SE9500589A0 - Nukleinsyremolekyl-detektion på fasta substrat vid enmolekyl-DNA-sekvensering - Google Patents
Nukleinsyremolekyl-detektion på fasta substrat vid enmolekyl-DNA-sekvenseringInfo
- Publication number
- SE9500589A0 SE9500589A0 SE9500589A SE9500589A SE9500589A0 SE 9500589 A0 SE9500589 A0 SE 9500589A0 SE 9500589 A SE9500589 A SE 9500589A SE 9500589 A SE9500589 A SE 9500589A SE 9500589 A0 SE9500589 A0 SE 9500589A0
- Authority
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- Sweden
- Prior art keywords
- nucleic acid
- dna
- acid bases
- solid substrate
- base
- Prior art date
Links
- 108020004707 nucleic acids Proteins 0.000 title claims abstract description 48
- 102000039446 nucleic acids Human genes 0.000 title claims abstract description 48
- 150000007523 nucleic acids Chemical class 0.000 title claims abstract description 48
- 239000000758 substrate Substances 0.000 title claims abstract description 39
- 239000007787 solid Substances 0.000 title claims abstract description 27
- 238000001712 DNA sequencing Methods 0.000 title claims abstract description 12
- 238000001514 detection method Methods 0.000 title claims description 16
- 238000000034 method Methods 0.000 claims abstract description 15
- 230000002255 enzymatic effect Effects 0.000 claims abstract description 4
- 238000000151 deposition Methods 0.000 claims abstract 5
- 238000003776 cleavage reaction Methods 0.000 claims description 14
- 230000007017 scission Effects 0.000 claims description 14
- 108020004414 DNA Proteins 0.000 claims description 13
- 230000005284 excitation Effects 0.000 claims description 11
- 102000004190 Enzymes Human genes 0.000 claims description 8
- 108090000790 Enzymes Proteins 0.000 claims description 8
- 230000008021 deposition Effects 0.000 claims description 8
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 claims description 7
- 229910052710 silicon Inorganic materials 0.000 claims description 7
- 239000010703 silicon Substances 0.000 claims description 7
- 230000005684 electric field Effects 0.000 claims description 6
- 150000003212 purines Chemical class 0.000 claims description 6
- 150000003230 pyrimidines Chemical class 0.000 claims description 6
- 238000005370 electroosmosis Methods 0.000 claims description 5
- 238000012163 sequencing technique Methods 0.000 claims description 5
- KDCGOANMDULRCW-UHFFFAOYSA-N 7H-purine Chemical compound N1=CNC2=NC=NC2=C1 KDCGOANMDULRCW-UHFFFAOYSA-N 0.000 claims description 4
- 238000004458 analytical method Methods 0.000 claims description 4
- 238000003491 array Methods 0.000 claims description 4
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 3
- NYHBQMYGNKIUIF-UUOKFMHZSA-N Guanosine Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O NYHBQMYGNKIUIF-UUOKFMHZSA-N 0.000 claims 2
- MIKUYHXYGGJMLM-GIMIYPNGSA-N Crotonoside Natural products C1=NC2=C(N)NC(=O)N=C2N1[C@H]1O[C@@H](CO)[C@H](O)[C@@H]1O MIKUYHXYGGJMLM-GIMIYPNGSA-N 0.000 claims 1
- NYHBQMYGNKIUIF-UHFFFAOYSA-N D-guanosine Natural products C1=2NC(N)=NC(=O)C=2N=CN1C1OC(CO)C(O)C1O NYHBQMYGNKIUIF-UHFFFAOYSA-N 0.000 claims 1
- -1 Guanosine 5-methyl-2-pyrimidinone Chemical compound 0.000 claims 1
- 229940029575 guanosine Drugs 0.000 claims 1
- 239000011159 matrix material Substances 0.000 claims 1
- 239000007795 chemical reaction product Substances 0.000 abstract 1
- 239000002773 nucleotide Substances 0.000 description 15
- 125000003729 nucleotide group Chemical group 0.000 description 15
- 108060002716 Exonuclease Proteins 0.000 description 6
- 102000013165 exonuclease Human genes 0.000 description 6
- 102000053602 DNA Human genes 0.000 description 5
- 239000000975 dye Substances 0.000 description 5
- 238000004557 single molecule detection Methods 0.000 description 4
- 235000012431 wafers Nutrition 0.000 description 4
- 239000012634 fragment Substances 0.000 description 3
- 238000006862 quantum yield reaction Methods 0.000 description 3
- 239000010453 quartz Substances 0.000 description 3
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicon dioxide Inorganic materials O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 3
- 230000001036 exonucleolytic effect Effects 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 238000010348 incorporation Methods 0.000 description 2
- 150000002500 ions Chemical class 0.000 description 2
- PYWVYCXTNDRMGF-UHFFFAOYSA-N rhodamine B Chemical compound [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- MWBWWFOAEOYUST-UHFFFAOYSA-N 2-aminopurine Chemical compound NC1=NC=C2N=CNC2=N1 MWBWWFOAEOYUST-UHFFFAOYSA-N 0.000 description 1
