CN1230255A - Methods and materials for optimization of electronic hybridization reactions - Google Patents
Methods and materials for optimization of electronic hybridization reactions Download PDFInfo
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- CN1230255A CN1230255A CN97197960A CN97197960A CN1230255A CN 1230255 A CN1230255 A CN 1230255A CN 97197960 A CN97197960 A CN 97197960A CN 97197960 A CN97197960 A CN 97197960A CN 1230255 A CN1230255 A CN 1230255A
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- nucleic acid
- damping fluid
- hybridization
- electric conductivity
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- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
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- 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
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- H01L29/66409—Unipolar field-effect transistors
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Abstract
The inventions relate to discoveries concerning the various parameters, electrolytes (buffers), and other conditions which improve or optimize the speed of DNA transport, the efficiency of DNA hybridization reactions, and the overall hybridization specificity in microelectronic chips and devices. In particular, this invention relates to the discovery that low conductance zwitterionic buffer solutions, especially those containing the amino acid Histidine prepared at concentrations of SIMILAR 50 mM and at or near the pI (isoelectric point SIMILAR pH 7.47), provide optimal conditions for both rapid electrophoretic DNA transport and efficient hybridization reactions. Hybridization efficiencies of at least a factor of 10 relative to the next best known buffer, Cysteine, are achieved. Test data demonstrate an approximately 50,000 fold increase in hybridization efficiency compared to Cysteine.
Description
Field of the present invention
The present invention relates to be adapted to medical diagnosis, the damping fluid that is used for electronic installation and the electrolyte of biology and other purposes.More particularly, the present invention relates to for the DNA hybridization analysis that on the microelectronics medical diagnostic apparatus, carries out favourable damping fluid and electrolyte.
Background of the present invention
Recently, increase gradually for interest in conjunction with microelectronics and molecular biological device.In the applying date is the series number 08/146,504 on November 1st, 1993, and the electronic installation of the active processization of molecular biological analysis and diagnostics " but be used for " discloses a kind of this type systematic, the document is now as U.S. Patent number 5,605,662 publish, and this paper quotes as a reference.Wherein disclosed system will be called the APEX system.The APEX system can carry out many functions, and such as nucleic acid hybridization, the antibody/antigen reaction is used in the synthetic molecular biology reaction of clinical diagnosis and biopolymer to have advantage.
APEX type device utilizes damping fluid and electrolyte to be used for its operation.Damping fluid is defined as when adding acid or alkali pH is changed the chemical solution with resistance.For example, referring to, the biotechnology dictionary, second edition, James Coombs, Stockton publishes.As described therein, " say traditionally, be used for biological experiment for basic damping fluid with inorganic salts (phosphate, carbonate) and acylate (acetate, citrate, succinate, glycocoll, maleate, barbiturate etc.).”
The objective of the invention is to have found hybridizing, reaction is used damping fluid and the electrolyte that has superiority in diagnosis or the synthetic molecular biology electronic installation.
The present invention's general introduction
Following invention relates to us about improve or optimization DNA migration velocity the specific various parameters of the efficient of DNA hybridization reaction and total hybridization, the discovery of electrolyte (damping fluid) and other condition in APEX microelectronics sheet and device.Specifically, the discovery that the present invention relates to is, low electric conductivity zwitter-ion buffer solution, particularly those contain at 10-100mM, preferably approximately 50mM concentration and near pI (the about pH7.47 of isoelectric point) down the solution of the amino acid histidine of preparation be provided for the top condition of rapid DNA migration and efficient hybridization reaction.Realized with respect to inferior good known damping fluid, halfcystine, at least 10 times hybridization efficiency.Test figure confirms that comparing hybridization efficiency with halfcystine increases about 50,000 times.
Brief description of the drawings
Fig. 1 is the planimetric map that is used for the chessboard arrangement of histidine buffering liquid.
