EP1409721A2 - Detection of nucleic acid polymorphisms - Google Patents

Detection of nucleic acid polymorphisms

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Publication number
EP1409721A2
EP1409721A2 EP20010993709 EP01993709A EP1409721A2 EP 1409721 A2 EP1409721 A2 EP 1409721A2 EP 20010993709 EP20010993709 EP 20010993709 EP 01993709 A EP01993709 A EP 01993709A EP 1409721 A2 EP1409721 A2 EP 1409721A2
Authority
EP
European Patent Office
Prior art keywords
primer
ad
method according
nucleic acid
rch
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP20010993709
Other languages
German (de)
French (fr)
Inventor
Lars Edman
Zeno Clinical Immunology FÖLDES-PAPP
Rudolf Rigler
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Gnothis Holding SA
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Gnothis Holding SA
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority to DE10056226 priority Critical
Priority to DE10056226 priority
Priority to DE10065631 priority
Priority to DE10065631A priority patent/DE10065631A1/en
Application filed by Gnothis Holding SA filed Critical Gnothis Holding SA
Priority to PCT/EP2001/013120 priority patent/WO2002038806A2/en
Publication of EP1409721A2 publication Critical patent/EP1409721A2/en
Application status is Withdrawn legal-status Critical

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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING 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
    • C12Q1/00Measuring 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
    • C12Q1/6827Hybridisation assays for detection of mutation or polymorphism

Abstract

The invention relates to methods that function at an individual molecule level, for characterising nucleotide polymorphisms.

Description

Detection of polymorphisms IMukleinsäure

description

The present invention relates to a method for the detection of single or multiple nucleic acid polymorphisms by detection of individual fluorescently labeled Desoxyribonucleinsäuremoleküle.

Between the genomes of the individuals of a species there are sequence differences by nucleic acid insertions and deletions, differences in the number of repetitions of short, repetitive sequence motifs (so-called microsatellites and minisatellite) and variations in individual base pairs (as single nucleotide polymorphisms SNPs, engl. Single nucleotide fiolymorphisms be) called and (with about one base pair per 1,000 base pairs in humans see WO 00/18960) are most common.

Such variations in the genome can be associated in many cases with the occurrence of hereditary diseases. Classic examples are Huntington, cystic fibrosis, Duchenne muscular dystrophy, and certain forms of breast cancer (see WO 00/18960). More recently, diseases such as Alzheimer's and Parkinson's have been associated with individual mutations on the molecular level.

Usually it concerns with these mutations to single nucleotide polymorphisms (SNPs). On finding new positions in the genome where SNPs occur, so there is considerable interest in medical research. In the investigation of SNPs, their position in the overall nom is accurately known to the nucleotide, however, is primarily a concern for the diagnosis of molecular diseases. A number of methods for routine investigation of such SNPs at a known position in the genome have therefore been developed in recent years.

Thus miniaturized Oligonukleo- were prepared tidarrays high density by photolithographic synthesis. On these arrays, there is a complementary probe for each possible allele. Prototypes with such chips for genotyping of up to 3,000 SNPs can be assayed simultaneously (Sapolsky et al, Genet Anal 1999, 14:.. 187-192).

A similar method, which is also based on the hybridization of the searching to sub allele with a complementary oligonucleotide probe was developed by Axys Pharmaceuticals. This method uses oligonucleotide probes gekop- of fluorescently labeled microspheres pelt. These probes are hybridized directly also with fluorescently labeled polymerase chain reaction (PCR) products. Detection then takes place in a conventional flow cytometer. In this way, up to eight polymorphic genes could be tested simultaneously (Armstrong et al, Cytometry, 2000, 40: 102-108).

While in these methods, the hybridization of a possible DNA amplification step is carried out with PCR, go Lake et al. the opposite way. Their process uses primers with different fluorophores at the 5'-nucleotides, the 3'-end lies with the to be examined nucleotide. Only with the primer that is also complementary to the 3 'end to the to be examined .Nukleotid, entseht a PCR product. The samples are then analyzed by electrophoresis according to size and fluorescence analyzed (See et al, Biotechniques, 2000, 28: 710-714).

A very elegant method for characterizing SNPs does not use a complete PCR, but only the extension of a primer by a single, fluorescence-labeled Didesoxyribonucleinsäuremolekül (ddNTP) that is complementary to the nucleotide to be investigated. By detecting the extended by one base and thus fluorescently labeled primer can be on the nucleotide at the polymorphic site include (Kobayashi et al, Mol Cell Probes, 1995, 9:.. 175-182). A disadvantage of this method is, however, that in a reaction only a single polymorphism can be studied.

A possible solution to this problem is to incorporate a ZipCoded as designated uniquely identifiable sequence in the primer. This ZipCode is detected (the cZipCode) of a complementary ZipCode, which is covalently bound to a fluorescent microspheres. • • The microspheres decoding and SNP typing is then in a conventional flow cytometer. The ZipCode system allows analysis of a large number of SNPs with a limited amount coupled to ZipCode M-krokügelchen (Chen et al, Genome Res, 2000, 10. 549-557).

The latter two methods, which are based on the extension of a primer with a fluorescently labeled dideoxynucleotide, have a significant advantage: Fluorescence-labeled dideoxynucleotides, which are optimized for high fluorescence yield and for incorporation into DNA by naturally occurring or genetically modified polymerases are due to their use for the Sanger method of DNA sequencing by chain termination (Sanger et al, Proc Nat Acad Sci USA, 1977, 74:.... 5463) available inexpensively. Like the other methods but also the two latter methods, which are based on the extension of a primer with a fluorescently labeled dideoxynucleotide consuming to carry out.

