JP2001501301A - Methods and materials for optimization of electronic hybridization reactions - Google Patents
Methods and materials for optimization of electronic hybridization reactionsInfo
- Publication number
- JP2001501301A JP2001501301A JP10512676A JP51267698A JP2001501301A JP 2001501301 A JP2001501301 A JP 2001501301A JP 10512676 A JP10512676 A JP 10512676A JP 51267698 A JP51267698 A JP 51267698A JP 2001501301 A JP2001501301 A JP 2001501301A
- Authority
- JP
- Japan
- Prior art keywords
- nucleic acid
- buffer
- hybridization
- target nucleic
- histidine
- 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.)
- Granted
Links
- 238000009396 hybridization Methods 0.000 title claims abstract description 40
- 238000000034 method Methods 0.000 title claims description 45
- 238000006243 chemical reaction Methods 0.000 title abstract description 13
- 239000000463 material Substances 0.000 title description 2
- 238000005457 optimization Methods 0.000 title description 2
- 239000000872 buffer Substances 0.000 claims abstract description 60
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 claims abstract description 31
- 235000018417 cysteine Nutrition 0.000 claims abstract description 25
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 claims abstract description 25
- 238000012360 testing method Methods 0.000 claims abstract description 11
- 238000004377 microelectronic Methods 0.000 claims abstract description 7
- 230000003139 buffering effect Effects 0.000 claims description 18
- 230000005684 electric field Effects 0.000 claims description 8
- 150000001875 compounds Chemical class 0.000 claims description 7
- 238000009825 accumulation Methods 0.000 claims description 5
- 230000015572 biosynthetic process Effects 0.000 claims description 4
- 229930014626 natural product Natural products 0.000 claims description 3
- 230000000087 stabilizing effect Effects 0.000 claims description 3
- 150000007523 nucleic acids Chemical class 0.000 claims 21
- 102000039446 nucleic acids Human genes 0.000 claims 21
- 108020004707 nucleic acids Proteins 0.000 claims 21
- 230000002708 enhancing effect Effects 0.000 claims 1
- 235000001014 amino acid Nutrition 0.000 abstract description 15
- 150000001413 amino acids Chemical class 0.000 abstract description 15
- 239000003792 electrolyte Substances 0.000 abstract description 13
- 239000007853 buffer solution Substances 0.000 abstract description 7
- 238000012546 transfer Methods 0.000 abstract description 3
- 108020004414 DNA Proteins 0.000 description 42
- 239000000243 solution Substances 0.000 description 10
- 238000001962 electrophoresis Methods 0.000 description 8
- 241000894007 species Species 0.000 description 8
- 230000035945 sensitivity Effects 0.000 description 7
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- UCMIRNVEIXFBKS-UHFFFAOYSA-N beta-alanine Chemical compound NCCC(O)=O UCMIRNVEIXFBKS-UHFFFAOYSA-N 0.000 description 6
- 239000001488 sodium phosphate Substances 0.000 description 6
- 229910000162 sodium phosphate Inorganic materials 0.000 description 6
- XOAAWQZATWQOTB-UHFFFAOYSA-N taurine Chemical compound NCCS(O)(=O)=O XOAAWQZATWQOTB-UHFFFAOYSA-N 0.000 description 6
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 6
- 235000018102 proteins Nutrition 0.000 description 5
- 108090000623 proteins and genes Proteins 0.000 description 5
- 102000004169 proteins and genes Human genes 0.000 description 5
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 4
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 description 4
- 239000004472 Lysine Substances 0.000 description 4
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 4
- 150000002500 ions Chemical class 0.000 description 4
- 235000018977 lysine Nutrition 0.000 description 4
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 description 3
- 239000006173 Good's buffer Substances 0.000 description 3
- 239000012491 analyte Substances 0.000 description 3
- 229940000635 beta-alanine Drugs 0.000 description 3
- 229920001222 biopolymer Polymers 0.000 description 3
- 150000001768 cations Chemical class 0.000 description 3
- 230000007423 decrease Effects 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 239000008151 electrolyte solution Substances 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 229960003080 taurine Drugs 0.000 description 3
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 2
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 2
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 2
- 102000053602 DNA Human genes 0.000 description 2
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 2
- 239000004471 Glycine Substances 0.000 description 2
- 239000007995 HEPES buffer Substances 0.000 description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 2
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 2
- SEQKRHFRPICQDD-UHFFFAOYSA-N N-tris(hydroxymethyl)methylglycine Chemical compound OCC(CO)(CO)[NH2+]CC([O-])=O SEQKRHFRPICQDD-UHFFFAOYSA-N 0.000 description 2
- 229910019142 PO4 Inorganic materials 0.000 description 2
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 2
- 108091036333 Rapid DNA Proteins 0.000 description 2
- 108010090804 Streptavidin Proteins 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 230000002378 acidificating effect Effects 0.000 description 2
- 235000004279 alanine Nutrition 0.000 description 2
- 150000001450 anions Chemical class 0.000 description 2
- 229960002433 cysteine Drugs 0.000 description 2
- -1 dansyl amino acids Chemical class 0.000 description 2
- 238000001502 gel electrophoresis Methods 0.000 description 2
- 235000013922 glutamic acid Nutrition 0.000 description 2
- 239000004220 glutamic acid Substances 0.000 description 2
- 125000002883 imidazolyl group Chemical group 0.000 description 2
- 238000001155 isoelectric focusing Methods 0.000 description 2
- 230000003287 optical effect Effects 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 2
- 239000010452 phosphate Substances 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 239000000523 sample Substances 0.000 description 2
- 239000001509 sodium citrate Substances 0.000 description 2
- 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 2
- KDYFGRWQOYBRFD-UHFFFAOYSA-L succinate(2-) Chemical compound [O-]C(=O)CCC([O-])=O KDYFGRWQOYBRFD-UHFFFAOYSA-L 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- ZSLUVFAKFWKJRC-IGMARMGPSA-N 232Th Chemical compound [232Th] ZSLUVFAKFWKJRC-IGMARMGPSA-N 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 1
- 108020003215 DNA Probes Proteins 0.000 description 1
- 239000003298 DNA probe Substances 0.000 description 1
- RWSOTUBLDIXVET-UHFFFAOYSA-N Dihydrogen sulfide Chemical compound S RWSOTUBLDIXVET-UHFFFAOYSA-N 0.000 description 1
- 241000255925 Diptera Species 0.000 description 1