- YCIPQJTZJGUXND-UHFFFAOYSA-N Aglaia odorata Alkaloid Natural products C1=CC(OC)=CC=C1C1(C(C=2C(=O)N3CCCC3=NC=22)C=3C=CC=CC=3)C2(O)C2=C(OC)C=C(OC)C=C2O1 YCIPQJTZJGUXND-UHFFFAOYSA-N 0.000 description 1
- 241000283160 Inia Species 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 238000000149 argon plasma sintering Methods 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- 239000013626 chemical specie Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000005137 deposition process Methods 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- 230000005281 excited state Effects 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 239000000990 laser dye Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 238000001782 photodegradation Methods 0.000 description 1
- VTGOHKSTWXHQJK-UHFFFAOYSA-N pyrimidin-2-ol Chemical compound OC1=NC=CC=N1 VTGOHKSTWXHQJK-UHFFFAOYSA-N 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 230000002285 radioactive effect Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000007306 turnover Effects 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
Classifications
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- 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
- C12Q1/6869—Methods for sequencing
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
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- G01—MEASURING; TESTING
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
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- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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- B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
- B01J2219/00277—Apparatus
- B01J2219/00351—Means for dispensing and evacuation of reagents
- B01J2219/00378—Piezoelectric or ink jet dispensers
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- B01J2219/00583—Features relative to the processes being carried out
- B01J2219/00603—Making arrays on substantially continuous surfaces
- B01J2219/00659—Two-dimensional arrays
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- B01J2219/00722—Nucleotides
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- C12Q2565/00—Nucleic acid analysis characterised by mode or means of detection
- C12Q2565/10—Detection mode being characterised by the assay principle
- C12Q2565/125—Electrophoretic separation
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- C40—COMBINATORIAL TECHNOLOGY
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- C40B40/00—Libraries per se, e.g. arrays, mixtures
- C40B40/04—Libraries containing only organic compounds
- C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof
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Abstract
SAMMANDRAG
_
Forfarande fOr att analysera reaktionsprodukten (nukleinsyrebassekvens) av enzymatisk enmolekyl-DNA-sekvensering genom att deponera enstaka nukleinsyrebaser pa fasts substrat och anslutande tvadimensionell scanning av substratet, kannetecknat av att deponeringen av reaktionsprodukten sker genom tokala elektriska faltgradienter mellan kapillaren och det fasta substratet samt att detektionen av nukleinsyrebasers fluorescensemission sker
I sma konfokala volymelement.
Description
R.Rigler/Single nucleic acid base detection
SINGLE NUCLEIC ACID BASE DETECTION ON SOLID SUBSTRATES IN SINGLE MOLECULE DNA SEQUENCING
SPECIFICATION
Field
The analysis of the sequence of nucleic acid bases, the elementary building blocks of DNA, is of praramount importance for understanding the genomic code. Presently two methods are being used based on specific cleavage (Maxam-Gilbert) or chain termination (Sanger) of the DNA chain after A,T,G and C. Separation of the fragments is carried out by gel electrophoresis and detection of radioactive or fluorescence marked fragments by appropriate means.The present technology allows a sequencing speed of a few thousand nucleic acid bases per day.
Background
The present technology has a throughput of about 3 to 4 bases per day and requires the sequencing of many overlapping fragments which usivally are cloned in various vectors .The demand for DNA sequencing particularly with medical perspectives in mind is very much higher as exemplified by the human genome project with the aim of sequencing 3x9 bases. In order to reach this goal radically new concepts for DNA sequencing are necessary.
