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Sequencing method

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Publication number
WO1990013666A1
WO1990013666A1 PCT/GB1990/000726 GB9000726W WO1990013666A1 WO 1990013666 A1 WO1990013666 A1 WO 1990013666A1 GB 9000726 W GB9000726 W GB 9000726W WO 1990013666 A1 WO1990013666 A1 WO 1990013666A1
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Prior art keywords
template
dna
nucleotide
copy
primer
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PCT/GB1990/000726
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French (fr)
Inventor
Peter Bryan Garland
Paul James Heaney
Denise Vera Pollard-Knight
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Amersham International Plc
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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813Hybridisation assays
    • C12Q1/6816Hybridisation assays characterised by the means of detection
    • C12Q1/6825Nucleic acid detection involving sensors
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869Methods for sequencing

Abstract

A method of sequencing a single-stranded nucleic acid chain involves providing an immobilised complex of a template to be sequenced and a primer. This complex is exposed to flow containing only one dNTP at a time. The primer is extended only if the dNTP is complementary to and can hybridise to the next free nucleotide of the template. Each such polymerisation event is detected, preferably directly and by spectroscopic means. Preferred detection techniques include fluorescence detection and absorption spectroscopy (non-evanescent wave) and particularly evanescent spectroscopy e.g. surface plasmon resonance spectroscopy.

Description

SEQUENCING METHOD

Introduction

Determination of the sequence of nucleotide bases in 2 ' -deoxyribonucleic acid (DNA) and to a lesser extent ribonucleic acid (RNA) is a central technology for much of present day molecular biology and its many applications. There are currently two main methods available for sequencing DNA, and both involve using a DNA polymerase enzyme to make a copy of a suitably primed strand of the unknown DNA which acts as a template. In the so-called chain-termination method of Sanger et al (1977), the growing copy strand is randomly (at least, randomness is intended) terminated by incorporation of a 2' , 3 ' -dideoxyribonucleotide (ddNTP) competing with the normal 2'- deoxyribonucleotide (dNTP) for addition to the 3'-end of the growing copy strand. By using suitable conditions and four separate preparations corresponding to dATP with ddATP, dCTP with ddCTP, dGTP with ddGTP and dTTP with ddTTP, all possible lengths of terminated copy strands are available. They are separated by size by gel electrophoresis, and detected by means of an incorporated label, commonly radioactive but more recently fluorescent (Smith et al , 1986; Ansorge et al , 1987). The DNA sequence of the copy, and therefore the complementary sequence of the template, is derived from the pattern of chain lengths detected for the separate A, C, G and T termination mixture.

The chemical degradation method of Maxa and Gilbert (1977) also uses DNA polymerase catalysed formation cf a template-copy. The dNTP ' s are radio-

32 35 active with P (or S) at the α-position, so the copy is also radioactive. Chemical treatments specific for different bases cleave the copy into sets of fragments which, as with the Sanger dideoxy chain termination method, can be separated by gel electrophoresis and detected (by autoradiography) to yield patterns from which the DNA sequence can be deduced. Variants of these methods have been described (Church & Kieffer- Higgins, 1988; Gish and Eckstein, 1988), but are still dependent upon gel electrophoresis to separate DNA fragments, and upon such methods such as radiolabel ling for detection of the separated fragments.

All of these methods are slow, complex and expensive in relation to the needs for DNA sequencing. For example, the best rates of DNA sequencing to date are about 600 nucleotides in 12 hours, using an automated instrument (Landegran et al, 1988). This corresponds to 50 bases per hour or 0.014 bases per sec. This rate is approximately two-orders of magnitude lower than that thought necessary for large- scale sequencing in the Human Genome Project (Alberts et al, 1988).

Three laboratories have reported attempts to avoid the needs for copy-strand fragments and their length-determination by gel electrophoresis. Hyman (1988) immobilised the DNA template primer with polymerase on an ion-exchange material and exposed the complex to a flow wherein only one of the four dNTP's was present at a time. A downstream detection system then detected the pyrophosphate liberated in the DNA polymerase reaction:- template - primer + dNTP -> template - (primer + dNMP) + PPi where dNMP stands for the added nucleotide residue, PPi for pyrophosphate. The sequencing rate was approximately 1 base every 10 minutes. Melamede (1987) also proposed the use of an immobilised DNA template, primer and polymerase complex exposed to a flow containing only one species of dNTP at a time. However, incorporation of dNTP into the copy was calculated not from measurements of pyrophosphate release but from the difference in dNTP concentrations entering and leaving the flow cell which contains the complex of DNA template and polymerase. No experimental data were presented.

Jett (1989) provided a single stranded DNA or RNA sequence of labelled nucleotides, complementary to the sequence to be determined, suspended in a moving flow stream. An exonuclease sequentially cleaved individual basis from the end of the suspended sequence. The resulting train of individual labelled nucleotides was passed to a downstream location for analysis of the individual nucleotides. This invention

As with the methods of Melamede (1987) and Hyman (1988), we also use an immobilised complex of template and primer, exposed to a flow containing only one dNTP at a time. We differ however in that we directly measure the growth of the template copy, rather than infer it indirectly from changes in the composition of the flow medium. The methods of detection are preferably spectroscopic and do not require the further addition of chemical reagents to effect measurement of template-copy growth. The spectroscopic methods are:- i) Evanescent wave spectroscopy ii) Fluorescence detection (non-evanescent wave) iii) Absorption spectroscopy (non-evanescent wave) Alternatively the individual nucleotides may be labelled, e.g. radioactively. Attachment of each successive labelled nucleotide to the immobilised complex is then detected by an increase in total radioactivity. Irrespective of which detection method is used, the time-dependent signal arising from polymerase catalysed template copy growth depends on the availability of the correct (i.e. complementary) nucleotide for base-pairing with the template at the growing point of the copy. If all four nucleotides are absent, there is no polymerase activity. If only one nucleotide is present, then extension of the copy will occur only as far as base-pairing with the template is possible in the absence of the other three nucleotides. Preferably a continuous flow of pulses of deoxynucleotides (dATP, dCTP, dGTP, dTTP) or ribonucleotides (ATP, CTP, GTP, UTP)or analogues of these nucleotides, each separated by a wash pulse, is passed over the tethered template DNA or RNA (with any extension and linkers, and the primer oligonucleotide). This is done under conditions, e.g. in the presence of a suitable polymerase enzyme, to cause an extension of the primer by addition of the nucleotide (or analogue) to the 3'-end of the primer if the nucleotide (or analogue) is complementary to and can hybridize with the next free nucleotide of the template (going from the 3'-end towards the 5'-end). Conditions of solvent, temperatures, ionic strength and concentrations of any polymerase enzyme activating cations and anioπs are maintained such that the polymerase is enzymatical ly active in the presence of its substrates. Because only one of the four nucleotides is present at a time, extension of the copy strand occurs in jumps depending on whether the appropriate nucleotide for pairing with the next unpaired base of the template strand is present or not. Each such jump is herein designated a polymerisation event. Thus the signal also exhibits time-dependent jumps that reflect the extension jumps (polymerisation events) of the copy strand. Furthermore, the size of the signal jumps is proportional to the number of bases added in each extension jump (i.e. proportional to the number of polymerisation events), e.g. the addition of say three adenine residues gives three times the signal given by the addition of only one such residue. Because the copy strand can be extended by only an integral number of bases (0, 1 , 2, 3— ) during any extension jump, then the optical signals are quantised. This property of the signals is highly advantageous for calibration and for discrimination against noise. For simplicity, this invention is hereafter described in terms of DNA sequencing, using a DNA template with DNA polymerase and deoxynucleotides (dNTP's) or analogues to make a DNA copy. It should be noted that RNA technologies could be used : e.g. (i) DNA as the template, RNA polymerase to make an RNA copy (ii) RNA as the template, reverse transcriptase to make a DNA copy, and (iii) RNA as the template, RNA replicase to make an RNA copy. ( ) Evanescent wave spectroscopy (EWS) Evanescent wave spectroscopy is defined in the present context as embracing three related methods: (i) attenuated total reflection (ATR) spectroscopy (ii) total internal reflectance fluorescence (TIRF) spectroscopy, and (iii) surface plasmon resonance (SPR) spectroscopy. Each of these three spectroscopic techniques examines an optical property of a solution bordering a surface where total internal reflection of a light beam has occurred. In each case the incident and reflected beams are on the side of the surface distal to (i.e. remote from) the solution under study, whereas the evanescent wave is established on the solution side of the surface but extends into that solution for a very short distance, typically less than the wavelength of the incident/reflected beam. Spectroscopy by ATR or TIRF requires only a transparent material such as glass or quartz to create the interfacial surface with the solution, whereas SPR spectroscopy requires that the glass or quartz surface be coated with a thin (e.g. 50nm) metal layer of, for example, silver. All three methods can detect the exchange of solute molecules between the bulk phase of the solution and the interfacial surface, albeit by different means. ATR spectroscopy detects the absorption of evanescent wave light by molecules, with appropriate absorption spectra, that lie within the evanescent wave region. If the absorbed light is re- emitted as fluorescence then the emission can be measured with a suitable detector, such as a photo- multiplier tube, leading to TIRF spectroscopy. Thus both ATR and TIRF spectroscopy measure the absorption of light by molecules at or close to the interfacial surface, the difference being that ATR measures the absorption directly whereas TIRF measures it indirectly, as re-emitted fluorescence. By contrast, SPR spectroscopy measures changes of refractive index that may occur in the SPR evanescent wave region, but, just as with ATP and TIRF, those changes arise due to redistribution of molecules between the bulk phase of the solution and the evanescent wave region. For a review of these methods, see Sutherland and Dahne, 1987.