- 206010013457 Dissociation Diseases 0.000 description 1
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 1
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 1
- WHXSMMKQMYFTQS-UHFFFAOYSA-N Lithium Chemical compound [Li] WHXSMMKQMYFTQS-UHFFFAOYSA-N 0.000 description 1
- 239000007993 MOPS buffer Substances 0.000 description 1
- 241001460678 Napo <wasp> Species 0.000 description 1
- UZMAPBJVXOGOFT-UHFFFAOYSA-N Syringetin Natural products COC1=C(O)C(OC)=CC(C2=C(C(=O)C3=C(O)C=C(O)C=C3O2)O)=C1 UZMAPBJVXOGOFT-UHFFFAOYSA-N 0.000 description 1
- 229910052776 Thorium Inorganic materials 0.000 description 1
- ATJFFYVFTNAWJD-UHFFFAOYSA-N Tin Chemical compound [Sn] ATJFFYVFTNAWJD-UHFFFAOYSA-N 0.000 description 1
- 239000007997 Tricine buffer Substances 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 208000034953 Twin anemia-polycythemia sequence Diseases 0.000 description 1
- 238000002479 acid--base titration Methods 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 239000002249 anxiolytic agent Substances 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 235000003704 aspartic acid Nutrition 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 229940125717 barbiturate Drugs 0.000 description 1
- HNYOPLTXPVRDBG-UHFFFAOYSA-N barbituric acid Chemical compound O=C1CC(=O)NC(=O)N1 HNYOPLTXPVRDBG-UHFFFAOYSA-N 0.000 description 1
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 1
- 229960002685 biotin Drugs 0.000 description 1
- 235000020958 biotin Nutrition 0.000 description 1
- 239000011616 biotin Substances 0.000 description 1
- 239000004327 boric acid Substances 0.000 description 1
- 239000008364 bulk solution Substances 0.000 description 1
- 238000005251 capillar electrophoresis Methods 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- KCFYHBSOLOXZIF-UHFFFAOYSA-N dihydrochrysin Natural products COC1=C(O)C(OC)=CC(C2OC3=CC(O)=CC(O)=C3C(=O)C2)=C1 KCFYHBSOLOXZIF-UHFFFAOYSA-N 0.000 description 1
- URSLCTBXQMKCFE-UHFFFAOYSA-N dihydrogenborate Chemical compound OB(O)[O-] URSLCTBXQMKCFE-UHFFFAOYSA-N 0.000 description 1
- 238000010494 dissociation reaction Methods 0.000 description 1
- 230000005593 dissociations Effects 0.000 description 1
- 208000018459 dissociative disease Diseases 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 230000005611 electricity Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000012145 high-salt buffer Substances 0.000 description 1
- 230000036571 hydration Effects 0.000 description 1
- 238000006703 hydration reaction Methods 0.000 description 1
- 238000002218 isotachophoresis Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 229910052744 lithium Inorganic materials 0.000 description 1
- VZCYOOQTPOCHFL-UPHRSURJSA-N maleic acid Chemical compound OC(=O)\C=C/C(O)=O VZCYOOQTPOCHFL-UPHRSURJSA-N 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000001823 molecular biology technique Methods 0.000 description 1
- 238000007899 nucleic acid hybridization Methods 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 239000001103 potassium chloride Substances 0.000 description 1
- 235000011164 potassium chloride Nutrition 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 125000003396 thiol group Chemical group [H]S* 0.000 description 1
- VZCYOOQTPOCHFL-UHFFFAOYSA-N trans-butenedioic acid Natural products OC(=O)C=CC(O)=O VZCYOOQTPOCHFL-UHFFFAOYSA-N 0.000 description 1
- 238000000539 two dimensional gel electrophoresis Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
-
- H—ELECTRICITY
- H01—ELECTRIC ELEMENTS
- H01L—SEMICONDUCTOR DEVICES NOT COVERED BY CLASS H10
- H01L29/00—Semiconductor devices specially adapted for rectifying, amplifying, oscillating or switching and having potential barriers; Capacitors or resistors having potential barriers, e.g. a PN-junction depletion layer or carrier concentration layer; Details of semiconductor bodies or of electrodes thereof ; Multistep manufacturing processes therefor
- H01L29/66—Types of semiconductor device ; Multistep manufacturing processes therefor
- H01L29/66007—Multistep manufacturing processes
- H01L29/66075—Multistep manufacturing processes of devices having semiconductor bodies comprising group 14 or group 13/15 materials
- H01L29/66227—Multistep manufacturing processes of devices having semiconductor bodies comprising group 14 or group 13/15 materials the devices being controllable only by the electric current supplied or the electric potential applied, to an electrode which does not carry the current to be rectified, amplified or switched, e.g. three-terminal devices
- H01L29/66409—Unipolar field-effect transistors
- H01L29/66477—Unipolar field-effect transistors with an insulated gate, i.e. MISFET
- H01L29/6656—Unipolar field-effect transistors with an insulated gate, i.e. MISFET using multiple spacer layers, e.g. multiple sidewall spacers
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6832—Enhancement of hybridisation reaction
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Microelectronics & Electronic Packaging (AREA)
- Power Engineering (AREA)
- Microbiology (AREA)
- General Physics & Mathematics (AREA)
- Biophysics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Manufacturing & Machinery (AREA)
- Ceramic Engineering (AREA)
- Condensed Matter Physics & Semiconductors (AREA)
- Electrochemistry (AREA)
- Pathology (AREA)
- Computer Hardware Design (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analyzing Materials By The Use Of Electric Means (AREA)
Abstract
(57)【要約】 以下の発明は、超小型電子チップおよび装置において、ＤＮＡ輸送速度、ＤＮＡハイブリダイゼーション反応効率および全ハイブリダイゼーション特異性を改良または最適化する種々のパラメーター、電解液（緩衝液）および他の条件に関係する発見に関する。詳細には、本発明は、低導電率の双性イオン緩衝溶液、とりわけ約５０ｍＭの濃度並びにｐＩ（等電点約ｐＨ ７.４７）およびその付近で調製されたアミノ酸ヒスチジンを含有するそれらが、迅速な電気泳動ＤＮＡ輸送および効率的なハイブリダイゼーション反応について共に、最適条件を提供するという発見に関する。次の最良の既知緩衝剤、システインに関して少なくとも１０倍のハイブリダイゼーション効率を達成する。試験データは、システインに比較してハイブリダイゼーション効率において約５０,０００倍の増大を示す。 (57) [Summary] The following invention relates to various parameters, electrolytes (buffers) and other conditions that improve or optimize DNA transport rate, DNA hybridization reaction efficiency and overall hybridization specificity in microelectronic chips and devices. Regarding discovery. In particular, the present invention relates to low conductivity zwitterionic buffer solutions, especially those containing the amino acid histidine prepared at and near the concentration of about 50 mM and the pI (isoelectric point about pH 7.47). Both the rapid electrophoretic DNA transfer and the efficient hybridization reaction relate to the discovery that they provide optimal conditions. Achieve at least a 10-fold hybridization efficiency with the next best known buffer, cysteine. The test data shows an approximately 50,000-fold increase in hybridization efficiency compared to cysteine.