Detailed description of the present invention
There are various physical parameters to relate to DNA and the electrophoretic transfer of other charged analyte in various types of electrolyte/damping fluids.Some device, for example, at the U.S. Patent number of above quoting 5,605, the APEX device of the applicant described in 662 is DC (direct current) electric installation basically, it produces electric field on the surface of device.These electric fields and cause charged molecule this apparatus surface relatively (+/-) have between the film micro area of bias voltage and electrophoretic migration occurs.On the contrary, so-called Genosensor (impedance transducer), referring to, for example, Hollis etc., " optics and electrical method and the device that are used for Molecular Detection ", W093/22678 and dielectrophoresis device, referring to, for example, Washizu25 electrostatics magazine, 109-123,1990 relate to use AC electric field.The important difference that relates to these devices is, when using the AC electric field, do not have net current basically in any this type systematic,, do not make the electrophoresis propelling power of charged molecule migration that is.APEX type device produces significantly clean direct current (DC) electricity when applied voltage, and it is recognized as " feature of electrophoresis ".In electrophoresis, the migration of ion and charged particle is produced by the electrical forces along the electric-force gradient direction, and the relation of electric current and voltage is important for this technology.Electrophoretic migration this in showing as under applied voltage influence the conduction of current in the solution on the macroscopic view and deferring to Ohm law:
V=RxI
V is an electromotive force
R is electrolytical resistance [VxA
-1=R (Ω)]
I is electric current [A].
The resistance of solution is the inverse of electric conductivity, and electric conductivity can be measured with diagometer.Electric conductivity depends primarily on ionic species and its concentration in damping fluid/electrolyte; Therefore these parameters are extremely important for the Protocols in Molecular Biology that relates to electric field.Base current/voltage relationship for the APEX technology be identical basically for any other electrophoresis system, although the electric field that produces in fact is a micro.
The APEX system has unique feature about the variety of way that produces electric current and voltage, and has found how electric current and voltage schemes improve the effect of this system.Specifically, as if various DC pulse schemes (linear and logarithm gradient) have improved the hybridization severity.
Electrophoretic migration and ionic strength
Be fully recognized that charged analyte kind (protein in the electrophoresis field, DNA etc.) mobility logarithm reduces, the square root of it and electrolyte solution ionic strength is inversely proportional to (referring to " electrocapillary phoresis: principle and put into practice ", R.Kuhn and S.Hoffstetter, Springer-Verlag, 1993 the 83rd pages and Fig. 3 .16).Under any given steady electric field intensity, along with the minimizing of electrolyte concentration with respect to analyte kind (protein, DNA etc.), analyte moves with faster rate.The similar result of this effect who confirms dansyl amino acid is by J.J.Issaq etc., chromatogram, and the 32nd volume, #3/4, in August, 1991,155 pages to 161 pages (particularly referring to the 157th page of Fig. 3) shows.The result of this effect of confirmation DNA is at P.D.Ross and R.L in different electrolyte solutions.Scruggs, biopolymer, the 2nd volume, shows among 1964 (particularly referring to 232 pages of Fig. 1) by the 231st to 236 page.
