ ＤＰ４ＴＧＧＡＧＣＣＡ
ＧＡＴＧＣＴＡＡＣＧＡ ＤＰ５ＣＴＧＴＣＧＡＡＧＣ
ＧＣＡＣＧＡＡＣＴ ＤＰ６ＣＣＡＧＴＡＣＴＣＣＧＣ
ＡＧＣＡＧＧＣ ＤＰ７[Claims] 1. An oligonucleotide probe that detects the presence or absence of at least one nucleotide change in the nucleic acid sequence of the HLA (human leukocyte membrane antigen) DP antigen gene, which includes DPw2, DPw3, DPw4. , the above-mentioned oligonucleotide probe used when performing Cp63 type determination. 2. The oligonucleotide probe according to claim 1, which is a 19-base nucleic acid that encodes the β-chain gene of the DP antigen and contains part or all of the following base sequence. GGAACCACTACTTAACAAA DP1AG
TATCCAATTTGGAAGTT DP2TGGA
ACCAGGTGCTAACAA DP3AGTATC
CTTTTTGGAAGTT DP4TGGAGCCA
GATGCTAACGA DP5CTGTCGAAGC
GCACGAACT DP6CCAGTACTCCGC
AGCAGGC DP7
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP63239823A JPH0288967A (en) | 1988-09-27 | 1988-09-27 | Novel oligonucleotide probe |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP63239823A JPH0288967A (en) | 1988-09-27 | 1988-09-27 | Novel oligonucleotide probe |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH0288967A true JPH0288967A (en) | 1990-03-29 |
Family
ID=17050382
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP63239823A Pending JPH0288967A (en) | 1988-09-27 | 1988-09-27 | Novel oligonucleotide probe |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0288967A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0584094A4 (en) * | 1991-05-08 | 1997-07-02 | Univ Minnesota | Dna sequence-based hla class i typing method |
| JP2008050108A (en) * | 2006-08-24 | 2008-03-06 | Kobayashi Create Co Ltd | Concealing label peeling-off device |
-
1988
- 1988-09-27 JP JP63239823A patent/JPH0288967A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0584094A4 (en) * | 1991-05-08 | 1997-07-02 | Univ Minnesota | Dna sequence-based hla class i typing method |
| JP2008050108A (en) * | 2006-08-24 | 2008-03-06 | Kobayashi Create Co Ltd | Concealing label peeling-off device |
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