Description
【発明の詳細な説明】 エレクトロニック・ハイブリダイゼーション反応の 最適化のための方法および物質発明の分野 本発明は、医学診断用途、生物学的用途および他の用途に適合する電子装置に 用いる緩衝液および電解液に関する。より詳細には、超小型電子医学診断装置で 行うＤＮＡハイブリダイゼーション分析での有利な使用を目的とした緩衝液およ び電解液に関する。発明の背景 近年、超小型電子技術および分子生物学を結合させた装置への関心が増大して いる。かかるシステムの１つは、１９９３年１１月１日付で出願され、現在米国 特許第５,６０５,６６２号として公開されたシリアル番号第０８/１４６,５０４ 号の「ＡＣＴＩＶＥ ＰＲＯＧＲＡＭＭＡＢＬＥ ＥＬＥＣＴＲＯＮＩＣ ＤＥ ＳＶＩＣＥ ＦＯＲ ＭＯＬＥＣＵＬＡＲ ＢＩＯＬＯＧＩＣＡＬ ＡＮＡＬＹ ＳＩＳ ＡＮＤ ＤＩＡＧＮＯＳＴＩＣＳ」に開示され、ここに出典明示して本 明細書の一部とみなす。そこに開示されたシステムは、ＡＰＥＸシステムと言わ れる。ＡＰＥＸシステムは、核酸ハイブリダイゼーション、抗体/抗原反応、臨 床診断および生体高分子合成のごとき分子生物学的反応において、有利に用いら れ、広範な機能を果たすことができる。 ＡＰＥＸ型装置は、それらの操作に緩衝液および電解液を利用する。緩衝液は 、酸またはアルカリの添加に対してｐＨ変化に抵抗性のある化学溶液として定義 される。例えば、Ｄｉｃｔｉｏｎａｒｙ ｏｆ Ｂｉｏｔｅｃｈｎｏｌｏｇｙ、２ 版、Ｊａｍｅｓ Ｃｏｏｍｂｓ、ｓｔｏｃｋｔｏｎ Ｐｒｅｓｓ参照。そこに記載 されたごとく、「伝統的に、無機塩（リン酸塩、炭酸塩）および有機塩（酢酸塩 、クエン酸塩、コハク酸塩、グリシン、マレイン酸塩、バルビツール酸塩等）に 基づく緩衝液 を生物学実験において用いた。」 ハイブリダイゼーション、反応、診断および合成を行う分子生物学的電子装置 において有利に用いられる緩衝液および電解液を見出すことが本発明の目的であ る。発明の概要 下記の発明は、本発明者らのＡＰＥＸ超小型電子チップおよび装置におけるＤ ＮＡ輸送速度、ＤＮＡハイブリダイゼーション反応の効率および全ハイブリダイ ゼーション特異性を改良または最適化する種々のパラメーター、電解液（緩衝液 ）および他の条件に関連する発見に関する。詳細には、本発明は低導電率双性イ オン緩衝溶液、とりわけ１０−１００ｍＭ、好ましくは約５０ｍＭの濃度または ｐＩ（等電点約ｐＨ７.４７）またはその付近にて調製されたアミノ酸ヒスチジ ンを含有するものが、迅速なＤＮＡ輸送および効率的なハイブリダイゼーション 反応について最適化条件を提供するという発見に関する。次の最もよく知られた 緩衝液、システインに対して少なくとも１０倍のハイブリダイゼーション効率が 達成される。試験データは、システインに比較してハイブリダイゼーション効率 においておよそ５０,０００倍の増大を示す。図表の簡単な記載 図１は、ヒスチジン緩衝液を利用したチェッカーボード配列の平面図である。発明の詳細な記載 さまざまなタイプの電解液/緩衝溶液において、ＤＮＡの電気泳動輸送および 他の荷電分析物に関係する種々の物理的パラメーターがある。ある種の装置、例 えば、前記の米国特許第５,６０５,６６２号に記載のごとき出願人のＡＰＥＸ装 置は、基本的に装置表面に電場を生成するＤＣ（直流）電気装置である。次いで 、これらの場は、装置表面に反対に（＋／−）偏った微小位置（ｍｉｃｒｏｌｏ ｃａｔｉｏｎ）間に荷電分子の電気泳動輸送を引き起こす。対照的に、いわゆる ジノセンサー（Ｇｅｎｏｓｅｎｓｏｒ）（インピーダンス・センサー）（例えば 、ＷＯ９３/２２６ ７８のＨｏｌｌｅｓら、「Ｏｐｔｉｃａｌ ａｎｄ Ｅｌｅｃｔｒｉｃａｌ Ｍ ｅｔｈｏｄｓ ａｎｄ Ａｐｐａｒａｔｕｓ ｆｏｒ Ｍｏｌｅｃｕｌａｒ Ｄ ｅｔｅｃｔｉｏｎ」参照）、および二次元電気泳動（ｄｉｅｌｅｃｔｒｏｐｈｏ ｒｅｓｉｓ）装置（例えば、Ｗａｓｈｉｚｕ ２５ Ｊｏｕｒｎａｌ ｏｆ Ｅ ｌｅｃｔｒｏｓｔａｔｉｃｓ）１０９−１２３、１９９０参照）は、ＡＣ電場で の使用を含む。これらの装置に関する重要な相違点は、これらのＡＣ場が印加さ れる場合、これらのシステムのいずれにおいても本質的に正味の電流はない、す なわち、荷電分子輸送用の電気泳動の推進力はない点である。ＡＰＥＸ型装置は 、電圧が印加される場合、かなりの正味の直流電流（ＤＣ）を生じさせ、それは 「電気泳動のサイン」として認識される。電気泳動において、イオンまたは荷電 粒子の移動は、電場勾配の方向に沿った電気力によって生じ、電流および電圧の 関係は、この技術に重要である。この電気泳動的移動は、印加された電圧の影響 下、溶液中の電流伝導性としてそれ自体肉眼的に示され、 Ｖ＝Ｒ×Ｉ Ｖは電位である Ｒは電解液の電気抵抗である［Ｖ×Ａ-1＝Ｒ（Ω）］ Ｉは電流である［Ａ］。 のオームの法則に従う。 溶液の抵抗は、伝導率測定器によって測定できる導電率の逆数である。導電率 は、主に緩衝液/電解液のイオン種およびそれらの濃度に依存し；従って、これ らのパラメーターは電場関連分子生物学的技術に非常に重要である。生じた電場 は本当に顕微鏡的環境におけるものであるが、基本的な電流/電圧関係は、いず れの他の電気泳動システムについてとも同様にＡＰＥＸ技術につき本質的に同じ である。 電流および電圧を供給する種々の方法および電流および電圧の筋書が、いかに してかかるシステムの性能を改良するのに見出されたかに関して、ＡＰＥＸシス テムはユニークな特徴がある。詳細には、いくつかのＤＣパルス手法（線形およ び対数勾配）が、改良されたハイブリダイゼーションストリンジェンシーを提供 するらしい。電気泳動輸送−対−イオン強度 荷電分析物種（蛋白質、ＤＮＡ等）の移動度において対数的減少があり、電解 溶液のイオン強度の平方根に反比例することが、電気泳動の分野においてよく確 立されている（「Ｃａｐｉｌｌａｒｙ Ｅｌｅｃｔｒｏｐｈｏｒｅｓｉｓ：Ｐｒ ｉｎｃｉｐｌｅｓ ａｎｄ Ｐｒａｃｔｉｃｅ」、Ｒ．ＫｕｈｎおよびＳ．Ｈｏ ｆｆｓｔｅｔｔｅｒ、Ｓｐｒｉｎｇｅｒ−Ｖｅｒｌａｇ、１９９３の８３頁およ び図３．１６参照）。いずれかの所与の一定の電場強度にて、電解液濃度は分析 物種（蛋白質、ＤＮＡ等）に対して減少するにつれ、分析物はより速い速度にて 輸送されるであろう。ダンシルアミノ酸について、この効果を示す同様の結果が 、Ｊ．Ｊ．Ｉｓｓａｑら、Ｃｈｒｏｍａｔｏｇｒａｐｈｉａ Ｖｏｌ．３２、＃ ３/４、１９９１年８月、１５５ないし１６１頁（詳細には、１５７頁図３参照 ）によって示されている。ＤＮＡについてこの効果は異なる電解溶液であること を示す結果が、Ｐ．Ｄ．ＲｏｓｓおよびＲ．Ｌ．Ｓｃｒｕｇｇｓ、Ｂｉｏｐｏｌ ｙｍｅｒｓ Ｖｏｌ．２、２３１ないし２３６頁、１９６４（詳細には、２３２ 頁図１参照）に示されている。イオン強度/導電率の関係 カチオンを含む非緩衝性電解液（塩化ナトリウム、塩化カリウム等）について、 イオン強度および導電率は等価である、すなわち導電率は通常イオン強度に比例 する （リン酸塩、酢酸塩、クエン酸塩、コハク酸塩等）について、イオン強度および 導電率は、通常等価になるであろう、すなわち導電率はイオン強度に比例する。 双性イオン種（それらのｐＩにて正味の荷電をしない）を有することができる緩 衝性電解液［グッド緩衝液（ＭＯＰＳ、ＨＥＰＥＳ、ＴＡＰＳ、トリシン、ビシ ン）、アミノ酸緩衝液、両性電解液等］について、導電率は、等電点（ｐＩ）お よび（ｐＫａ）間の毎ｐＨ単位差につきおよそ１０倍減少するであろう。例えば 、その双性イオン状態（-ＯＯＣ−ＣＨ(Ｒ)−ＮＨ3 +）にあるアミノ酸は、「ア ミノ酸部分」が００倍低い導電率値を有するであろう。従って、そのｐＩから離れるにつれ、形 式的な負または正の電荷がアミノ酸部分で発生し、導電率およびイオン強度は関 連しだすであろう。しかしながら、ｐＩまたはその付近にて、導電率は所与のイ オン強度または濃度で予期されるより大変低いであろう。それらのｐＩまたはそ の付近にて用いられる場合、電気泳動のテキストは、グッド緩衝液およびアミノ 酸緩衝液が「高イオン強度または濃度にて低導電率」を有すると言及する（Ｃａ ｐｉｌｌａｒｙ Ｅｌｅｃｔｒｏｐｈｏｒｅｓｉｓ：Ｐｒｉｎｃｉｐｌｅｓ ａ ｎｄ Ｐｒａｃｔｉｃｅ」、Ｒ．ＫｕｈｎおよびＳ．Ｈｏｆｆｓｔｅｔｔｅｒ、 Ｓｐｒｉｎｇｅｒ−Ｖｅｒｌａｇ、１９９３の８８頁参照）。通常用いられる電 気泳動緩衝液「トリス−ホウ酸」は、実際上、そのイオン強度または濃度から予 期されるよりかなり低い導電率を有する。これは、溶液中で比較的安定した双性 イオン複合体を形成する「トリス・カチオン」および「ホウ酸アニオン」のため かも知れない。１００ｍＭトリス−ホウ酸溶液の導電率は６９４μＳ/ｃｍであ ると測定され、これはそのイオン強度から予期されるよりおよそ２０倍低く、５ ｍＭリン酸ナトリウムまたは塩化ナトリウム溶液とおよそ等価であった。表１は 、多数の輸送緩衝液の導電率測定値を示す。 双性イオン緩衝液/導電率/輸送速度 それらのｐＩまたはその付近にて、双性イオン緩衝液（グッド緩衝液、アミノ 酸緩衝液）またはトリス−ホウ酸緩衝液を用いた場合、ある有利さがＤＮＡの電 気泳動輸送の割合および速度に関して存在する。これらは、１）かかる緩衝液は 、比較的高濃度にて用いることができ、緩衝能を増大させ、２）それらの導電率 が同一濃度にて他のタイプの緩衝液よりかなり低く、また、３）注目する分析物 （ＤＮＡ）について高い電気泳動輸送率の有利さを得ることである。等電点（ｐＩ）での双性イオン緩衝能 アミノ酸緩衝液は、それらのplにて緩衝特性を有する。所与のアミノ酸は、そ のｐＩにてその「最高緩衝能」を有するかまたは有しないかもしれないので、そ れはいくらか緩衝能を有するであろう。緩衝能は、ｐＩおよびｐＫａの間の毎ｐ Ｈ単位差について１０倍減少し、３種のイオン化可能基（ヒスチジン、システイ ン、リジン、グルタミン酸、アスパラギン酸等）を有するアミノ酸は、一般的に ２種の解離のみを有するアミノ酸よりそれらのｐＩにて高い緩衝能を有する。例 えば、それらのｐＩにて比較的低い緩衝能を有するアラニンまたはグリシンに比 較して、ヒスチジンｐＩ＝７.４７、リジンｐＩ＝９.７４およびグルタミン酸ｐ Ｉ＝３.２２の全ては、それらのｐＩにて比較的良好な緩衝能を有する（Ａ．Ｌ ．Ｌｅｈｎｉｇｅｒ、Ｂｉｏｃｈｅｍｉｓｔｒｙ、２版、Ｗｏｒｔｈ Ｐｕｂｌ ｉｓｈｅｒｓ、Ｎｅｗ Ｙｏｒｋ、１９７５；詳細には７９頁の図４−８および ８０頁図４−９参照）。ヒスチジンは、ゲル電気泳動で使用される緩衝液として 推奨されてきたが、ハイブリダイゼーションは、かかるシステムにおいては行わ れない。米国特許第４,９３６,９６３号参照。システインは、緩衝能に関してよ り中間的な位置にある。システインのｐＩは５.０２、αカルボキシル基のｐＫ ａは１.７１、スルフヒドリルのｐＫａは８.３３、またαアミノ基のｐＫａは１ ０.７８である。２５０ｍＭシステインの酸/塩基滴定曲線は、システインが２０ ｍＭリン酸ナトリウムより約ｐＨ５にて良好な「緩衝能」を有することを示す。 ｐＨ４ないし６の範囲にて、システインの緩衝能は、とりわけ高いｐＨにて、２ ０ｍＭリン酸ナトリウムより有意に優れ ている。しかしながら、これらのｐＨ範囲において、２５０ｍＭシステイン溶液 の導電率は、１００倍大きい約２.９ｍＳ/ｃｍの値を有する２０ｍＭリン酸ナト リウムに比較して非常に低い約２３μＳ/ｃｍである。図１は、種々の輸送用緩 衝液の導電率測定値を示す。 ２０年以上前に開発されたいくつかの電気泳動技術は、「それらのｐＩにて」 双性イオン緩衝液中の蛋白質を分離する能力に基づいている。これらの技術は、 等電点電気泳動法、イソタコフォレシス（Ｉｓｏｔａｃｈｏｐｈｏｒｅｓｉｓ） および等電点分画法と呼ばれる（Ｂ．Ｄ．ＨａｍｅｓおよびＤ．Ｒｉｃｋｗｏｏ ｄによる「Ｇｅｌ Ｅｌｅｃｔｒｏｐｈｏｒｅｓｉｓ ｏｆ ｐｒｏｔｅｉｎｓ ：Ａ Ｐｒａｃｔｉｃａｌ Ａｐｐｒｏａｃｈ」の第３章および第４章、ＩＲＬ Ｐｒｅｓｓ １９８１参照）。これらの適用について、全てそれらのｐＩにて 、いくつかのアミノ酸緩衝液およびグッド緩衝液が用いられた（前記引用文献の １６８頁表２参照）。低イオン強度および低導電率緩衝液におけるＤＮＡ輸送 ２.５％アガロースをコートした５５８０チップおよびＢｙＴｒ−ＲＣＡ５蛍 光性プローブを用いて、一連の蛍光性チェッカーボード試験を行った。以下のシ ステム：（１）２５０ｍＭ ＨＥＰＥＳ（低導電率）、（２）１０μＭ コハク 酸ナトリウム、（３）１０μＭ クエン酸ナトリウムおよび（４）蒸留水の全て に迅速な（６秒）チェッカーボード・アドレッシングを達成できた。