Ionic strength/electric conductivity relation
Relate to negative ion and the cation type (Na that in solution, dissociates fully for those
+← → CL
-, K
+← → CL
-, etc.) non-buffering electrolyte, ionic strength and electric conductivity are suitable, that is, electric conductivity usually and ionic strength proportional.For those dissociated states (for example, 2Na
+← → PO
4 -2) buffering electrolyte (phosphate, acetate, citrate, succinates etc.), ionic strength and electric conductivity are normally suitable, promptly, electric conductivity and ionic strength are proportional. have the buffering electrolyte [Good damping fluid (MOPS of zwitter-ion kind (under its pI, not having net charge) for those, HEPES, TAPS, Tricine, Bicine), buffered with amino acid liquid, ampholyte etc.], every pH unit difference will reduce about 10 times to electric conductivity in isoelectric point (pI) with (pKa).For example, amino acid its zwitterionic state (
-OOC-CH (R)-NH
3 +) the electric conductivity value has all clean positive charge (HOOC-CH (R)-NH than " amino acid group "
2 +← → X
-), or whole negative charge (Y
+← →
-OOC-CH (R)-NH
2) time low about 1000 times.Therefore, outside negative or positive electric charge forms on amino acid group along with it leaves pI, and electric conductivity begins relevant with ionic strength.Yet, be in or during near pI, more much lower for given ionic strength or concentration electric conductivity than expection.When being in or using near its pI place, the electrophoresis test relates to Good damping fluid and buffered with amino acid liquid, because " when high ionic strength or concentration, have low electric conductivity " (referring to " electrocapillary phoresis: principle and put into practice " the 88th page, R.Kuhn and S.Hoffstetter, Springer-Verlag, 1993).Electrophoretic buffer " Tris-borate " commonly used is in fact than having significantly lower electric conductivity from its ionic strength and concentration are desired.This may be because " Tris kation " and " acid anion " forms metastable zwitter-ion compound in solution.The electric conductivity of having measured 100mM Tris-borate solution is 694 μ S/cm, than desired approximately low 20 times from its ionic strength, and is about as much as 5mM sodium phosphate or sodium chloride solution.Table 1 has shown the conductivity measurements of many migration damping fluids.
Table 1
| Solution/damping fluid | Measured value | Measured value | Measured value | On average/standard deviation |
| ?10mM ?MgCl 2 | 1.95mS/cm | ?2.02mS/cm | 2.13mS/cm | ?2.03+/-0.09 ?mS/cm |
| ?1mM?MgCl 2 | 174μS/cm | ?208μS/cm | 177μS/cm | ?186+/-18.8 ?μS/cm |
| ?0.1mM ?MgCl 2 | 16.9μS/cm | ?16.7μS/cm | 18.3μS/cm | ?17.3+/-0.87 ?μS/cm |
| ?10mM?NaCl | 1.07mS/cm | ?1.10mS/cm | 1.18mS/cm | ?1.12+/-0.057 ?mS/cm |
| ?1mM?NaCl | 112μS/cm | ?115μS/cm | 111μS/cm | ?112.7+/-2.08 ?μS/cm |
| ?0.1mM ?NaCl | 8.80μS/cm | ?8.98μS/cm | 10.5μS/cm | ?9.43+/-0.93 ?μS/cm |
| ?20mM ?NaPO 4 | 2.90mS/cm | ?2.79mS/cm | 3.00mS/cm | ?2.90+/-0.11 ?mS/cm |
| ?10mM ?NaPO 4 | 1.40mS/cm | ?1.44mS/cm | 1.48mS/cm | ?1.44+/-0.04 ?mS/cm |
| ?1mM?NaPO 4 | 122μS/cm | ?128μS/cm | 136μS/cm | ?128.7+/-7.0 ?μS/cm |
| ?50mM?TRIS | 3.50mS/cm | ?3.14mS/cm | 3.40mS/cm | ?3.35+/-0.19 ?mS/cm |
| ?10mM?TRIS | 572μS/cm | ?562μS/cm | 583μS/cm | ?572+/-10.5 ?μS/cm |
| ?250mM ?HEPES | 141μS/cm | ?144μS/cm | 158μS/cm | ?147.6+/-9.07 ?μS/cm |
| ?25mM ?HEPES | 916μS/cm | ?9.44μS/cm | 10.5μS/cm | ?9.7+/-0.71 ?μS/cm |
| 3.3mM sodium citrate | 964μS/cm | ?964μS/cm | 1.03mS/cm | ?986+/-3?8.1 ?μS/cm |
| The 5mM sodium succinate | 1.05mS/cm | ?960μS/cm | 1.01mS/cm | ?1.01+/-0.045 ?mS/cm |
| The 5mM sodium oxalate | 1.02mS/cm | ?1.03mS/cm | 1.12mS/cm | ?1.06+/-0.055 ?mS/cm |
| The 10mM sodium acetate | 901μS/cm | ?917μS/cm | 983μS/cm | ?934+/-43.5 ?μS/cm |
| The 250mM halfcystine | 274μS/cm | ?17.3μS/cm | 23.5μS/cm | ?22.7+/-5.09 ?μS/cm |
| Mil1i-Q water | ＜0.5μS/cm | Detection limit | ||
| 0.1 the unit is too low |
Zwitterionic buffer/electric conductivity/mobility
When use be in or during near the zwitterionic buffer (Good damping fluid, buffered with amino acid liquid) of its pI or Tris-borate buffer solution speed or the speed for the DNA electrophoretic migration have advantage.These advantages have: the concentration that 1) these damping fluids can be quite high is used to improve surge capability, 2) its electric conductivity damping fluid than other type under same concentrations is obviously lower, and 3) can obtain the more advantage of high voltage electrophoresis migration rate for interested analyte (DNA).