A proposal suggested by the Los Alamos National Laboratory (Jett et al., 1989 ) is based on the processive cleavage of a single DNA strand by an exonuclease.This will result in the succesive liberation of nucleic acid bases,which have to be detected
a t the level of a single molecule.The Los Alamos group has
Ela SOW 41 Tea tithatit* tef ttl?e tagged nucleotides and skessive cleavage.As detection system a sheet flow system has been devised. (Davies et al.1991 ).The main idea is to use the high clevage rate (100 s-1) of the exonuclease combined with rapid single molecule detection to increase the speed of the sequencing process by orders of magnitude.
To be succesfull this approach requires the ability to detect single molecules in a time intervall which is comparable to the turnover number of the enzyme (ca 100 s-1) as well as enzymes which are able to cope with nucleotides tagged with bulky aromatic dye molecules. It has been shown that Rhodamine-labelled nucleotide bases can be detected within 100 s (Rigler&Mets,1992, Mets&Rigler,1994) when excited to fluorescence by a laser beam focussed in extremely small volume elements (2x-16L). The second requirement ,however ,i.e the faithful incorporation of dye tagged nucleotides has yet to be demonstrated.
While for dye tagged nucleotides the possibility of single molecule detection has been demonstrated it appears to be very difficult for natural nucleotides due to their very low quantum yield which is is of order -3 to -4- at room temperature for regular nucleotides (Callis,1979),If this would be possible,e.g. at conditions with enhanced fuoescence yields , the problem connected with enzymes which are not designed by nature to handle dye tagged compounds would be avoided.
Even if both requirements i.e. faithful incorporation of all dye tagged nucleotides as well as single molecule detection can be met an important issue is how many nucleotides can be detected successively before one will be lost.This will depend on the flow system to be used i.e. sheet flow ( Davies et.al.,1991.) or electrophoretic flow (Eigen &Rigler,1994).The problem here is to avoid losses of molecules during the detection process which will occurr if turbulence exists in the sheet flow system or the electric field gradients are not properly designed.A third component, probably the most important is the resistence of the molecules to photodegradation during the detection process which even with laser dyes like Rhodamine is a matter of concern.
R.Rigler/Singie nucleic acid base detectWE INVENTION3
Objects
It is amongst the objects of the invention to provide methods for the detection of nucleic acid bases in single molecule DNA sequencing which are not based on flow devices, at both high or low (cryo) temperatures.
It is another object of the invention to provide
substrates for the deposition of single nucleotides after their cleavage from the DNA chain which are based oon solid substrates (e.g.silicon or quartz wafers),
It is another object of the invention to provide a process which Heads to the generation of single nucleotides in glass capillaries or silicon wafer structures and simultaneous deposition on a solid substrate.
It is another object of the invention to introduce electric fields eletroosmotic flow or flow introducd by pressure gradients for the deposirion of single nucleotides after their cleavage on tthe substrate.
It is another object of the invention to avoid concomitant deposition of single nucleotides with excess enzyme (exonuclease) by a process which deposits the charged nucleic acid base molecule on the substrate by an electric field gradient while the excess enzyme is moved in the opposite direction by the same field and/or electroosmotic flow due to differences in the surface charge of nucleotide and enzyme.
It is another object of the invention to analyse the presence and location of single nucleic acid bases by excitation with a laser beam focussed into a confocal volume element and detection of the fluorescence emission or ligth scatter.
It is another object of the invention to analyse the presence and location of single nucleic acid bases by scanning the substrate or its image by a contincyGus or step scanning device.
R.RigierlSingle nucleic acid base detection4
It is another object of the invention to analyse the presence and location of single nucleic acid bases by scanning the substrate or its image by a continouus or step scanning device.
It is another object of the invention to analyse the substrate for the presence and location of single nucleic acid bases on the substrate by exciting and detecting multiple volume elements using laser and detector arrays.
It is another object cf the i,rnfenti,c2 tc 2r2,,,a! tIft, presence and location of the nuleotide bases by scanning of the object or image plane in all temperatures including cryo temperatures.