Having given a brief outline of evanescent wave spectroscopies , we can describe their application to DNA sequencing. The DNA molecule of unknown sequence, derived from biological techniques such as DNA cloning or polymerase chain reactions must (i) be single stranded (ii) be attached either directly or indirectly by either its 31 or 5' end to the interfacial surface of an EWS device, and (iii) possess a known sequence, either naturally occurring or added, at its 3' end such that a complementary oligonucleotide (primer) can be hybridised to create a run of double stranded DNA. Thus in the presence of DNA polymerase and deoxyribonucleotide triphosphates (dNTP) the unknown DNA acts as a template for the synthesis of a complementary copy extending by growth from the 3' end of the primer. DNA polymerase catalysed growth of the template copy results in recruitment of dNTP's from the bulk phase to the interfacial region where the template is tethered. It is assumed that the copy strand remains base-paired to the template strand, and does not diffuse away. Template copy growth can therefore be measured by EWS spectroscopy. ATR spectroscopy requires that the incident beam be in the wavelength region of a major absorption band of the dNTP's, typically about 260nm. TIRF spectroscopy requires that the nucleosides incorporated into the template copy can be excited to fluoresce by the evanescent wave : fluorescent DNTP analogues are required. SPR spectroscopy has fewer limitations : dNTP analogues are not required (although substitution with refractive index enhancing atoms such as Br, I or Hg might be advantageous for sensitivity) and the choice of wavelength is not critical.

Figure 1 illustrates the experimental set up, shown for the specific case of SPR spectroscopy. The 3' end of the template DNA (or its extension) is attached to the interfacial silver surface, and the primer oligonucleotide is hybridised to the template. Alternatively the primer could be attached to the surface and the template DNA hybridised to the primer. Clearly the polarity of attachment can be reversed:- the 5' end of the template bound to the surface, and the primer hybridised to the free 3' end of the template or extension.

Figure 2 shows idealised experimental results using SPR spectroscopy. The sequence of the template being copied is read directly from the associations between jumps in the EWS signal and the particular dNTP pulse present at the template site, (ii ) Fluorescence detection

Because each cycle of dNTP (or rather its fluorescent analogue) is preceded and followed by a wash, EWS (TIRF) is not mandatory to distinguish the fluorescence signal arising from the template-copy from free nucleotides in the medium. Thus a more conventional optical geometry can be used, and immobilisation of the template-primer-polymerase complex can be on beads, fibres or membranes rather than an evanescent wave surface.

A serious drawback of the fluorescent method for DNA sequencing, irrespective of whether or not it uses an evanescent wave, is the fact that the absolute size of the signal grows as the copy strand grows. This makes it harder to detect the increments (jumps) of fluorescence resulting from strand extension. Even under ideal conditions (Poissonian photon-counting statistics, zero background and noise-less excitation source), it is necessary to increase the measurement time for each increment of nucleotide addition in proportion to the number of such increments in order to maintain constant sensitivity for the detection of each successive fluorescence increment. For example, if it took 1 sec to measure the fluorescence of the first added nucleotide to a satisfactory accuracy, then 10 seconds would be needed to measure the tenth added nucleotide, 100 seconds for the hundredth, and so on. Thus what is intended to be a fast DNA sequencing method becomes progressively slower as it proceeds.

There are two instrumental solutions to this problem. The first is to increase not the duration of measurement for successive increments of fluorescence, but the excitation intensity. By this means the accumulation of photon counts required for accuracy can be achieved in a constant time. Technically this can be arranged with a CW laser excitation source equipped with an acousto-optic or electro-optic modulator to modify the beam intensity transmitted to the sample. The second solution is to irreversibly photobleach the sample fluorescence with an intense light pulse at periodic intervals, thereby resetting the sample fluorescence to zero before it can significantly degrade the detection of successive increments.

Technically this can also be achieved with a CW laser equipped with a modulator (Garland, 1981).

The fluorescent probe itself can be attached to each of the four dNTP's. As each dNTP is presented separately to the template-primer-polymerase complex, the same fluorophoric group can be used for each dNTP. Alternatively, fluorescent dideoxy chain terminators can be used in competition with unlabelled dNTP's, under conditions where the number of terminated chains s not so large as to deplete the number of unterminated chains to the point where fluorescence increments associated with chain termination become difficult to detect. Each dNTP is presented with its corresponding fluorescently-label led ddNTP to the template-primer-polymerase complex, and a single fluorophore is sufficient, (iii) Absorption spectroscopy

The direct measurement of template copy growth by detecting the increase in ultraviolet light absorption at approximately 250-260nm caused by addition of new nucleotides to the copy strand is not sensitive. However, the availability of relatively large amounts of starting material (template) through DNA cloning or polymerase chain methods, allied to powerful U-V laser sources such as f equency-tripled Nd-Yag lasers, makes it technically possible to measure template-copy growth directly by absorption spectroscopy.

The advantages of these optical methods over existing methods are very large. They include:- - potential for high speed (e.g. 10 bases/sec) miniaturisation and parallel flow lanes no need for gel electrophoresis direct signal transfer to computer no need for radioactive nucleotides - greater chance for complete automation due to absence of gel separation steps.

The following Examples illustrate the invention.

EXAMPLE 1

Detection of Polymerase Catalysed Nucleotide Addition on a Silver Slide using Surface Plasmon Resonance

Materials and Methods Reagents

1. 0.25pmole/μl solutions of hybrid DNA (consisting of a 17 nucleotide primer sequence hybridised to a 20, 27 or a 97 nucleotide template sequence) prepared in 10 mM phosphate buffer pH 7.4 containing 10mM phosphate 5 buffer pH 7.4 containing 10mM MgCl and 50mM NaCl.

2. 10mM phosphate buffer pH 7.4 containing 10mM MgCl2 and 50mM NaCl (sequencing phosphate).

3. 10mM phosphate buffer pH 7.4.

4. DNA Polymerase 1 "Klenow fragment" diluted in Q sequencing phosphate containing 250μM dATP, dCTP, dGTP and dTTP.

5. Block solution of 0.05% BSA in sequencing phosphate.

5 Equipment

Rig

A single beam direct reference rig using a 632.8nm wavelength helium neon laser which interrogated two spots and subtracted one signal from the other, was used. Pump and flow cell

A modified step motor pump which held 2 syringes was used to deliver different or identical solutions to the 2 segregated areas of the flow cell. Si 1ver SI ides

Silver coating thickness on the slides was greater than that normally used for SPR work i.e. 60nm to achieve an SPR width of approximately 0.6 degrees at approximately 5% from the reflectance minimum.