Zwitter-ion is in the surge capability of isoelectric point (pI)
Buffered with amino acid liquid has damping characteristics at its pI place.Although given amino acid has or do not have its " the highest surge capability " at its pI place, it has surge capability to a certain degree.The difference of each pH unit descends 10 times surge capability between pI and pK; Those have amino acid (histidine, halfcystine, the lysine of three ionogens, glutamic acid, aspartic acid etc.) generally has higher surge capability at its pI place than those amino acid (glycocoll, alanine, leucine etc.) that only have two groups that dissociate.For example, histidine pI=7.47, lysine pI=9.74 all has relative preferably surge capability at its pI place with respect to alanine or glycocoll with glutamic acid pI=3.22, and the latter have at its pI place relatively low surge capability (referring to, A.L.Lehninger, biological chemistry, second edition, Worth Publishers, New York, 1975; Particularly the 79th page of Fig. 4-8 and the 80th page Fig. 4-9 page).Histidine has been proposed as damping fluid and has been used for gel electrophoresis, referring to, for example, United States Patent (USP) 4,936,963, but in this system, hybridize.The surge capability of halfcystine is positioned at more position intermediate.The pI of halfcystine is 5.02, and the pKa of α carboxyl is 1.71, and the pKa of sulfydryl is 8.33, and the pKa of α amino is 10.78.The acid of 250mM halfcystine/alkalimetric titration curve shows that halfcystine has better " surge capability " at～pH5 than 20mM sodium phosphate.In the scope of pH4 to 6, the surge capability of halfcystine is obviously better than 20mM sodium phosphate, yet particularly at higher pH., in these pH scopes, the electric conductivity of 250mM halfcystine solution with have～the 20mM sodium phosphate of 2.9mS/cm value is compared extremely lowly, is～23 μ S/cm, low 100 times.Fig. 1 has shown the conductivity measurement of various migration damping fluids.
Surpass some electrophoretic techniquess of forming in the past in 20 years ability based on isolated protein in the zwitterionic buffer of " its pI ", these technology are called the isoelectric point electrophoresis, and isotachophoresis and isoelectric focusing are (referring to by B.D.Hames﹠amp; The 3rd and 4 chapters among " gel electrophoresis of protein: hands-on approach " IRL Press1981 that D.Rickwood edits).In using, all these have used various buffered with amino acid liquid and Good damping fluid, all at its pI point (referring to above-mentioned list of references the 168th page table 2).
DNA migration in low ionic strength and low electric conductivity damping fluid
5580 small pieces and the ByTr-RCA5 fluorescence probe that use 2.5% agarose to cover carry out a series of fluorescence chessboard experiments.We can realize in all following systems that the chessboard of (6 seconds) loads rapidly: (1) 250mM HEPES (low electric conductivity), (2) 10 μ M sodium succinates, (3) 10 μ M sodium citrates and (4) distilled water.The result of sodium citrate shows in Fig. 1.Although the low electric conductivity of some type or low ionic-strength solution have some better characteristic, use all these systems to realize that all chessboard loads and DNA migration (DNA runs up on the bar of 80 μ m in 6 to 12 seconds) rapidly.In addition, DNA can load the APEX sheet in distilled water, because DNA (itself being a kind of polyanion) is the electrolyte that electric conductivity is provided that is present in the mixed solution.Fig. 1 has shown the planimetric map that uses the APEX small pieces of histidine.