クエン酸ナ トリウムの結果を図１に示す。一方、いくつかのタイプの低導電率または低イオ ン強度溶液は、幾分良好な特質であるチェッカーボード・アドレッシングを有し 、迅速なＤＮＡ輸送（８０μｍパッドの６ないし１２秒のＤＮＡ蓄積）がこれら の全てのシステムを用いて達成された。さらに、ＤＮＡ（それ自体がポリアニオ ン）は、導電率を提供するバルク溶液中に存在する電解質であるので、蒸留水中 のＤＮＡアドレッシングＡＰＥＸチップが可能である。図１は、ヒスチジンを用 いたＡＰＥＸチップの平面図を示す。電気泳動輸送速度およびカチオン/アニオン種の関係 荷電分析物種（ＤＮＡ、蛋白質等）の移動度が電解溶液のイオン強度に関係す るという事実に加えて、また、その移動度は、電解溶液中のカチオンおよびアニ オン種の性質によって大きく影響される（「Ｃａｐｉｌｌａｒｙ Ｅｌｅｃｔｒ ｏｐｈｏｒｅｓｉｓ：Ｐｒｉｎｃｉｐｌｅｓ ａｎｄ Ｐｒａｃｔｉｃｅ」参照 ）。前記Ｂｉｏｐｏｌｙｍｅｒ、Ｖｏｌ．２、ｐｐ．２３１−２３６、１９６４ 文献に、ＤＮＡ輸送についてこの特殊な点が示されている。この文献の２３２頁 の図１は、同一のイオン強度にて、異なる１価アニオン（Ｌｉ+＞Ｎａ+＞Ｋ+＞ ＴＭＡ ）を有する電解液を用いる場合のＤＮＡ移動度の変化を示す。基本的に は、異なるカチオンは、ＤＮＡリン酸基で異なる会合定数を有し、および/また はＤＮＡ分子周囲の水和領域を変化させ、これは輸送速度の変化に導くことがで きる。 本発明は、電場分子生物学的装置、とりわけＡＰＥＸ微小電子チップおよび装 置において、ＤＮＡ輸送速度、ＤＮＡハイブリダイゼーション反応効率および全 ハイブリダイゼーション特異性を改良または至適化する種々のパラメーター、電 解液（緩衝液）および他の条件に関連する本発明者らの発見に関する。詳細には 、本発明は、ｐＩ（等電点約７.４７）またはその付近で１０−１００ｍＭ、と りわけ約５０ｍＭの濃度にて調製されたアミノ酸ヒスチジンを含有する低導電率 の双性イオン緩衝溶液が、迅速な電気泳動ＤＮＡ輸送および効率的なハイブリダ イゼーション反応について共に至適条件を提供するという本発明者の発見に関す る。ヒスチジン緩衝液のこの有利さは、特にＡＰＥＸチップ型装置に重要である 。 これらの特別な装置（ミクロ機械加工型装置とは対照的に）は、印加できる電 流および電圧の量に関して限界を有する。この限界は、同一緩衝液システムを用 いて迅速な輸送および効率の良いハイブリダイゼーションを共に達成するのを困 難にする。これらの場合、限られた電流/電圧が迅速な輸送を依然として引き起 こす低導電率緩衝液（システインまたはアラニン）中で、ＤＮＡ輸送を行った。 これらの条件下、ＤＮＡは試験部位で蓄積したが、効率的にハイブリダイズしな かった。これらの低導電率緩衝液中の輸送の後、溶液を高い塩緩衝液（＞１００ ｍＭ 塩化ナトリウムまたはリン酸ナトリウム）に変更し、これは次いで試験部 位で効率的なハイブリダイゼーションを引き起こした。 表２は、緩衝能、ｐＨおよび導電率のパラメーターと、ＡＰＥＸチップ装置を 用いたＤＮＡ蓄積およびハイブリダイゼーション感度（効率）とを関連付ける一 連試験についての結果を示す。 詳細には、表２は、試験部位で特異的捕獲ＤＮＡへの輸送された標的ＤＮＡの ハイブリダイゼーションに対する種々の双性イオンアミノ酸緩衝液[β−アラニ ン、タウリン、システイン、ヒスチジン、リジンおよびリン酸ナトリウム（双性 イオン緩衝液ではない）]の効果を示す。輸送に関して、一般的に導電率は同一 場条件下で輸送と相関する。β−アラニン、タウリンおよびシステインは、優れ た輸送を示し、ヒスチジンは良好な輸送を示し、また、リジンおよびＮａＰＯ4 はかなりの輸送を示す。ＤＮＡハイブリダイゼーション感度は、負に荷電したポ リアニオン性リン酸骨格を有する「標準的ＤＮＡ」について報告されている。 1 / ｐＨ７.４に合わせた２０ｍＭ ＮａＰＯ4 また、ハイブリダイゼーション感度に加えて、表２はストレプトアビジン/ビオ チンＤＮＡプローブ捕獲親和性に関する感度を示す。 表２は、ＤＮＡ輸送（蓄積）と低導電率（β−アラニン、タウリン、システイ ン、ヒスチジン）との相関関係を明確に示す。表は、β−アラニン、システイン およびヒスチジンを用いるストレプトアビジン/ビオチンプローブの親和性反応 について優れた感度を示す。表２の感度データに反映されたごとく、ヒスチジン は、システインまたは２０ｍＭ ＮａＰＯ4のごとき他の緩衝液のいずれかより良 好なハイブリダイゼーション効率を４オーダー以上大きく提供する。システイン に対する改善は、少なくとも１０倍で、より詳細には１０2倍であり、最も詳細 には、少なくとも１０4倍である。最も重要なことには、表２は、ＤＮＡハイブ リダイゼーション感度(効率)がヒスチジン緩衝液について非常に良好であること を示すことである。従って、現在試験された双性イオンアミノ酸緩衝液のうち、 ヒスチジンが良好な輸送および良好なＤＮＡ/ＤＮＡハイブリダイゼーション効 率を共に提供する唯一のものである。 ヒスチジン緩衝液系の低導電率は、迅速な輸送（蓄積）の原因であると信じら れている。なぜヒスチジン緩衝液が、比較的有効なＤＮＡ/ＤＮＡハイブリダイ ゼーションを引き起こすのかに関していくつかの可能な説明がある。一つの長所 が、ヒスチジンの優れた緩衝能であるのかもしれない。７.４７のそのｐＩにて 、ヒスチジンは、酸性または塩基性条件下で優れた緩衝液である(Ａ．Ｌ．Ｌｅ ｈｎｉｎｇｅｒ、Ｂｉｏｃｈｅｍｉｓｔｒｙ、２版、ｗｏｒｔｈ Ｐｕｂｌｉｓ ｈｅｒｓ、Ｎｅｗ Ｙｏｒｋ、１９７５、８０頁図４−９参照)。ＤＮＡがハイ ブリダイゼーションのために蓄積され、ヒスチジンがこれらの条件を効率的に緩 衝するかもしれない正電極にて、ＡＰＥＸチップは酸を生成する。より重要なこ とは、これらの酸性条件（ｐＨ＜５）下、ヒスチジンのイミダゾール基のプロト ン化が分子のジカチオン種への転化を開始する。正に荷電したα−アミノ基およ び正に荷電したイミダゾール基を有するこのジカチオン種は、ハイブリダイゼー ションを促進し、ＡＰＥＸチップの正電極にて形成されたＤＮＡ/ＤＮＡハイブ リッドを安定化させるのを助けるかも知れないのが当てはまる場合であろう。カ チオン、ジカチオンおよびポリカチ オンは、二本鎖ＤＮＡ構造上の負に荷電したリン酸骨格の斥力を低下させること によって、ＤＮＡ/ＤＮＡハイブリッドの安定化を助けることが知られている。 また、ＤＮＡ/ＤＮＡ/ヒスチジンは、正電極にて生成する他の電気化学的生成物 (過酸化水素等)からのいくつかのタイプの安定化付加物を形成するかも知れない という可能性がある。 本具体例は、天然に生じたヒスチジンを利用するが、本発明は良好な緩衝能、 低導電率（または双性イオン特性）を有し、電荷安定化または付加物形成によっ て、ＤＮＡハイブリダイゼーションを安定化させる特性を有する他の天然または 合成化合物に十分に適用できる。 前記発明は、明確化および理解を目的とする図示および例の方法によって、い くらか詳細に記載したが、ある種の変更および修飾が、添付された請求の範囲の 精神または範囲から逸脱することなしに成され得ることは、本発明の教授の知識 において当業者には容易に明らかであろう。DETAILED DESCRIPTION OF THE INVENTION Electronic hybridization reaction Methods and materials for optimizationField of the invention The present invention relates to electronic devices adapted for medical diagnostic, biological and other applications. It relates to the buffer and electrolyte used. More specifically, with microelectronic medical diagnostic equipment Buffers and buffers for advantageous use in DNA hybridization assays And electrolytes.Background of the Invention In recent years, interest in devices that combine microelectronics and molecular biology has increased I have. One such system was filed on November 1, 1993 and is currently in the United States. Serial No. 08 / 146,504 published as Patent No. 5,605,662 No. "ACTIVE PROGRAMMABLE ELECTRONIC DE" SVICE FOR MOLECULAR BIOLOGICAL ANALY SIS AND DIAGNOSTICS ”, and hereby explicitly Considered as part of the specification. The system disclosed therein is called the APEX system It is. The APEX system is used for nucleic acid hybridization, antibody / antigen reaction, Advantageously used in molecular biological reactions such as bed diagnostics and biopolymer synthesis Can perform a wide range of functions. APEX-type devices utilize buffers and electrolytes for their operation. The buffer is Defined as a chemical solution that is resistant to pH changes to the addition of acids or alkalis Is done. For example, Dictionary of Biotechnology, 2 See, Editions, James Coombs, stockton Press. Listed there As has been said, "traditionally, inorganic salts (phosphate, carbonate) and organic salts (acetate) , Citrate, succinate, glycine, maleate, barbiturate, etc.) Based buffer Was used in biological experiments. " Molecular biological electronics for hybridization, reactions, diagnostics and synthesis It is an object of the present invention to find buffers and electrolytes that are advantageously used in You.Summary of the Invention The following invention relates to the DEX in our APEX microelectronic chip and device. NA transport rate, efficiency of DNA hybridization reaction and total hybridization Various parameters to improve or optimize the specificity of the ) And other conditions related findings. Specifically, the present invention provides a low conductivity zwitterionic On buffer solution, especially at a concentration of 10-100 