It is another object of the invention to analyse the presence and location of individual nucleotide bases by analyzing the image of the substrate in a multielement detector ( e.g. CCD or APD) in combination with time gating.
Definitions
The terminology used herein is not intended to vary from the terminology used in the field.However the meaning of some terms used in the field is not necessarily uniform,and the following definitions will be of help in this case.
"Substrates" refers to extremely pure chips made from silicon or quart wafers.
"Scanning" refers to a motion of the substrate relative to the exciting laser beam in two dimensions.
"CCD" refers to solid state detectors which built up in 2 dimensional arrays of charged coupled devices.
"APD" refers to solid state detectors which are built up bysingle elements or 2 dimensional arrays of avalanche photo diodes.
R.Rigler/Single nucleic acid base detection
"Confocal" refers to a pinhole positioned in the image plane which is conjugated with the object plane.The purpose of the confocal pinhole is to delimit the volume element of excitation in the z-direction.
"Electroosmosis" refers to a process wich moves the solvens including solvated ions in one direction of the field gradient which can be opposite to the motion of charged ions,i.e. nucleic acid bases.
"CW" refers to a process ,where a stationary laser source is used for exciting the fluorescence of nucleic acid bases.
"Pulsed" refers to a process where a pulsed laser source is used for exciting th fluorescence of nucleic acid bases.
"Time gating" refers to a process where the difference in the time constant for emissions is used to discriminate ligth scattering as well as Ramanscattering from fluorescence.
Drawings
which: The disclosures herein have referencs to the drawings in
Figure 1 is a schematic of the exonuclease based cleavage process of the DNA molecule postioned on a support in a capillary and of the deposition of single DNA bases onto the solid substrate (silicon or quartz wafer).The transfer of the charged base from the capillary to the substrate is due to eletric field gradients, elctro-osmotic flow, or mechanical deposition .The substrate moves relatively to the capillary with a speed comparable to the cleavage rate.
Figure 2 is a schematic of the single DNA base detection in cryo temperature under high vaccuum.The substrate with the deposited single DNA bases is scanned through the volume element of excitation . This is imaged over a confocal pinhole on the detector . Exciting and emitted radiation are separated by a dichroic mirror. instead of the substrate itsself also its image can be scanned.
R.Rigier/Singie nucleic acid base detection
Figure 3 is an example of nucleic acid base analogs: 2 amino-purine 5-methyl-2 pyrimidinone and 2-pyrimidinone which emitt at room temperature and can act as A and C analogs respectively
Fig 4 is an example of a sequence identified by DNA base emission at cryo temperature and base analog emission at room temperature.(a) use of one purine and one pyrimidine analog,(b) use of two purine analogs
Summary
A new way to analyse the tkA RACIIIPtIA ni IniA■rt ne-t■rl;;;747.-.ZQ deposited on solid substrates generated by exonucleolytic cleavage of a single DNA molecule is discribed.
Detailed description of the Best Mode of Carrying Out the Invention.
The following detailed description is by way of example and not by way of limitation of the principle of this investigation and has reference to specific examples in which DNA sequencing is carried out by single molecule detection on a solid substrate.
Generation of single nucleic acid bases by
enzymatic cleavage and transfer to a solid substrate.
The enzymatic cleavage of a single DNA molecule bound to a spherical support is carried out in a small glass capillary.The spherical support carrying a single DNA molecule is introduced into the capillary or a microstructure with equivalent function.The processive exonucleolytic cleavage is started by addition of an excess of exonuclease which will cleave each nucleic acid base in a processive manner (Figure 1).
Due to the generation of a local electric field gradient between the capillary and the solid substrate alternatively by generation of a
R.Rigier/Single nucleic acid base detection7
pressure gradient by piezoelectric constriction or other means the single nucleic acid bases are deposited in tiny droplets (pL ) on the surface of the solid support. Chips of silicon or quartz whih can be produced with extreme purity are preferred.During the deposition process the chip is scanned in two dimensions with a speed which is compatible with the rate of base cleavage with the exonuclease. If
one nucleic acid base is deposited i310
bases can be hold on a chip of size lcm x 1 cm.
Solid substrates will have the important advantage over flow detection systems that they will keep a permanent record of the nucleic acid base sequence and can be analyses over and ove again.