The relation of electrophoretic mobility and anionic/cationic kind
Except charged analyte kind (DNA, protein etc.) outside the mobility fact relevant with the ionic strength of electrolyte solution, mobility also greatly is subjected to the influence (referring to the 89th page of " electrocapillary phoresis: principle and put into practice " list of references) of the characteristic of kation and anion species in the electrolyte solution.Biopolymer above, the 2nd volume, the 231-236 page or leaf, this that has confirmed the DNA migration in 1964 the list of references is specific.The 232nd page of Fig. 1 of this list of references shown when using to have different monovalent anion (Li under same ion intensity
+＞Na
+＞K
+＞TMA
+) electrolyte the time DNA mobility variation.Basically, different kations has different association constants with the DNA phosphate group, and/or changes the aquation ball around the dna molecular, causes its mobility to change.
The present invention relates to us about at electric field molecular biology device, particularly improve or optimization DNA migration velocity the specific various parameters of the efficient of DNA hybridization reaction and total hybridization in APEX microelectronics sheet and the device, the discovery of electrolyte (damping fluid) and other condition.Specifically, the discovery that the present invention relates to is, contain at 10-100mM, particularly approximately 50mM concentration and be in or near pI (the about pH7.47 of isoelectric point) down the low electric conductivity zwitter-ion buffer solution of the amino acid histidine of preparation be provided for the top condition of rapid electrophoresis DNA migration and efficient hybridization reaction.This advantage of histidine buffering liquid is for the device particular importance of APEX platelet-type.It is restricted for applicable electric current and voltage that these specifically install (opposite with micromechanics type device).These restrictions make and are difficult to use identical buffer system to realize migration rapidly and efficient hybridization simultaneously.In these cases, carry out the DNA migration with low electric conductivity damping fluid (halfcystine or alanine), wherein Xian Zhi current/voltage still produces rapid migration.Under these conditions, DNA accumulates in the test site, but can not effectively hybridize.In these low electric conductivity damping fluids, after the migration, this solution is become high-salt buffer (＞100mM sodium chloride or sodium phosphate), produce effectively hybridization in the test site then.
Table 2 has shown that use APEX die devices determines surge capability, a series of result of experiment of pH and electric conductivity parameter and DNA accumulation and hybridization sensitivity (efficient) relation.
Table 2
| Solution | Surge capability pH4-10 | PH is at PI | Electric conductivity (μ S) | Relative DNA mobility | SA biotin T12 sensitivity | The hybridization sensitivity of DNA | |
| Beta-alanine | ?pK 1-3.6 ?pK 2-10.2 | ???+ | ????7.3 | ???10.0 | The fastest ++ +++ | ????3×10 6 | |
| Taurine | ?pK 1-1.5 ?pK 2-8.7 | ???+/- | ????4.6 | ???4.5 | ???++++ | ??＞7.5×10 10 | |
| Halfcystine | ?pK 1-1.7 ?pK 2-8.3 ?pK 3-10.8 | ???+/- | ????5.2 | ???25.0 | ???++++ | ????3×10 7 | ???7.5×10 10 |
| Histidine | ?PK 1-1.8 ?pK 1-6.0 ?pK 3-9.0 | ???+++ | ????7.6 | 212.0 (172.0) high-purity | ???+++ | ????3×10 6 | ???3×10 6 |
| Lysine | ?PK 1-2.2 ?pK 2-8.9 ?pK 3-10.3 | ???++ | ????9.6 | ???477.0 | ???++ | ??＞7.5×10 10 | |
| ???NaPO 4 | Complex | ???+ | ????7.4 1/ | ?1,400.0 | + the slowest | ||
Specifically; table 2 has shown various zwitterionic amino acid damping fluid [Beta-alanine, taurine, halfcystines; histidine, lysine and sodium phosphate (not being zwitterionic buffer)] target DNA and the specificity of migration are caught the influence of DNA in the hybridization ability in test site.For migration, electric conductivity is generally relevant with migration under the same electrical field condition.Beta-alanine, taurine, halfcystine demonstrate fabulous migration, and histidine demonstrates migration preferably, and lysine and sodium phosphate demonstrate general migration.Reported the DNA hybridization sensitivity of " normal DNA " with electronegative polyanion phosphoric acid skeleton.Except the hybridization sensitivity, table 2 has also been reported streptavidin/biology
Plain dna probe is caught the sensitivity of compatibility.