mM, preferably about 50 mM or Amino acid histidine prepared at or near the pI (isoelectric point about pH 7.47) Containing DNA for rapid DNA transport and efficient hybridization It concerns the discovery of providing optimized conditions for the reaction. Next best known At least 10 times more efficient hybridization with buffer and cysteine Achieved. Test data show hybridization efficiency compared to cysteine Shows an increase of approximately 50,000-fold.Brief description of charts FIG. 1 is a plan view of a checkerboard array using a histidine buffer.Detailed description of the invention Electrophoretic transport and transfer of DNA in various types of electrolytes / buffers There are various physical parameters related to other charged analytes. Certain devices, examples For example, the applicant's APEX device as described in the aforementioned US Patent No. 5,605,662. The device is basically a DC (direct current) electrical device that generates an electric field on the device surface. Then , These fields are oppositely (+/-) biased microlo citation) during the electrophoretic transport of charged molecules. In contrast, the so-called Genosensor (impedance sensor) (for example, , WO93 / 226 78 Holes et al., "Optical and Electric M". methods and Apparatus for Molecular D selection ”), and two-dimensional electrophoresis (dielectrophoro). (e.g., Washiz 25 Journal of E) electro-statics) 109-123, 1990), in an AC electric field. Including the use of An important difference with these devices is that these AC fields are not If there is, there is essentially no net current in any of these systems, That is, there is no driving force of electrophoresis for charged molecule transport. APEX-type device , When a voltage is applied, produces a considerable net direct current (DC), which Recognized as a "sign of electrophoresis". In electrophoresis, ions or charged The movement of particles is caused by electric forces along the direction of the electric field gradient, Relationships are important to this technology. This electrophoretic movement is affected by the applied voltage. Below, visually shown itself as current conductivity in solution, V = R × I V is the potential R is the electric resistance of the electrolyte [V × A-1= R (Ω)] I is the current [A]. Obeys Ohm's law. The resistance of a solution is the reciprocal of conductivity that can be measured by a conductivity meter. conductivity Depends mainly on the ionic species of the buffer / electrolyte and their concentration; These parameters are very important for electric field-related molecular biology techniques. The resulting electric field Is really in a microscopic environment, but the basic current / voltage relationship is Essentially the same for APEX technology as for any other electrophoresis system It is. How various methods of supplying current and voltage and the scenario of current and voltage are APEX system has been found to improve the performance of such systems. Tem has unique features. In detail, several DC pulse techniques (linear and And logarithmic gradient) provide improved hybridization stringency It seems to do.Electrophoretic transport-vs. ionic strength There is a logarithmic decrease in the mobility of charged analyte species (proteins, DNA, etc.) It is well established in the field of electrophoresis that it is inversely proportional to the square root of the ionic strength of the solution. ("Capillary Electrophoresis: Pr Includes and Practice ", R.C. Kuhn and S.M. Ho ffsetter, Springer-Verlag, 1993, p. And Figure 3.16). At any given constant electric field strength, the electrolyte concentration is analyzed As it decreases relative to species (proteins, DNA, etc.), analytes will move at a faster rate Will be transported. Similar results showing this effect for dansyl amino acids J. J. Issaq et al., Chromatographia Vol. 32, # 3/4, August 1991, pages 155 to 161 (for details, see FIG. 3 on page 157) ). For DNA this effect is a different electrolytic solution Indicates that P.S. D. Ross and R.M. L. Scruggs, Biopol ymers Vol. 2, 231-236, 1964 (for details, 232 Page 1).Relationship between ionic strength and conductivity For non-buffer electrolytes containing cations (sodium chloride, potassium chloride, etc.) Ionic strength and conductivity are equivalent, ie conductivity is usually proportional to ionic strength Do (Phosphate, acetate, citrate, succinate, etc.) The conductivity will usually be equivalent, ie the conductivity is proportional to the ionic strength. Relaxants that can have zwitterionic species (no net charge at their pI) Impulsive electrolyte [Good buffer (MOPS, HEPES, TAPS, Tricine, ), Amino acid buffers, amphoteric electrolytes, etc.] Will decrease approximately 10-fold for every pH unit difference between and (pKa). For example , Its zwitterionic state (-OOC-CH (R) -NHThree +The amino acids in parentheses are The amino acid moietyIt will have a conductivity value that is 00 times lower. Thus, as one moves away from its pI, the shape A formal negative or positive charge is generated at the amino acid moiety, and conductivity and ionic strength are related. It will start running. However, at or near the pI, the conductivity is Will be much lower than expected in on-strength or concentration. Their pI or their Electrophoresis text when used near Good Buffer and amino acid It is noted that the acid buffer has "low conductivity at high ionic strength or concentration" (Ca pillary Electrophoresis: Principles