Slide Tolerance Limits

The slides were rejected if the SPR profiles on both areas of the silver slide were not similar in shape and minimum.

Experimental Procedure

1. 1ml 10mM phosphate buffer ph 7.4 was added at 4μl/second to the silver slide.

2. 1ml sequencing phosphate was added at 4μl/second.

3. 1ml of hybrid (17/20 was used as the reference DNA) 17/27 or 17/97 was added at 2μl/second followed by 3 washes in sequencing phosphate. 4- 1ml of BSA block solution was added at 2μl/second followed by 3 x 0.3ml washes in sequencing phosphate. 5. 1ml of Klenow plus nucleotides was added at 2μl/second followed by 3 x 0.3ml washes in sequencing phosphate . Angle and time scans were obtained at strategic points during the experiment. The overall shift difference caused by the addition of extra nucleotides (3 for the reference, 10 for the 17/27 and 80 for the 17/97) was recorded.

Results

The figures in the last column are additional changes over and above those of the 17/20 reference.

Conclusion

The SPR signal obtained from the addition of ten nucleotides to a primer-template complex by the Klenow fragment of DNA polymerase could be measured above the signal obtained from a three nucleotide addition in the reference sample. This clearly demonstrates the detection by SPR of the addition of seven nucleotides.

If the following change is made to the equipment then single nucleotide addition will be detected by SPRS.

The experiments were conducted by recording one reflectivity measurement per second. This gives signal to noise resolution down to seven nucleotides. However by increasing the measurement rate to 1000 per second an improvement by a factor of 30 can be achieved which will permit detection of single nucleotide addition. Noise is proportional to the square root of the number of readings taken, therefore the square root of 1000 equals 30.

EXAMPLE 2

Polymerase Catalysed Single Nucleotide Addition to template DNA Immobilised on Silver Slides

Method 1. Template-primer DNA (a 17 nucleotide primer annealed to a 97 nucleotide template) was immobilised

- 13 on the silver slides at a coverage of 1.25 x 10 moles/silver slide.

2. The 4 nucleotide / polymerase mixes were made up as follows in 10mM phosphate buffer pH 7.4 containing 10mm

MgCl and 50mm NaCl:- a) 5μM deoxyadenosine 5 ' -triphosphate + 200U/ml Klenow fragment (Amersham T2141Z), b) 5μM deoxycytidine 5 ' -triphosphate + 200U/ml Klenow fragment, c) 5μM deoxyguanosine 5 ' -triphosphate + 200U/ml Klenow fragment, d) 5μM deoxythymidine 5 ' -triphosphate + 200U/ml Klenow fragment, To each mix 100μCi/ml of the corresponding

32

P labelled deoxynucleotide triphosphate was added

(Amersham PB204, 205, 206 and 207).

The mixtures a-d were added singly in the sequence in which they appeared in the template strand as follows:- Single nucleotide additions

1. The silver slide was placed on the biosensor he icylinder and its position marked. The flow cell was placed on top of the slide. 2. 3ml of 10mM phosphate buffer pH 7.4 was flowed over the silver slide.

3. 1ml of 10mM phosphate buffer pH 7.4 containing 10mm MgCl and 50mm NaCl was flowed over the silver slide.

4. 100μl of the nucleotide / enzyme mix was injected and left on the silver slide for 5 minutes at room temperature.

5. The silver slide was washed with 5ml of 10mM phosphate buffer pH 7.4 containing 10mm MgCl- and 50mm NaCl + 0.005% Tween 20. 6. The silver slide was removed and blotted to remove any surface fluid then counted on the tritium channel of a scintillation counter.

Incorporation of the deoxynucleotide was determined from the increase in counts on the silver slides and calculated from the specific activity of the nucleotide/polymerase mixture used at that step. The results are shown in Table 1.

7. The silver slide and flow cell were replaced on the biosensor in the same position and steps 3-6 were repeated with the next nucleotide/polymerase mixture until the predetermined nucleotide addition sequence was completed.

Control silver slides were subjected to the same nucleotide addition procedure. Results

Correct Sequence Single Nucleotide Addition to Silver Slide Immobilised DNA

Conclusion

The addition of successive single nucleotides by the Klenow fragment of DNA polymerase 1 to this primer template hybrid immobilised at the silver surface can be observed with radioactively labelled nucleotides.

In the absence of the primer template hybrid no successive increase in nucleotide addition is observed.

References

Alberts B M, Botstein D, Brenner S, Cantor C R,

Doolittle R F, Hood L, McKusick V A , Nathans D, Olson

M V, Orkin S, Rosenberg L E, Ruddle F H, Tilghman S, Tooze J & Watson J D, (1988). Report of the Committee on Mapping and Sequencing the Human Genome. National

Academy Press, Washington DC.

Ansorge W, Sproat B, Stegemann J, Schwager C & Zenke M,

(1987). Nucleic Acid Res J_5, 4593-4602. Church G M & Kieffer-Higgins S (1988) Science 240,

185-188.

Garland P B (1987) Biophys. J. 33, 481-482.

Gish G & Eckstein F (1988) Science 2^0, 1520-1522.

Hy an E D (1988) Anal. Biochem. V74-, 423-436. Landegran U, Kaiser R, Caskey CT & Hood L (1988).

Science 242., 229-237.

Maxam A M & Gilbert W (1977). Proc. Natl. Acad. Sci.

(USA) 74., 560-564.

Melamede J (1987) European Patent Application. Publication Number 0 223 618 A2.

Sanger F, Nickless S & Coulson A R (1977). Proc. Natl.

Acad. Sci. (USA) (1987), 7 , 5463-5467.

Sutherland R M & Dahne C (1987) in "Biosensors -

Fundamentals and Applications" (A P F Turner, I Kamber, G S Wilson, eds) pp 655-678. Oxford University Press,

Oxford.

Smith L M, Sanders J Z, Kaiser R J, Hughes P, Dodd C, connell C R, Heiner C, Kent S B H & Hood L E. (1986)

Nature 3jLi» 674-679. Jett J H et al, International Patent Application

WO 89/03432 published 20 April 1989 under the Patent

Co-operation Treaty.