Table 2 has clearly illustrated the relation of DNA migration (accumulation) and low electric conductivity (Beta-alanine, taurine, halfcystine, histidine).Shown the use Beta-alanine in the table, taurine, halfcystine and histidine are for streptavidin/biotinylated probes compatibility sensitivity preferably.Reacted as sensitivity data in the table 2, histidine is than halfcystine or other damping fluid, as 20mNaPO
44 orders of magnitude of hybridization efficiency.At least improving 10 times with respect to halfcystine, more particularly is 10
2Doubly, most particularly at least 10
4Doubly.The most important thing is that table 2 has shown for histidine buffering liquid DNA hybridization susceptibility (efficient) fabulous.Therefore, in present all zwitterionic amino acid damping fluids of testing, histidine is a unique existing migration preferably, and the damping fluid of DNA/DNA hybridization efficiency is preferably arranged again.
The low electric conductivity that it is believed that the histidine buffer system is the reason of rapid DNA migration (accumulation).Why can produce DNA/DNA hybridization quite efficiently for histidine buffering liquid some possible explanations are arranged.An advantage may be a histidine surge capability preferably.Because its pI is 7.47, therefore histidine can both cushion under acidity or alkali condition preferably (referring to A.L.Lehninger, biological chemistry, second edition, Worth Publisher, New York, 1975, the 80th page of Fig. 4-9) the .APEX small pieces produce acid on the positive electrode that accumulation DNA is used to hybridize, and histidine can effectively cushion these conditions.The more important thing is that under these acid conditions (pH＜5), the protonated beginning of the imidazole radicals on the histidine becomes the dication kind with molecular conversion.The alpha-amido that may thisly have positive electric charge can help to promote to hybridize and be stabilized in the DNA/DNA crossbred that APEX small pieces anode forms with the dication kind that has the imidazole radicals of positive electric charge.Kation, the known process of dication and polycation reduce the mutual exclusion of electronegative phosphoric acid skeleton on the double-stranded DNA structure and help stabilized DNA/DNA crossbred.Also probable dna/DNA/ histidine also forms some stable adduct types from other galvanochemistry product (hydrogen peroxide etc.) that produces at anode.
Although the present embodiment has been utilized naturally occurring histidine, but the present invention can be applicable to other natural or synthetic compound fully, this compound has surge capability preferably, lower electric conductivity (or zwitter-ion feature) and have and allow DNA hybridization to be formed institute's stable properties by charge stable or adduct.
Although for clear and understand in the mode of explanation and embodiment easily and on some details, described foregoing invention, those of ordinary skill in the art be it is evident that under instruction of the present invention and can make some change and modification and not depart from the essence or the scope of claims it.
Claims (32)
1. one kind is used for comprising the steps: having a method of carrying the microelectronic device migration in the test site of catching nucleic acid and hybridizing target nucleic acid at least
(1) use a kind of low electric conductivity damping fluid to this device,
(2) apply electric current to this device so that produce electric field in the test site,
(3) target nucleic acid is moved to the test site and
(4) make target nucleic acid at test site and seizure nucleic acid hybridization, its hybridization efficiency is higher at least 10 times with halfcystine than under the same conditions.
2. the process of claim 1 wherein that the low electric conductivity damping fluid is a zwitterionic buffer.