a nd Practice ", R.A. Kuhn and S.M. Hoffsetter, Springer-Verlag, 1993, p. 88). Normally used electricity The electrophoresis buffer “Tris-boric acid” is actually predicted based on its ionic strength or concentration. It has a significantly lower conductivity than expected. This is a relatively stable zwitter in solution For "Tris cation" and "Borate anion" to form ion complex May. The conductivity of the 100 mM Tris-borate solution is 694 μS / cm. Which is approximately 20 times lower than expected from its ionic strength. Approximately equivalent to mM sodium phosphate or sodium chloride solution. Table 1 Shows conductivity measurements for a number of transport buffers. Zwitterion buffer / conductivity / transport rate At or near their pI, a zwitterionic buffer (Good buffer, amino acid) Acid buffer) or Tris-borate buffer has certain advantages. It exists with respect to the rate and rate of electrophoretic transport. These are: 1) Such a buffer is Can be used at relatively high concentrations, increasing the buffer capacity and 2) their conductivity Is significantly lower than other types of buffers at the same concentration, and 3) the analyte of interest It is to obtain the advantage of high electrophoretic transport rates for (DNA).Zwitterion buffering capacity at isoelectric point (pI) Amino acid buffers have buffer properties at their pl. A given amino acid is It may or may not have that "highest buffering capacity" at a pI of It will have some buffer capacity. The buffer capacity is defined as the pI between pI and pKa. H unit difference is reduced by 10 times, and three types of ionizable groups (histidine, cysteine, , Lysine, glutamic acid, aspartic acid, etc.) It has a higher buffer capacity at their pI than amino acids having only two dissociations. An example For example, compared to alanine or glycine, which have relatively low buffer capacity at their pI. In comparison, histidine pI = 7.47, lysine pI = 9.74 and glutamic acid pI All of I = 3.22 have relatively good buffering capacity at their pI (AL . Lehniger, Biochemistry, 2nd Edition, Worth Publ ishers, New York, 1975; See FIG. 4-9 on page 80). Histidine is used as a buffer in gel electrophoresis. Although recommended, hybridization is performed in such systems. Not. See U.S. Pat. No. 4,936,963. Cysteine is good for buffering capacity It is in an intermediate position. The cysteine has a pI of 5.02 and the α carboxyl group has a pK of 5.02. a is 1.71, pKa of sulfhydryl is 8.33, and pKa of α-amino group is 1 0.78. The acid / base titration curve for 250 mM cysteine shows that It shows that it has better "buffering capacity" at about pH 5 than mM sodium phosphate. In the pH range 4 to 6, cysteine's buffer capacity is 2 Significantly better than 0 mM sodium phosphate ing. However, at these pH ranges, a 250 mM cysteine solution Is 20 mM sodium phosphate with a value of about 2.9 mS / cm, which is 100 times greater. It is about 23 μS / cm, which is much lower than that of lithium. FIG. 1 shows various transport buffers. The measured conductivity of the liquid impingement is shown. Some electrophoretic techniques, developed more than 20 years ago, are "at their pI" Based on the ability to separate proteins in zwitterionic buffers. These technologies are Isoelectric focusing, Isotachophoresis And the method of isoelectric focusing (BD Hames and D. Rickwood). d "Gel Electrophoresis of proteins" : A Practical Approach, Chapters 3 and 4, IRL Press 1981). For these applications, all at their pI Some amino acid buffers and good buffers were used (see above cited references). See Table 2 on page 168).DNA transport in low ionic strength and low conductivity buffers 5580 chip coated with 2.5% agarose and ByTr-RCA5 A series of fluorescent checkerboard tests were performed using an optical probe. The following Stem: (1) 250 mM HEPES (low conductivity), (2) 10 μM amber Sodium citrate, (3) 10 μM sodium citrate and (4) distilled water Quickly (6 seconds) checkerboard addressing. Na citrate The results for thorium are shown in FIG. On the other hand, some types of low conductivity or low ion The strength solution has checkerboard addressing which is a somewhat better attribute Rapid DNA transport (6-12 seconds of DNA accumulation on 80 μm pad) This was achieved with all systems. Furthermore, DNA (polyanio itself) Is an electrolyte present in the bulk solution that provides conductivity, DNA addressing APEX chip is possible. Figure 1 uses histidine FIG. 2 shows a plan view of the APEX chip.Electrophoretic transport rates and cation / anion species relationships The mobility of charged analyte species (DNA, proteins, etc.) is related to the ionic strength of the electrolytic solution. In addition to the fact that, in addition to the fact that It is greatly influenced by the nature of the on-species (“Capillary Electr. opforesis: Principles and Practice " ). Biopolymer, Vol. 2, pp. 231-236, 1964 The literature shows this particular point for DNA transport. Page 232 of this document FIG. 1 shows that different monovalent anions (Li+> Na+> K+> 3 shows changes in DNA mobility when an electrolytic solution having TMA) is used. fundamentally Different cations have different association constants at the DNA phosphate group, and / or Changes the hydration region around the DNA molecule, which can lead to changes in transport rates. Wear. The present invention relates to an electric field molecular biological device, especially an APEX microelectronic