Claims

1. A method of sequencing a single-stranded nucleic acid chain, which method comprises:- a) providing a template-primer complex comprising the single-stranded chain (the template) and at its 3'-end a double stranded chain (comprising the primer), the complex being attached at one end to a solid surface, b) presenting a single nucleotide or nucleotide analogue to the complex in the presence of a polymerase under conditions to cause an extension of the primer (a polymerisation event) by addition of the nucleotide (or analogue) to the 3'-end of the primer if the nucleotide (or analogue) is complementary to and can hybridize to the next free nucleotide at the 5'-end of the template, and c) repeating step b) successively using different nucleotides or nucleotide analogues, d) the method being characterized by detecting a change in the template-extended primer complex resulting from each polymerisation event that occurs during performance of steps b) and c).
2. A method as claimed in claim 1, characterised by detecting, directly and by spectroscopic means, each polymerisation event that occurs during performance of steps b) and c).
3. A method as claimed in claim 2, wherein each polymerisation event is detected by evanescent wave spectroscopy.
4. A method as claimed in claim 2, wherein each polymerisation event is detected by attenuated total reflection spectroscopy.
5- A method as claimed in claim 2, wherein fluorescent nucleotide analogues are used in steps b) and c) and each polymerisation event is detected by total internal reflectance fluorescence spectroscopy.
6. A method as claimed in claim 2, wherein each polymerisation event i s detected by surface plasmon
5 resonance spectroscopy.
7. A method as claimed in claim 6, wherein high refractive index nucleotide analogues are used in steps b) and c) .
8. A method as claimed in claim 2, wherein 0 fluorescent nucleotide analogues are used in steps b) and c) and each polymerisation event is detected by fluorescence spectroscopy.
9. A method as claimed in claim 2, wherein each polymerisation event is detected by absorption spectroscopy.
10. A method as claimed in any one of claims 1 to 9, wherein the single-stranded nucleic acid chain is DNA.
11. A method as claimed in any one of claims 1 to o 10. wherein a continuous flow of pulses of deoxynucleotides (dATP, dCTP, dGTP, dTTP) or ribonucleotides (ATP, CTP, GTP, UTP) or analogues of these nucleotides, each separated by a wash pulse, is passed over the complex attached to the solid surface. 5
0
5
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Cited By (78)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991006678A1 (en) * 1989-10-26 1991-05-16 Sri International Dna sequencing
WO1993006241A1 (en) * 1991-09-16 1993-04-01 The Secretary Of State For Defence In Her Britannic Majesty's Government Of The United Kingdom Of Great Britain And Northern Ireland Gene probe biosensor method
WO1993021340A1 (en) * 1992-04-22 1993-10-28 Medical Research Council Dna sequencing method
US5547839A (en) * 1989-06-07 1996-08-20 Affymax Technologies N.V. Sequencing of surface immobilized polymers utilizing microflourescence detection
WO1997004129A1 (en) * 1995-07-14 1997-02-06 Biacore Ab Method for nucleic acid sequencing
US5641634A (en) * 1995-11-30 1997-06-24 Mandecki; Wlodek Electronically-indexed solid-phase assay for biomolecules
WO1997027331A2 (en) * 1996-01-23 1997-07-31 Rapigene, Inc. Methods and compositions for determining the sequence of nucleic acid molecules
WO1997047761A1 (en) * 1996-06-14 1997-12-18 Sarnoff Corporation Method for polynucleotide sequencing
US5736332A (en) * 1995-11-30 1998-04-07 Mandecki; Wlodek Method of determining the sequence of nucleic acids employing solid-phase particles carrying transponders
WO1998039485A2 (en) * 1997-03-05 1998-09-11 The Regents Of The University Of Michigan Compositions and methods for analysis of nucleic acids
WO1999005315A2 (en) * 1997-07-28 1999-02-04 Medical Biosystems Ltd. Nucleic acid sequence analysis
WO1999005321A1 (en) * 1997-07-22 1999-02-04 Rapigene, Inc. Amplification and other enzymatic reactions performed on nucleic acid arrays
WO1999005320A1 (en) * 1997-07-22 1999-02-04 Rapigene, Inc. Multiple functionalities within an array element and uses thereof
WO1999054497A1 (en) * 1998-04-22 1999-10-28 Stefan Seeger Method and device for determining the concentration, adsorption and binding kinetics and equilibrium and binding constants of molecules by luminescence measurements
US5981166A (en) * 1997-04-23 1999-11-09 Pharmaseq, Inc. Screening of soluble chemical compounds for their pharmacological properties utilizing transponders
WO1999057321A1 (en) * 1998-05-01 1999-11-11 Arizona Board Of Regents Method of determining the nucleotide sequence of oligonucleotides and dna molecules
US6027890A (en) * 1996-01-23 2000-02-22 Rapigene, Inc. Methods and compositions for enhancing sensitivity in the analysis of biological-based assays
WO2000018956A1 (en) * 1998-09-30 2000-04-06 Molecular Machines & Industries Gmbh Method for dna- or rna-sequencing
US6051377A (en) * 1995-11-30 2000-04-18 Pharmaseq, Inc. Multiplex assay for nucleic acids employing transponders
WO2000053805A1 (en) * 1999-03-10 2000-09-14 Asm Scientific, Inc. A method for direct nucleic acid sequencing
WO2000058507A1 (en) * 1999-03-30 2000-10-05 Solexa Ltd. Polynucleotide sequencing
WO2000060114A2 (en) * 1999-04-06 2000-10-12 Medical Biosystems Ltd. Polynucleotide sequencing using a helicase
US6136543A (en) * 1997-01-31 2000-10-24 Hitachi, Ltd. Method for determining nucleic acids base sequence and apparatus therefor
US6153379A (en) * 1993-06-22 2000-11-28 Baylor College Of Medicine Parallel primer extension approach to nucleic acid sequence analysis
US6197557B1 (en) 1997-03-05 2001-03-06 The Regents Of The University Of Michigan Compositions and methods for analysis of nucleic acids
WO2001025480A2 (en) * 1999-10-06 2001-04-12 Medical Biosystems Ltd. Dna sequencing method
WO2001032930A1 (en) * 1999-11-04 2001-05-10 California Institute Of Technology Methods and apparatuses for analyzing polynucleotide sequences
US6242193B1 (en) 1999-07-30 2001-06-05 Hitachi, Ltd. Apparatus for determining base sequence of nucleic acid
WO2001057248A2 (en) * 2000-02-01 2001-08-09 Solexa Ltd. Polynucleotide arrays and their use in sequencing