3. the method for claim 2, wherein zwitterionic buffer comprises histidine.
4. the method for claim 3, wherein histidine is prepared to the concentration of about 10-100mM.
5. the method for claim 3, wherein histidine is prepared at or about the isoelectric point place.
6. the process of claim 1 wherein that isoelectric point is about pH7.47.
7. the process of claim 1 wherein damping fluid entity stable target nucleic acid and catch hybridization between nucleic acid.
8. the method for claim 7, wherein the damping fluid entity is the native compound with low electric conductivity.
9. the method for claim 7, wherein the damping fluid entity is the natural amphoteric ionic compound.
10. the method for claim 7, wherein the damping fluid entity is the synthetic compound with low electric conductivity.
11. the method for claim 7, wherein the damping fluid entity is the zwitterionic compound that synthesizes.
12. the process of claim 1 wherein that hybridization efficiency is higher at least 100 times with halfcystine than under the same conditions.
13. the process of claim 1 wherein that hybridization efficiency is higher at least 1,000 times with halfcystine than under the same conditions.
14. the process of claim 1 wherein that hybridization efficiency is higher at least about 50,000 times with halfcystine than under the same conditions.
15. the process of claim 1 wherein that the damping fluid entity has reduced the repulsion of catching between nucleic acid and target nucleic acid.
16. the process of claim 1 wherein that damping fluid has reduced the formation of catching adduct between nucleic acid and target nucleic acid.
17. a method that is used at migration of microelectronics hybrid device intensifier target nucleic acid electrophoresis and hybridization efficiency, this device comprise a kind of film micro area test site of catching nucleic acid that has, and comprise the steps:
Use a kind of low electric conductivity damping fluid to this device,
To this device apply electric energy so as to cause the electrophoretic migration of target nucleic acid in this device film micro area test site and accumulation and
Following with the same terms than hybridizing target nucleic acid with the high at least 10 times efficient of halfcystine.
18. the method for claim 17, wherein the low electric conductivity damping fluid is a zwitterionic buffer.
19. the method for claim 18, wherein zwitterionic buffer comprises histidine.
20. the method for claim 19, wherein histidine is prepared to the concentration of about 10-100mM.
21. the method for claim 19, wherein histidine is prepared at or about the isoelectric point place.
22. the method for claim 17, wherein isoelectric point is about pH7.47.
23. the method for claim 17, the wherein hybridization between the entity stable target nucleic acid of damping fluid and seizure nucleic acid.
24. the method for claim 23, wherein the damping fluid entity is the native compound with low electric conductivity.
25. the method for claim 23, wherein the damping fluid entity is the natural amphoteric ionic compound.
26. the method for claim 23, wherein the damping fluid entity is the synthetic compound with low electric conductivity.
27. the method for claim 23, wherein the damping fluid entity is the zwitterionic compound that synthesizes.
28. the method for claim 17, wherein hybridization efficiency is higher at least 100 times with halfcystine than under the same conditions.
29. the method for claim 17, wherein hybridization efficiency is higher at least 1,000 times with halfcystine than under the same conditions.
30. the method for claim 17, wherein hybridization efficiency is higher at least about 50,000 times with halfcystine than under the same conditions.
31. the method for claim 17, wherein the damping fluid entity has reduced the repulsion of catching between nucleic acid and target nucleic acid.
32. the method for claim 17, wherein damping fluid has reduced the formation of catching adduct between nucleic acid and target nucleic acid.
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| US70826296A | 1996-09-06 | 1996-09-06 | |
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| 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 |
| AU783169B2 (en) * | 1999-09-27 | 2005-09-29 | 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 |
| US7314542B2 (en) * | 2004-09-23 | 2008-01-01 | Nanogen, Inc. | Methods and materials for optimization of electronic transportation and hybridization reactions |
| 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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| 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 |
| ATE277450T1 (en) * | 1994-11-10 | 2004-10-15 | Orchid Biosciences Inc | 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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