chip and device. The DNA transfer rate, DNA hybridization reaction efficiency and total Various parameters and voltages to improve or optimize hybridization specificity It relates to our findings relating to lysis (buffer) and other conditions. For details The present invention relates to the use of 10-100 mM at or near the pi (isoelectric point of about 7.47). Low conductivity containing the amino acid histidine prepared at a concentration of about 50 mM Zwitterionic buffer solution provides rapid electrophoretic DNA transport and efficient hybridisation Concerning the inventor's discovery that both provide optimal conditions for the You. This advantage of histidine buffer is particularly important for APEX chip-type devices. . These special devices (as opposed to micro-machined devices) are capable of applying There are limits on the amount of current and voltage. This limit is based on the same buffer system. To achieve both rapid transport and efficient hybridization. Make it difficult. In these cases, limited current / voltage still causes rapid transport DNA transport was performed in a low conductivity buffer (cysteine or alanine). Under these conditions, DNA accumulated at the test site but did not hybridize efficiently. won. After transport in these low conductivity buffers, the solution was transferred to a high salt buffer (> 100 mM sodium chloride or sodium phosphate). Caused efficient hybridization. Table 2 shows the parameters of buffer capacity, pH and conductivity, and the APEX chip device. A link between DNA accumulation used and hybridization sensitivity (efficiency) The results for the serial test are shown. In particular, Table 2 shows the transported target DNA to the specific capture DNA at the test site. Various zwitterionic amino acid buffers for hybridization [β-alani , Taurine, cysteine, histidine, lysine and sodium phosphate Not an ion buffer)]. Transport is generally the same for transport Correlates with transport under field conditions. β-alanine, taurine and cysteine are excellent Histidine shows good transport, and lysine and NaPOFour Indicates considerable transport. DNA hybridization sensitivity is negatively charged "Standard DNA" having a lianionic phosphate backbone has been reported. 1 / 20 mM NaPO adjusted to pH 7.4Four In addition to the hybridization sensitivity, Table 2 shows streptavidin / bio 9 shows the sensitivity with respect to tin DNA probe capture affinity. Table 2 shows DNA transport (accumulation) and low conductivity (β-alanine, taurine, cysteine). Histidine). The table shows β-alanine, cysteine Reaction of streptavidin / biotin probe using histidine and histidine Shows excellent sensitivity. As reflected in the sensitivity data in Table 2, histidine Is cysteine or 20 mM NaPOFourBetter than any of the other buffers It provides good hybridization efficiency by 4 orders or more. Cysteine Is at least a factor of 10, and more specifically 10TwoDouble and most detailed Has at least 10FourIt is twice. Most importantly, Table 2 shows the DNA hive Very good redidation sensitivity (efficiency) for histidine buffer It is to show. Thus, of the currently tested zwitterionic amino acid buffers, Histidine has good transport and good DNA / DNA hybridization effect It is the only one that offers rates together. It is believed that the low conductivity of the histidine buffer system is responsible for rapid transport (accumulation). Have been. Why histidine buffer is relatively effective for DNA / DNA hybridization There are several possible explanations for what causes the stigma. One advantage May be the superior buffer capacity of histidine. At its pI of 7.47 , Histidine is an excellent buffer under acidic or basic conditions (AL Le. hninger, Biochemistry, 2nd edition, worth Publis hers, New York, 1975, p. 80, FIG. 4-9). DNA is high Histidine accumulates for hybridization and effectively relaxes these conditions. At the positive electrode that may strike, the APEX tip produces acid. More important Under these acidic conditions (pH <5), the protocol of imidazole group of histidine is Conversion initiates the conversion of the molecule to the dicationic species. A positively charged α-amino group and This dicationic species, which has a positively charged imidazole group, DNA / DNA hybrid formed at the positive electrode of the APEX chip It may be the case that may help stabilize the lid. Mosquito Thion, dication and polycation ON reduces the repulsion of the negatively charged phosphate backbone on double-stranded DNA structures Is known to help stabilize DNA / DNA hybrids. Also, DNA / DNA / histidine is another electrochemical product generated at the positive electrode. May form some types of stabilizing adducts (e.g., hydrogen peroxide) There is a possibility. Although this embodiment utilizes naturally occurring histidine, the present invention provides a good buffering capacity, It has low electrical conductivity (or zwitterionic properties) and is And other natural or natural compounds having the property of stabilizing DNA hybridization. Applicable to synthetic compounds. The above invention is illustrated and illustrated by way of illustration for purposes of clarity and understanding. Although described in some detail, certain changes and modifications may be made in the appended claims. What can be done without departing from the spirit or scope is that of the teachings of the present invention. Will be readily apparent to those skilled in the art.