Analysis of single nucleic acid bases on solid substrates
Once the chip with the array of single nucleic acid bases which constitute an imprint of the DNA sequence are generated the chip will be tranferred to special measuring device which is outlined as follows (Figure 2).
The detection of the presence of the nucleic acid base
is carried out preferentially at cryo temperature (10-1000K)
where natural bases show an increase in quantum yield by
orders of magnitude (Morgan and Callis,1973,1979).With
excitation in extremely small confocal volume elements (0.2 fL) very
high signal to background ratios can be obtained (Rigler &
Mets ,1992,Rigler et al.,1993,Mets & Rigler,1994) already
with CW excitation. This ratio can be improved further by the
application of pulsed excitation and time gating (Rigler et al.,
1984)
For the actual measurement the laser beam is deflected
by a dichroic mirror onto the solid substrate and the emission
of single nucleic acid bases is detected passing the dichroic
mirror the confocal pinhole and impinging on the detector.
The presence and location of the nucleic acid bases is obtained
by scanning the chip in 2 dimensions or an optical element
generating the image in the plane of the pinhole.The pinhole
can also be substitutet by a combination of an array of lasers and an
tarray of detetors .Provided the signal to background is sufficient
R.Rigler/Single nucleic acid base detection8
pulsed excitation and time gating in combination with array detectors (CCD od APD) can be used.
An important advantage in using cryo temperatures is the reduction and virtual absence of photodestruction during the analysis.since generation and diffusion of chemical species like radicals involved in photodestructions is inhibited.
Application of nucleic acid base analogs for DNA sequencing.
It can be shown that purines (A,G) can be differentiated from pyrimidines (C,T) by the lifetime of the excited state
(Rigler etal.1984) .With the introduction of nucleic acid base analogs (Figure 3) which exhibit high quantum yield and can base pair with natural nucleic acid bases .The presence and location of theseanalogs can be measured at high temperatures where natural nucliec acid bases do not fluoresce. At low temperatures the position of purines and pyrimidines can be evaluated and thus the whole sequence .Two examples for the combination of
analogs with natural nucleic acid bases are given (Figure 4 a,b)
References
Callis,P.R.,Chemical Physics Letters,61,563-567,1979.
Davies, L. M. ,Fairfield, F. R. , Harger, C.A. ,Jett,J. H. ,Keller, R.A. , Hahn , J. H., Krakowski, L.A. , Marrone, B. L., Martin , J. C., Nutter, H. L. , Ratliff, R. L., Brooks Shera, E. , Simpson, D.J. and Soper, S.A. , GATA, 81-7.1991.
Eigen,M.and Rigler,R.,Proc.NatlAcad.Scie.U.S:91,5740-5747,1994.
Jett, J. Keller, R.A. , Martin, J. C , Marrone, B. L. , Moyzis, R. K, Rarliff, R. L., Seitzinger,N.K.,Brooks Shera,E. and Stewart,C.C.J.Biomolecular Structure and Dynamics 7,301-309,1989.
Mets,O. and Rigler,R.J.Fluorescence,4,259-264,1994.
Morgan,J.P. and Callis,P.R.,Photochemistry and Photobiology, 23,131-134,1976.
Claims (2)
- R.Rigler/Single nucleic acid base detection 9 Morgan,.l.P.and Callis,P.R.Photochemistry and Photobiology, 29,1107-1113.1979. Rigler,R.,Claesens,F .and Lomakka,G.,ln Ultrafast Phenomena, ed.Auston, D. H,and Eisenthal,K.B.(Springer, Berlin)Vol 4,pp 472-476, 1984. Rigler,R.and Mets,Ü,Soc.Photo,Opt.lnstr.Eng.(SPlE)1921 239-248, 1992. Rigier,R.,mets,ü,widengren,.i.ana Kasiæiëursiophysics 4.22, 1ee17s,1993. CLAlM 1.A new way to analyse the product of enzymatic single molecule DNA sequencing by depositing indivual nucleic acid bases on a solid substrate .