WO2001057249A1 (en) * 2000-02-02 2001-08-09 Solexa Ltd. Synthesis of spatially addressed molecular arrays
WO2001073121A1 (en) * 2000-03-30 2001-10-04 Toyota Jidosha Kabushiki Kaisha Method of determining base sequence of single nucleic acid molecule
US6312893B1 (en) 1996-01-23 2001-11-06 Qiagen Genomics, Inc. Methods and compositions for determining the sequence of nucleic acid molecules
EP1163369A1 (en) * 1999-02-23 2001-12-19 Caliper Technologies Corporation Sequencing by incorporation
WO2002002813A2 (en) * 2000-07-05 2002-01-10 Amersham Biosiences Uk Ltd. Sequencing method
EP1179085A1 (en) * 1999-05-19 2002-02-13 Cornell Research Foundation, Inc. Method for sequencing nucleic acid molecules
US6355433B1 (en) 2000-06-02 2002-03-12 Dna Sciences, Inc. Determination of nucleotide sequence variations through limited primer extension
US6361950B1 (en) 1995-11-30 2002-03-26 Pharmaseq, Inc. Multiplex assay for nucleic acids employing transponders
US6365349B1 (en) 1997-07-22 2002-04-02 Qiagen Genomics, Inc. Apparatus and methods for arraying solution onto a solid support
US6387623B1 (en) 1995-11-30 2002-05-14 Pharmaseq Screening of drugs from chemical combinatorial libraries employing transponders
US6458544B1 (en) 1999-12-02 2002-10-01 Dna Sciences, Inc. Methods for determining single nucleotide variations and genotyping
US6511803B1 (en) * 1997-10-10 2003-01-28 President And Fellows Of Harvard College Replica amplification of nucleic acid arrays
US6573047B1 (en) 1999-04-13 2003-06-03 Dna Sciences, Inc. Detection of nucleotide sequence variation through fluorescence resonance energy transfer label generation
US6613508B1 (en) 1996-01-23 2003-09-02 Qiagen Genomics, Inc. Methods and compositions for analyzing nucleic acid molecules utilizing sizing techniques
WO2003073088A2 (en) * 2001-03-09 2003-09-04 Christofer Toumazou Apparatus and method for the detection of localised fluctuactions of ionic charge by ion sensitive field effect transistors during a chemical reaction
US6653082B2 (en) 2001-05-17 2003-11-25 Baylor College Of Medicine Substrate-bound cleavage assay for nucleic acid analysis
WO2004029294A1 (en) * 2002-09-27 2004-04-08 Biotage Ab New sequencing method for sequencing rna molecules
WO2004072294A2 (en) * 2003-02-12 2004-08-26 Genizon Svenska Ab Methods and means for nucleic acid sequencing
US6787308B2 (en) 1998-07-30 2004-09-07 Solexa Ltd. Arrayed biomolecules and their use in sequencing
US6818395B1 (en) 1999-06-28 2004-11-16 California Institute Of Technology Methods and apparatus for analyzing polynucleotide sequences
WO2004106545A1 (en) * 2003-05-28 2004-12-09 Innogenetics N.V. Methods for enhanced detection using surface sensitive techniques.
US6908736B1 (en) 1999-10-06 2005-06-21 Medical Biosystems, Ltd. DNA sequencing method
WO2005118871A1 (en) * 2004-05-28 2005-12-15 The Arizona Board Of Regents Surface plasmon resonance sensor for detecting changes in polynucleotides mass
US7001722B1 (en) 1993-06-22 2006-02-21 Baylor College Of Medicine Parallel primer extension approach to nucleic acid sequence analysis
EP1681356A1 (en) * 1999-05-19 2006-07-19 Cornell Research Foundation, Inc. Method for sequencing nucleic acid molecules
US7170050B2 (en) 2004-09-17 2007-01-30 Pacific Biosciences Of California, Inc. Apparatus and methods for optical analysis of molecules
US7270958B2 (en) 1998-09-10 2007-09-18 The Regents Of The University Of Michigan Compositions and methods for analysis of nucleic acids
EP1837407A2 (en) * 2001-09-24 2007-09-26 Intel Corporation Nucleic acid sequencing by monitoring of uptake of precursors during molecular replication, with molecule dispenser
US7302146B2 (en) 2004-09-17 2007-11-27 Pacific Biosciences Of California, Inc. Apparatus and method for analysis of molecules
US7427673B2 (en) 2001-12-04 2008-09-23 Illumina Cambridge Limited Labelled nucleotides
US7501245B2 (en) 1999-06-28 2009-03-10 Helicos Biosciences Corp. Methods and apparatuses for analyzing polynucleotide sequences
US7541444B2 (en) 2002-08-23 2009-06-02 Illumina Cambridge Limited Modified nucleotides
US7592435B2 (en) 2005-08-19 2009-09-22 Illumina Cambridge Limited Modified nucleosides and nucleotides and uses thereof
US7772384B2 (en) 2001-12-04 2010-08-10 Illumina Cambridge Limited Labelled nucleotides
US8114591B2 (en) 2001-03-09 2012-02-14 Dna Electronics Ltd. Sensing apparatus and method
US8192961B2 (en) 1998-12-14 2012-06-05 Pacific Biosciences Of California, Inc. System and methods for nucleic acid sequencing of single molecules by polymerase synthesis
US8263365B2 (en) 1998-05-01 2012-09-11 Arizona Board Of Regents Method of determining the nucleotide sequence of oligonucleotides and DNA molecules
US8399188B2 (en) 2006-09-28 2013-03-19 Illumina, Inc. Compositions and methods for nucleotide sequencing
US8911972B2 (en) 2009-12-16 2014-12-16 Pacific Biosciences Of California, Inc. Sequencing methods using enzyme conformation
US8993230B2 (en) 2008-12-04 2015-03-31 Pacific Biosciences of Californ, Inc. Asynchronous sequencing of biological polymers
US9012144B2 (en) 2003-11-12 2015-04-21 Fluidigm Corporation Short cycle methods for sequencing polynucleotides
US9115163B2 (en) 2007-10-19 2015-08-25 The Trustees Of Columbia University In The City Of New York DNA sequence with non-fluorescent nucleotide reversible terminators and cleavable label modified nucleotide terminators
US9127314B2 (en) 2002-08-23 2015-09-08 Illumina Cambridge Limited Labelled nucleotides
US9133511B2 (en) 2000-10-06 2015-09-15 The Trustees Of Columbia University In The City Of New York Massive parallel method for decoding DNA and RNA
US9175342B2 (en) 2007-10-19 2015-11-03 The Trustees Of Columbia University In The City Of New York Synthesis of cleavable fluorescent nucleotides as reversible terminators for DNA sequencing by synthesis
US9243284B2 (en) 2000-12-01 2016-01-26 Life Technologies Corporation Enzymatic nucleic acid synthesis: compositions and methods for inhibiting pyrophosphorolysis
US9528151B2 (en) 2006-12-01 2016-12-27 The Trustees Of Columbia University In The City Of New York Four-color DNA sequencing by synthesis using cleavable fluorescent nucleotide reversible terminators
US9708358B2 (en) 2000-10-06 2017-07-18 The Trustees Of Columbia University In The City Of New York Massive parallel method for decoding DNA and RNA
US9868978B2 (en) 2005-08-26 2018-01-16 Fluidigm Corporation Single molecule sequencing of captured nucleic acids