───────────────────────────────────────────────────── フロントページの続き (51)Int.Cl.7 識別記号 ＦＩ テーマコート゛(参考） // Ｃ１２Ｍ 1/00 Ｃ１２Ｎ 15/00 Ａ (72)発明者 トゥー，ユージーン アメリカ合衆国92103カリフォルニア州サ ンディエゴ、ラーク・ストリート3527番 (72)発明者 ネレンバーグ，マイケル・アービング アメリカ合衆国92126カリフォルニア州サ ンディエゴ、カミニト・イノセンタ11256 番 (72)発明者 ヘラー，マイケル・ジェイムズ アメリカ合衆国92024カリフォルニア州エ ンシニタス、ホーク・ビュー・ドライブ 1614番──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. 7 Identification symbol FI Theme coat ゛ (Reference) // C12M 1/00 C12N 15/00 A (72) Inventor To, Eugene United States 92103 Lark, San Diego, California, USA Street No. 3527 (72) Inventor Nerenberg, Michael Irving 92126 USA San Diego, California, Caminito Innocenter 11256 (72) Inventor Heller, Michael James United States 92024 Encinitas, California, Hawk View Drive 1614
Claims (1)
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US70826296A | 1996-09-06 | 1996-09-06 | |
| US08/708,262 | 1996-09-06 | ||
| PCT/US1997/014489 WO1998010273A1 (en) | 1996-09-06 | 1997-08-18 | Methods and materials for optimization of electronic hybridization reactions |
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| JP2001501301A true JP2001501301A (en) | 2001-01-30 |
| JP2001501301A5 JP2001501301A5 (en) | 2005-04-07 |
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| EP (1) | EP1019711A4 (en) |
| JP (1) | JP4213216B2 (en) |
| KR (1) | KR100591626B1 (en) |
| CN (1) | CN1180248C (en) |
| AU (1) | AU723564B2 (en) |
| BR (1) | BR9712800A (en) |
| CA (1) | CA2264780C (en) |
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| WO (1) | WO1998010273A1 (en) |
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| JP2008514928A (en) * | 2004-09-23 | 2008-05-08 | ナノゲン・インコーポレイテッド | Methods and materials for optimization of electron transport and hybridization reactions |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US6051380A (en) * | 1993-11-01 | 2000-04-18 | Nanogen, Inc. | Methods and procedures for molecular biological analysis and diagnostics |
| US6468742B2 (en) | 1993-11-01 | 2002-10-22 | Nanogen, Inc. | Methods for determination of single nucleic acid polymorphisms using bioelectronic microchip |
| US6379897B1 (en) | 2000-11-09 | 2002-04-30 | Nanogen, Inc. | Methods for gene expression monitoring on electronic microarrays |
| US5964995A (en) | 1997-04-04 | 1999-10-12 | Caliper Technologies Corp. | Methods and systems for enhanced fluid transport |
| US6238909B1 (en) * | 1999-05-04 | 2001-05-29 | Motorola, Inc. | Method and apparatus for obtaining electric field-enhanced bioconjugation |
| EP1216415A1 (en) * | 1999-09-27 | 2002-06-26 | Monsanto Technology LLC | Methods for determining oils in seeds |
| US7309581B2 (en) * | 2000-11-01 | 2007-12-18 | Sysmex Corporation | Method of staining, detection and counting bacteria, and a diluent for bacterial stain |
| GB0205455D0 (en) | 2002-03-07 | 2002-04-24 | Molecular Sensing Plc | Nucleic acid probes, their synthesis and use |
| US7153687B2 (en) | 2002-08-13 | 2006-12-26 | Hong Kong Dna Chips Limited | Apparatus and methods for detecting DNA in biological samples |
| JP4464664B2 (en) | 2003-06-13 | 2010-05-19 | 独立行政法人理化学研究所 | Biomolecule microarray substrate, biomolecule microarray, device and method for promoting interaction, and method for detecting interaction |
| KR100785011B1 (en) * | 2006-04-07 | 2007-12-11 | 삼성전자주식회사 | Method for increasing the specificity of nucleic acid hybridization using zwitterionic compounds |
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| US4936963A (en) * | 1987-05-27 | 1990-06-26 | Abbott Laboratories | Polycationic buffers and method for gel electrophoresis of nucleic acids |
| US5188963A (en) * | 1989-11-17 | 1993-02-23 | Gene Tec Corporation | Device for processing biological specimens for analysis of nucleic acids |
| US6017696A (en) * | 1993-11-01 | 2000-01-25 | Nanogen, Inc. | Methods for electronic stringency control for molecular biological analysis and diagnostics |
| JP3398749B2 (en) * | 1994-11-10 | 2003-04-21 | オーキッド バイオ サイエンシズ， インコーポレイテッド | Liquid distribution system |
| US5585069A (en) * | 1994-11-10 | 1996-12-17 | David Sarnoff Research Center, Inc. | Partitioned microelectronic and fluidic device array for clinical diagnostics and chemical synthesis |
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| JP2008514928A (en) * | 2004-09-23 | 2008-05-08 | ナノゲン・インコーポレイテッド | Methods and materials for optimization of electron transport and hybridization reactions |
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| NZ334314A (en) | 2000-09-29 |
| CA2264780A1 (en) | 1998-03-12 |
| AU4071997A (en) | 1998-03-26 |
| KR100591626B1 (en) | 2006-06-20 |
| EP1019711A1 (en) | 2000-07-19 |
| BR9712800A (en) | 1999-11-23 |
| CA2264780C (en) | 2006-08-01 |
| AU723564B2 (en) | 2000-08-31 |
| JP4213216B2 (en) | 2009-01-21 |
| CN1180248C (en) | 2004-12-15 |
| WO1998010273A1 (en) | 1998-03-12 |
| KR20010029477A (en) | 2001-04-06 |
| CN1230255A (en) | 1999-09-29 |
| EP1019711A4 (en) | 2001-06-13 |
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