- 2. The process for deposition of the cleavage products on to the solid substrate by electric field gradients ,hydrodynamic or electroosmotic flow while the solid substrate is scanned in two dimension. 3.The process for deposition of the cleavage products but not the enzyme onto the solid substrate by a combination of electric field gradients ,electroosmotic flow and charge properties of enzyme and nucleic acid base. 4.The generation of tiny droplets by piezolectric constriction or bubble jets for the deposition of nucleic acid bases on solid substrates while the solid substrate or the droplet dispenser is scanned in 2 dimensions. 6.The generation of a solid substrate (chip)with a matrix of nucleic acid bases beiing a permanent record of the DNA- sequence. 7.The 'determination of the presence and location of RRigler/Single nuoleic acid base detection 10 individual nucleic acid bases on solid substrates at high and low (cryo) temperatures using laser excitation in tiny confocal volume elements and 2 dimensional scanning of object or image. 8.The determination of the presence and location of individual nucleic acid bases on solid substrates by CW excltation and /or pulsed excitation and time gating. 9.The determination of the presence and location of individual nucleic acid bases on solid substrates by excitation with a combination of laser and detector arrays. 10. The determinatlon of the DNA-sequence by a combination of natural nucleic acid bases (cryo temperature) and anlogs (high temperature) deposited on a solid substrate.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| SE9500589A SE9500589A0 (sv) | 1995-02-17 | 1995-02-17 | Nukleinsyremolekyl-detektion på fasta substrat vid enmolekyl-DNA-sekvensering |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| SE9500589A SE9500589A0 (sv) | 1995-02-17 | 1995-02-17 | Nukleinsyremolekyl-detektion på fasta substrat vid enmolekyl-DNA-sekvensering |
Publications (3)
| Publication Number | Publication Date |
|---|---|
| SE9500589D0 SE9500589D0 (sv) | 1995-02-17 |
| SE9500589L SE9500589L (sv) | 1996-08-18 |
| SE9500589A0 true SE9500589A0 (sv) | 1996-08-18 |
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| SE9500589A SE9500589A0 (sv) | 1995-02-17 | 1995-02-17 | Nukleinsyremolekyl-detektion på fasta substrat vid enmolekyl-DNA-sekvensering |
Country Status (1)
| Country | Link |
|---|---|
| SE (1) | SE9500589A0 (sv) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1808496A1 (en) * | 2001-01-30 | 2007-07-18 | Solexa Limited | Preparation of polynucleotide arrays |
| US7501245B2 (en) | 1999-06-28 | 2009-03-10 | Helicos Biosciences Corp. | Methods and apparatuses for analyzing polynucleotide sequences |
| US7645596B2 (en) | 1998-05-01 | 2010-01-12 | Arizona Board Of Regents | Method of determining the nucleotide sequence of oligonucleotides and DNA molecules |
| US7666593B2 (en) | 2005-08-26 | 2010-02-23 | Helicos Biosciences Corporation | Single molecule sequencing of captured nucleic acids |
| US7981604B2 (en) | 2004-02-19 | 2011-07-19 | California Institute Of Technology | Methods and kits for analyzing polynucleotide sequences |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7875440B2 (en) | 1998-05-01 | 2011-01-25 | Arizona Board Of Regents | Method of determining the nucleotide sequence of oligonucleotides and DNA molecules |
-
1995
- 1995-02-17 SE SE9500589A patent/SE9500589A0/sv not_active Application Discontinuation
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7645596B2 (en) | 1998-05-01 | 2010-01-12 | Arizona Board Of Regents | Method of determining the nucleotide sequence of oligonucleotides and DNA molecules |
| US7501245B2 (en) | 1999-06-28 | 2009-03-10 | Helicos Biosciences Corp. | Methods and apparatuses for analyzing polynucleotide sequences |
| EP1808496A1 (en) * | 2001-01-30 | 2007-07-18 | Solexa Limited | Preparation of polynucleotide arrays |
| US7981604B2 (en) | 2004-02-19 | 2011-07-19 | California Institute Of Technology | Methods and kits for analyzing polynucleotide sequences |
| US7666593B2 (en) | 2005-08-26 | 2010-02-23 | Helicos Biosciences Corporation | Single molecule sequencing of captured nucleic acids |
Also Published As
| Publication number | Publication date |
|---|---|
| SE9500589L (sv) | 1996-08-18 |
| SE9500589D0 (sv) | 1995-02-17 |
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