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2002171999A (en) * 2000-12-08 2002-06-18 Eiken Chem Co Ltd Method and apparatus for assaying nucleic acid amplification reaction

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0223618A2 (en) * 1985-07-18 1987-05-27 New York Medical College Automatable process for nucleotide sequencing
EP0233053A2 (en) * 1986-02-07 1987-08-19 Applied Biosystems, Inc. Method of detecting electrophoretically separated oligonucleotides
US4770992A (en) * 1985-11-27 1988-09-13 Den Engh Gerrit J Van Detection of specific DNA sequences by flow cytometry
WO1989003432A1 (en) * 1987-10-07 1989-04-20 United States Department Of Energy Method for rapid base sequencing in dna and rna

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0223618A2 (en) * 1985-07-18 1987-05-27 New York Medical College Automatable process for nucleotide sequencing
US4770992A (en) * 1985-11-27 1988-09-13 Den Engh Gerrit J Van Detection of specific DNA sequences by flow cytometry
EP0233053A2 (en) * 1986-02-07 1987-08-19 Applied Biosystems, Inc. Method of detecting electrophoretically separated oligonucleotides
WO1989003432A1 (en) * 1987-10-07 1989-04-20 United States Department Of Energy Method for rapid base sequencing in dna and rna

Cited By (185)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5902723A (en) * 1989-06-07 1999-05-11 Dower; William J. Analysis of surface immobilized polymers utilizing microfluorescence detection
US5547839A (en) * 1989-06-07 1996-08-20 Affymax Technologies N.V. Sequencing of surface immobilized polymers utilizing microflourescence detection
WO1991006678A1 (en) * 1989-10-26 1991-05-16 Sri International Dna sequencing
US7056666B2 (en) 1990-12-06 2006-06-06 Affymetrix, Inc. Analysis of surface immobilized polymers utilizing microfluorescence detection
GB2274710B (en) * 1991-09-16 1995-09-06 Secr Defence Detection of nucleic acid sequences in a sample using an oligonucleotide immobilised on a waveguide
US5750337A (en) * 1991-09-16 1998-05-12 The Secretary Of State For Defence In Her Britannic Majesty's Government Of The United Kingdom Of Great Britain And Northern Ireland Methods for detecting nucleic acid sequences using evanescent wave detection
WO1993006241A1 (en) * 1991-09-16 1993-04-01 The Secretary Of State For Defence In Her Britannic Majesty's Government Of The United Kingdom Of Great Britain And Northern Ireland Gene probe biosensor method
GB2274710A (en) * 1991-09-16 1994-08-03 Secr Defence Gene probe biosensor method
WO1993021340A1 (en) * 1992-04-22 1993-10-28 Medical Research Council Dna sequencing method
US6087095A (en) * 1992-04-22 2000-07-11 Medical Research Council DNA sequencing method
US7001722B1 (en) 1993-06-22 2006-02-21 Baylor College Of Medicine Parallel primer extension approach to nucleic acid sequence analysis
US6153379A (en) * 1993-06-22 2000-11-28 Baylor College Of Medicine Parallel primer extension approach to nucleic acid sequence analysis
WO1997004129A1 (en) * 1995-07-14 1997-02-06 Biacore Ab Method for nucleic acid sequencing
US6387623B1 (en) 1995-11-30 2002-05-14 Pharmaseq Screening of drugs from chemical combinatorial libraries employing transponders
US6046003A (en) * 1995-11-30 2000-04-04 Pharmaseq, Inc. Method of determining the sequence of nucleic acids employing solid-phase particles carrying transponders
US5736332A (en) * 1995-11-30 1998-04-07 Mandecki; Wlodek Method of determining the sequence of nucleic acids employing solid-phase particles carrying transponders
US6361950B1 (en) 1995-11-30 2002-03-26 Pharmaseq, Inc. Multiplex assay for nucleic acids employing transponders
US6686158B2 (en) 1995-11-30 2004-02-03 Pharma Seg, Inc. Electronically-indexed solid-phase assay for biomolecules
US6051377A (en) * 1995-11-30 2000-04-18 Pharmaseq, Inc. Multiplex assay for nucleic acids employing transponders
US5641634A (en) * 1995-11-30 1997-06-24 Mandecki; Wlodek Electronically-indexed solid-phase assay for biomolecules
US6376187B1 (en) 1995-11-30 2002-04-23 Pharmaseq, Inc. Electronically-indexed solid-phase assay for biomolecules
US6815212B2 (en) 1996-01-23 2004-11-09 Qiagen Genomics, Inc. Methods and compositions for enhancing sensitivity in the analysis of biological-based assays
US6613508B1 (en) 1996-01-23 2003-09-02 Qiagen Genomics, Inc. Methods and compositions for analyzing nucleic acid molecules utilizing sizing techniques
US6623928B2 (en) 1996-01-23 2003-09-23 Qiagen Genomics, Inc. Methods and compositions for determining the sequence of nucleic acid molecules
US7642344B2 (en) 1996-01-23 2010-01-05 Operon Biotechnologies, Inc. Methods and compositions for determining the sequence of nucleic acid molecules
US7052846B2 (en) 1996-01-23 2006-05-30 Operon Biotechnologies, Inc. Methods and compositions for analyzing nucleic acid molecules utilizing sizing techniques
US6027890A (en) * 1996-01-23 2000-02-22 Rapigene, Inc. Methods and compositions for enhancing sensitivity in the analysis of biological-based assays
WO1997027331A3 (en) * 1996-01-23 1998-04-02 Darwin Molecular Corp Methods and compositions for determining the sequence of nucleic acid molecules
WO1997027331A2 (en) * 1996-01-23 1997-07-31 Rapigene, Inc. Methods and compositions for determining the sequence of nucleic acid molecules
US6312893B1 (en) 1996-01-23 2001-11-06 Qiagen Genomics, Inc. Methods and compositions for determining the sequence of nucleic acid molecules
US7247434B2 (en) 1996-01-23 2007-07-24 Operon Biotechnologies, Inc. Methods and compositions for determining the sequence of nucleic acid molecules
WO1997047761A1 (en) * 1996-06-14 1997-12-18 Sarnoff Corporation Method for polynucleotide sequencing
US5908755A (en) * 1996-06-14 1999-06-01 Sarnoff Corporation Sequential step method for sequencing and identifying polynucleotides
US6136543A (en) * 1997-01-31 2000-10-24 Hitachi, Ltd. Method for determining nucleic acids base sequence and apparatus therefor
WO1998039485A3 (en) * 1997-03-05 1999-02-25 John P Langmore Compositions and methods for analysis of nucleic acids
US6762022B2 (en) 1997-03-05 2004-07-13 The Regents Of The University Of Michigan Compositions and methods for analysis of nucleic acids
US6197557B1 (en) 1997-03-05 2001-03-06 The Regents Of The University Of Michigan Compositions and methods for analysis of nucleic acids
US6117634A (en) * 1997-03-05 2000-09-12 The Reagents Of The University Of Michigan Nucleic acid sequencing and mapping
US6537757B1 (en) 1997-03-05 2003-03-25 The Regents Of The University Of Michigan Nucleic acid sequencing and mapping
WO1998039485A2 (en) * 1997-03-05 1998-09-11 The Regents Of The University Of Michigan Compositions and methods for analysis of nucleic acids
US5981166A (en) * 1997-04-23 1999-11-09 Pharmaseq, Inc. Screening of soluble chemical compounds for their pharmacological properties utilizing transponders
US6365349B1 (en) 1997-07-22 2002-04-02 Qiagen Genomics, Inc. Apparatus and methods for arraying solution onto a solid support
WO1999005321A1 (en) * 1997-07-22 1999-02-04 Rapigene, Inc. Amplification and other enzymatic reactions performed on nucleic acid arrays
US6248521B1 (en) 1997-07-22 2001-06-19 Qiagen Genomics, Inc. Amplification and other enzymatic reactions performed on nucleic acid arrays
WO1999005320A1 (en) * 1997-07-22 1999-02-04 Rapigene, Inc. Multiple functionalities within an array element and uses thereof
WO1999005315A3 (en) * 1997-07-28 1999-04-22 Daniel Henry Densham Nucleic acid sequence analysis
EP1229133A3 (en) * 1997-07-28 2004-10-13 Medical Biosystems Ltd. Nucleic acid sequence analysis
WO1999005315A2 (en) * 1997-07-28 1999-02-04 Medical Biosystems Ltd. Nucleic acid sequence analysis
EP1229133A2 (en) * 1997-07-28 2002-08-07 Medical Biosystems Ltd. Nucleic acid sequence analysis
EP2267165A3 (en) * 1997-07-28 2012-10-10 Gen-Probe Incorporated Nucleic acid sequence analysis
EP1017848B2 (en) 1997-07-28 2012-02-22 Gen-Probe Incorporated Nucleic acid sequence analysis
EP2267164A3 (en) * 1997-07-28 2012-08-15 Gen-Probe Incorporated Nucleic acid sequence analysis
US7008766B1 (en) 1997-07-28 2006-03-07 Medical Biosystems, Ltd. Nucleic acid sequence analysis
US6511803B1 (en) * 1997-10-10 2003-01-28 President And Fellows Of Harvard College Replica amplification of nucleic acid arrays
US7785790B1 (en) 1997-10-10 2010-08-31 President And Fellows Of Harvard College Replica amplification of nucleic acid arrays
WO1999054497A1 (en) * 1998-04-22 1999-10-28 Stefan Seeger Method and device for determining the concentration, adsorption and binding kinetics and equilibrium and binding constants of molecules by luminescence measurements
US8263365B2 (en) 1998-05-01 2012-09-11 Arizona Board Of Regents Method of determining the nucleotide sequence of oligonucleotides and DNA molecules
WO1999057321A1 (en) * 1998-05-01 1999-11-11 Arizona Board Of Regents Method of determining the nucleotide sequence of oligonucleotides and dna molecules
US9096898B2 (en) 1998-05-01 2015-08-04 Life Technologies Corporation Method of determining the nucleotide sequence of oligonucleotides and DNA molecules
US9540689B2 (en) 1998-05-01 2017-01-10 Life Technologies Corporation Method of determining the nucleotide sequence of oligonucleotides and DNA molecules
US7037687B2 (en) 1998-05-01 2006-05-02 Arizona Board Of Regents Method of determining the nucleotide sequence of oligonucleotides and DNA molecules
US9212393B2 (en) 1998-05-01 2015-12-15 Life Technologies Corporation Method of determining the nucleotide sequence of oligonucleotides and DNA molecules
US9458500B2 (en) 1998-05-01 2016-10-04 Life Technologies Corporation Method of determining the nucleotide sequence of oligonucleotides and DNA molecules
US8263364B2 (en) 1998-05-01 2012-09-11 Arizona Board Of Regents Method of determining the nucleotide sequence of oligonucleotides and DNA molecules
US9725764B2 (en) 1998-05-01 2017-08-08 Life Technologies Corporation Method of determining the nucleotide sequence of oligonucleotides and DNA molecules
US7232656B2 (en) 1998-07-30 2007-06-19 Solexa Ltd. Arrayed biomolecules and their use in sequencing
US6787308B2 (en) 1998-07-30 2004-09-07 Solexa Ltd. Arrayed biomolecules and their use in sequencing
US7270958B2 (en) 1998-09-10 2007-09-18 The Regents Of The University Of Michigan Compositions and methods for analysis of nucleic acids
WO2000018956A1 (en) * 1998-09-30 2000-04-06 Molecular Machines & Industries Gmbh Method for dna- or rna-sequencing
US8192961B2 (en) 1998-12-14 2012-06-05 Pacific Biosciences Of California, Inc. System and methods for nucleic acid sequencing of single molecules by polymerase synthesis
US9845501B2 (en) 1998-12-14 2017-12-19 Pacific of Biosciences of California, Inc. System and methods for nucleic acid sequencing of single molecules by polymerase synthesis
US8980584B2 (en) 1998-12-14 2015-03-17 Pacific Biosciences Of California, Inc. System and methods for nucleic acid sequencing of single molecules by polymerase synthesis
US7566538B2 (en) 1999-02-23 2009-07-28 Caliper Lifesciences Inc. Sequencing by incorporation
US7105300B2 (en) 1999-02-23 2006-09-12 Caliper Life Sciences, Inc. Sequencing by incorporation
EP2177627A1 (en) * 1999-02-23 2010-04-21 Caliper Life Sciences, Inc. Manipulation of microparticles in microfluidic systems
EP1163369A1 (en) * 1999-02-23 2001-12-19 Caliper Technologies Corporation Sequencing by incorporation
US7344865B2 (en) 1999-02-23 2008-03-18 Caliper Life Sciences, Inc. Sequencing by incorporation
EP1163369A4 (en) * 1999-02-23 2002-08-28 Caliper Techn Corp Sequencing by incorporation
US6613513B1 (en) 1999-02-23 2003-09-02 Caliper Technologies Corp. Sequencing by incorporation
WO2000053805A1 (en) * 1999-03-10 2000-09-14 Asm Scientific, Inc. A method for direct nucleic acid sequencing
EP1961826A3 (en) * 1999-03-10 2008-09-17 ASM Scientific, Inc. A method for direct nucleic acid sequencing
US7270951B1 (en) 1999-03-10 2007-09-18 Asm Scientific, Inc. Method for direct nucleic acid sequencing
WO2000058507A1 (en) * 1999-03-30 2000-10-05 Solexa Ltd. Polynucleotide sequencing
WO2000060114A2 (en) * 1999-04-06 2000-10-12 Medical Biosystems Ltd. Polynucleotide sequencing using a helicase
WO2000060114A3 (en) * 1999-04-06 2002-01-31 Daniel Henry Densham Polynucleotide sequencing using a helicase
US6573047B1 (en) 1999-04-13 2003-06-03 Dna Sciences, Inc. Detection of nucleotide sequence variation through fluorescence resonance energy transfer label generation
US7361466B2 (en) 1999-05-19 2008-04-22 Cornell Research Foundation, Inc. Nucleic acid analysis using terminal-phosphate-labeled nucleotides
EP1179085A4 (en) * 1999-05-19 2004-11-10 Cornell Res Foundation Inc Method for sequencing nucleic acid molecules
US7943305B2 (en) 1999-05-19 2011-05-17 Cornell Research Foundation High speed nucleic acid sequencing
US7943307B2 (en) 1999-05-19 2011-05-17 Cornell Research Foundation Methods for analyzing nucleic acid sequences
US7033764B2 (en) 1999-05-19 2006-04-25 Cornell Research Foundation, Inc. Method for sequencing nucleic acid molecules
EP1179085A1 (en) * 1999-05-19 2002-02-13 Cornell Research Foundation, Inc. Method for sequencing nucleic acid molecules
US7485424B2 (en) 1999-05-19 2009-02-03 Cornell Research Foundation, Inc. Labeled nucleotide phosphate (NP) probes
US7056661B2 (en) 1999-05-19 2006-06-06 Cornell Research Foundation, Inc. Method for sequencing nucleic acid molecules
EP1681356A1 (en) * 1999-05-19 2006-07-19 Cornell Research Foundation, Inc. Method for sequencing nucleic acid molecules
US7416844B2 (en) 1999-05-19 2008-08-26 Cornell Research Foundation, Inc. Composition for nucleic acid sequencing
US7052847B2 (en) 1999-05-19 2006-05-30 Cornell Research Foundation, Inc. Method for sequencing nucleic acid molecules
US7056676B2 (en) 1999-05-19 2006-06-06 Cornell Research Foundation, Inc. Method for sequencing nucleic acid molecules
US6818395B1 (en) 1999-06-28 2004-11-16 California Institute Of Technology Methods and apparatus for analyzing polynucleotide sequences
US7501245B2 (en) 1999-06-28 2009-03-10 Helicos Biosciences Corp. Methods and apparatuses for analyzing polynucleotide sequences
US6242193B1 (en) 1999-07-30 2001-06-05 Hitachi, Ltd. Apparatus for determining base sequence of nucleic acid
US7939264B1 (en) 1999-10-06 2011-05-10 Gen-Probe Incorporated DNA sequencing method
US6908736B1 (en) 1999-10-06 2005-06-21 Medical Biosystems, Ltd. DNA sequencing method
WO2001025480A3 (en) * 1999-10-06 2002-05-16 Daniel Henry Densham Dna sequencing method
WO2001025480A2 (en) * 1999-10-06 2001-04-12 Medical Biosystems Ltd. Dna sequencing method
WO2001032930A1 (en) * 1999-11-04 2001-05-10 California Institute Of Technology Methods and apparatuses for analyzing polynucleotide sequences
US6458544B1 (en) 1999-12-02 2002-10-01 Dna Sciences, Inc. Methods for determining single nucleotide variations and genotyping
WO2001057248A2 (en) * 2000-02-01 2001-08-09 Solexa Ltd. Polynucleotide arrays and their use in sequencing
WO2001057248A3 (en) * 2000-02-01 2002-02-14 Solexa Ltd Polynucleotide arrays and their use in sequencing
US7384737B2 (en) 2000-02-02 2008-06-10 Solexa Limited Synthesis of spatially addressed molecular arrays
WO2001057249A1 (en) * 2000-02-02 2001-08-09 Solexa Ltd. Synthesis of spatially addressed molecular arrays
US7223568B2 (en) 2000-03-30 2007-05-29 Toyota Jidosha Kabushiki Kaisha Methods for determining nucleotide sequences of single nucleic acid molecules
EP1182267A1 (en) * 2000-03-30 2002-02-27 Genesis Research Institute, Inc. Method of determining base sequence of single nucleic acid molecule
EP1182267A4 (en) * 2000-03-30 2004-09-15 Toyota Motor Co Ltd Method of determining base sequence of single nucleic acid molecule
WO2001073121A1 (en) * 2000-03-30 2001-10-04 Toyota Jidosha Kabushiki Kaisha Method of determining base sequence of single nucleic acid molecule
US6355433B1 (en) 2000-06-02 2002-03-12 Dna Sciences, Inc. Determination of nucleotide sequence variations through limited primer extension
WO2002002813A3 (en) * 2000-07-05 2003-03-13 Amersham Biosiences Uk Ltd Sequencing method
WO2002002813A2 (en) * 2000-07-05 2002-01-10 Amersham Biosiences Uk Ltd. Sequencing method
US7056670B2 (en) 2000-07-05 2006-06-06 Ge Healthcare Uk Limited Sequencing method
US9719139B2 (en) 2000-10-06 2017-08-01 The Trustees Of Columbia University In The City Of New York Massive parallel method for decoding DNA and RNA
US9725480B2 (en) 2000-10-06 2017-08-08 The Trustees Of Columbia University In The City Of New York Massive parallel method for decoding DNA and RNA
US9868985B2 (en) 2000-10-06 2018-01-16 The Trustees Of Columbia University In The City Of New York Massive parallel method for decoding DNA and RNA
US9708358B2 (en) 2000-10-06 2017-07-18 The Trustees Of Columbia University In The City Of New York Massive parallel method for decoding DNA and RNA
US9718852B2 (en) 2000-10-06 2017-08-01 The Trustees Of Columbia University In The City Of New York Massive parallel method for decoding DNA and RNA
US9133511B2 (en) 2000-10-06 2015-09-15 The Trustees Of Columbia University In The City Of New York Massive parallel method for decoding DNA and RNA
US9243284B2 (en) 2000-12-01 2016-01-26 Life Technologies Corporation Enzymatic nucleic acid synthesis: compositions and methods for inhibiting pyrophosphorolysis
US7686929B2 (en) 2001-03-09 2010-03-30 Dna Electronics Limited Sensing apparatus and method
US7888015B2 (en) 2001-03-09 2011-02-15 Dna Electronics Ltd. qPCR using solid-state sensing
US8114591B2 (en) 2001-03-09 2012-02-14 Dna Electronics Ltd. Sensing apparatus and method
GB2389424B (en) * 2001-03-09 2004-11-24 Christofer Toumazou Sensing apparatus and method
US8685228B2 (en) 2001-03-09 2014-04-01 Dna Electronics Limited Sensing apparatus and method
WO2003073088A3 (en) * 2001-03-09 2003-11-20 Christofer Toumazou Apparatus and method for the detection of localised fluctuactions of ionic charge by ion sensitive field effect transistors during a chemical reaction
WO2003073088A2 (en) * 2001-03-09 2003-09-04 Christofer Toumazou Apparatus and method for the detection of localised fluctuactions of ionic charge by ion sensitive field effect transistors during a chemical reaction
US8698211B2 (en) 2001-03-09 2014-04-15 Dna Electronics Ltd. Sensing apparatus and method
US6653082B2 (en) 2001-05-17 2003-11-25 Baylor College Of Medicine Substrate-bound cleavage assay for nucleic acid analysis
EP1837407A3 (en) * 2001-09-24 2007-11-21 Intel Corporation Nucleic acid sequencing by monitoring of uptake of precursors during molecular replication, with molecule dispenser
EP1837407A2 (en) * 2001-09-24 2007-09-26 Intel Corporation Nucleic acid sequencing by monitoring of uptake of precursors during molecular replication, with molecule dispenser
US9388463B2 (en) 2001-12-04 2016-07-12 Illumina Cambridge Limited Labelled nucleotides
US9121062B2 (en) 2001-12-04 2015-09-01 Illumina Cambridge Limited Labelled nucleotides
US7785796B2 (en) 2001-12-04 2010-08-31 Illumina Cambridge Limited Labelled nucleotides
US7772384B2 (en) 2001-12-04 2010-08-10 Illumina Cambridge Limited Labelled nucleotides
US9410200B2 (en) 2001-12-04 2016-08-09 Illumina Cambridge Limited Labelled nucleotides
US7427673B2 (en) 2001-12-04 2008-09-23 Illumina Cambridge Limited Labelled nucleotides
US8158346B2 (en) 2001-12-04 2012-04-17 Illumina Cambridge Limited Labelled nucleotides
US8394586B2 (en) 2001-12-04 2013-03-12 Illumina Cambridge Limited Labelled nucleotides
US8148064B2 (en) 2001-12-04 2012-04-03 Illumina Cambridge Limited Labelled nucleotides
US9605310B2 (en) 2001-12-04 2017-03-28 Illumina Cambridge Limited Labelled nucleotides
US7566537B2 (en) 2001-12-04 2009-07-28 Illumina Cambridge Limited Labelled nucleotides
US9121060B2 (en) 2002-08-23 2015-09-01 Illumina Cambridge Limited Modified nucleotides
US7541444B2 (en) 2002-08-23 2009-06-02 Illumina Cambridge Limited Modified nucleotides
US9410199B2 (en) 2002-08-23 2016-08-09 Illumina Cambridge Limited Labelled nucleotides
US8071739B2 (en) 2002-08-23 2011-12-06 Illumina Cambridge Limited Modified nucleotides
US9127314B2 (en) 2002-08-23 2015-09-08 Illumina Cambridge Limited Labelled nucleotides
US9388464B2 (en) 2002-08-23 2016-07-12 Illumina Cambridge Limited Modified nucleotides
WO2004029294A1 (en) * 2002-09-27 2004-04-08 Biotage Ab New sequencing method for sequencing rna molecules
US7771973B2 (en) 2002-12-23 2010-08-10 Illumina Cambridge Limited Modified nucleotides
US8597881B2 (en) 2002-12-23 2013-12-03 Illumina Cambridge Limited Modified nucleotides
WO2004072294A3 (en) * 2003-02-12 2005-03-10 Global Genomics Ab Methods and means for nucleic acid sequencing
WO2004072294A2 (en) * 2003-02-12 2004-08-26 Genizon Svenska Ab Methods and means for nucleic acid sequencing
WO2004106545A1 (en) * 2003-05-28 2004-12-09 Innogenetics N.V. Methods for enhanced detection using surface sensitive techniques.
US9012144B2 (en) 2003-11-12 2015-04-21 Fluidigm Corporation Short cycle methods for sequencing polynucleotides
US9657344B2 (en) 2003-11-12 2017-05-23 Fluidigm Corporation Short cycle methods for sequencing polynucleotides
WO2005118871A1 (en) * 2004-05-28 2005-12-15 The Arizona Board Of Regents Surface plasmon resonance sensor for detecting changes in polynucleotides mass
US9588051B2 (en) 2004-09-17 2017-03-07 Pacific Biosciences Of California, Inc. Apparatus and method for performing nucleic acid analysis
US7476503B2 (en) 2004-09-17 2009-01-13 Pacific Biosciences Of California, Inc. Apparatus and method for performing nucleic acid analysis
US7170050B2 (en) 2004-09-17 2007-01-30 Pacific Biosciences Of California, Inc. Apparatus and methods for optical analysis of molecules
US7302146B2 (en) 2004-09-17 2007-11-27 Pacific Biosciences Of California, Inc. Apparatus and method for analysis of molecules
US7906284B2 (en) 2004-09-17 2011-03-15 Pacific Biosciences Of California, Inc. Arrays of optical confinements and uses thereof
US9709503B2 (en) 2004-09-17 2017-07-18 Pacific Biosciences Of California, Inc. Apparatus and method for performing nucleic acid analysis
US7315019B2 (en) 2004-09-17 2008-01-01 Pacific Biosciences Of California, Inc. Arrays of optical confinements and uses thereof
US7313308B2 (en) 2004-09-17 2007-12-25 Pacific Biosciences Of California, Inc. Optical analysis of molecules
US7592435B2 (en) 2005-08-19 2009-09-22 Illumina Cambridge Limited Modified nucleosides and nucleotides and uses thereof
US8212015B2 (en) 2005-08-19 2012-07-03 Illumina Cambridge Limited Modified nucleosides and nucleotides and uses thereof
US7816503B2 (en) 2005-08-19 2010-10-19 Illumina Cambridge Limited Modified nucleosides and nucleotides and uses thereof
US9868978B2 (en) 2005-08-26 2018-01-16 Fluidigm Corporation Single molecule sequencing of captured nucleic acids
US8808988B2 (en) 2006-09-28 2014-08-19 Illumina, Inc. Compositions and methods for nucleotide sequencing
US8399188B2 (en) 2006-09-28 2013-03-19 Illumina, Inc. Compositions and methods for nucleotide sequencing
US9469873B2 (en) 2006-09-28 2016-10-18 Illumina, Inc. Compositions and methods for nucleotide sequencing
US9051612B2 (en) 2006-09-28 2015-06-09 Illumina, Inc. Compositions and methods for nucleotide sequencing
US9528151B2 (en) 2006-12-01 2016-12-27 The Trustees Of Columbia University In The City Of New York Four-color DNA sequencing by synthesis using cleavable fluorescent nucleotide reversible terminators
US9175342B2 (en) 2007-10-19 2015-11-03 The Trustees Of Columbia University In The City Of New York Synthesis of cleavable fluorescent nucleotides as reversible terminators for DNA sequencing by synthesis
US9115163B2 (en) 2007-10-19 2015-08-25 The Trustees Of Columbia University In The City Of New York DNA sequence with non-fluorescent nucleotide reversible terminators and cleavable label modified nucleotide terminators
US9670539B2 (en) 2007-10-19 2017-06-06 The Trustees Of Columbia University In The City Of New York Synthesis of cleavable fluorescent nucleotides as reversible terminators for DNA sequencing by synthesis
US8993230B2 (en) 2008-12-04 2015-03-31 Pacific Biosciences of Californ, Inc. Asynchronous sequencing of biological polymers
US8911972B2 (en) 2009-12-16 2014-12-16 Pacific Biosciences Of California, Inc. Sequencing methods using enzyme conformation

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GB8910880D0 (en) 1989-06-28 grant
EP0471732A1 (en) 1992-02-26 application
JPH04505251A (en) 1992-09-17 application
CA2045505A1 (en) 1990-11-12 application

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