In the above-explained procedure without ZipCode it is necessary to achieve optimal performance development of a clear signal to preclean the sample prior to application onto a denaturing gel. In order to separate the excess of unincorporated into the primer dideoxynucleotide is recommended to treat the sample with alkaline phosphatase and then to cut down the primer with isopropanol. Especially the precipitation step can be automated bad.

In the method using the ZipCode labor-intensive steps are eliminated, but this is a technically complicated flow cytometer that covers a wide wavelength range, is required. In addition, there is a danger of interpreting signals incorrectly because the spectra of different fluorescent dyes overlap at least partially.

Moreover, with the use of DNA from donors who have two different alleles of the polymorphic DNA to be examined portion, the difficulty that the two alleles must first be isolated individually or selectively amplified either.

Object of the present invention is to provide methods for the characterization of nucleic acid polymorphisms, which do not exhibit these disadvantages of the prior art.

This object is achieved by a method for the characterization of nucleic acid polymorphisms, comprising the steps of:

(A) providing a nucleic acid template to be examined,

(B) annealing of a starting .mindestens primers to the nucleic acid template, wherein the 3'-end of the primer is located upstream of a to be examined Nukleinsäurepolymorphismus,

(C) elongating the starting primer by at least one fluorescently labeled nucleotide, and

(D) detecting built into the starting primer by nucleotides single molecule determination.

In the Nukleinsäurepolymorphismus is the simplest case, a single nucleotide (single nucleotide polymorphism, SNP). However, the polymorphism can also be several nucleotides, for example up to 20 consecutive nucleotides, or even more groups of one or more consecutive nucleotides concern.

As nucleic acid template particular person can DNA from any source, Example, from prokaryotes, particularly pathogenic prokaryotes Archaeaen or Eu karyonten, especially mammals, are used. but it can also be recombinantly produced DNA or synthetic DNA itself. The DNA is preferably used in single-stranded form. Such DNA can, for example, by reverse transcription of an RNA molecule by a reverse transcriptase, such as AMV reverse transcriptase ■ (Avian myeloblastosis virus) or MMLV (Moloney Murine Leucaemia virus) can be produced. However, it is also possible to double-stranded DNA, such as genomic DNA, DNA of a plasma mids or an episomal genetic element separating by heating in single-stranded DNA, optionally purify or enrich a strand, and then to allow the primers to anneal. When RNA or DNA is preferably a homogeneous mixture as possible. But since the start primer has specificity for the DNA to be examined, can also be used with heterogeneous mixtures.

The start primer preferably consists of single-stranded DNA. but it is also possible of course to work with RNA molecules. The start primer may nucleic acid also be a nucleic acid analog, for example, a peptide, wherein the phosphate-sugar backbone of the nucleic acids is replaced by a peptide-like backbone, for example, consisting of 2-Aminoethylenglycin (Nielsen et al, Science,. 254: 1497-1500 ) as a support of the individual bases a, T, G, C. such a peptide nucleic acid primers must possess a 3 'end which allows elongation.

Preferably, the starting primer binds immediately upstream of the SNPs to be characterized. If working with deoxynucleotides and not break molecules with chain termination, but it is also possible to use a starting primer further upstream, preferably not more than 5 nucleotides upstream from the. to be examined polymorphism imagine binds.

The fluorescently labeled nucleotide can be a deoxynucleotide and a chain termination molecule. Fluorescence labeling groups can be selected from the known thereof, etc. for labeling of biopolymers such as nucleic acids, fluorescent labeling groups used, such as fluorescence CEin, rhodamine, phycoerythrin, Cy3, Cy5 or derivatives thereof. The distinction of the dyes can about the wavelengths •; ge, which take place over the lifetime of the excited states, or a combination.

If several, -with different fluorescence labels provided nucleotides are used, they can be distinguished by the wavelength of the exciting light, the emitted light or a combination thereof. A differentiation of Fluorezenzfarbstoffe can gen also by measuring the excited state lifetime of successes. It makes sense to combine the process. Thus, four fluorescent labels for the four different ligands bases may for example be selected, all of which can be excited at the same wavelength and emit different at two wavelengths, being different for the marks, whose emission wavelength is equal to the LE lifetimes of the excited states ,

The extension of the primer can be carried out using methods of nucleic acid chemistry, which are known in oligonucleotide synthesis. The extension reaction is preferably carried but by enzymatic catalysis. The polymerase is whether as a template RNA or DNA selected depending on. a polymerase without exonuclease activity is preferably selected. Examples of possible polymerases are T7 polymerase or thermostable polymerases such as Taq, Pfu, Pwo and the like, which are normally used for PCR reactions.

The detection of the fluorescence of a single molecule can be by any measuring method, for example, be carried out with location- or / and time-resolved fluorescence-spectroscopy, which is able, in a very small volume element, as is present in a microchannel, fluorescence signals down to single photon counting capture.

For example, the detection can tion by confocal Einzelmoleküldetek- be done such as by fluorescence correlation spectroscopy,

• wherein a very small, preferably confocal volume element, for example, 0.1 x 10 15 to 20 x 10 '12 I flowing through the microchannel Probefiüssigkeit is exposed to an excitation light of a laser ", that the fluorescent labels to emit fluorescent light located in this measuring volume stimulates, wherein the emitted fluorescent light from the measurement volume is measured by a photodetector, and a correlation between the temporal change in the measured emission and the relative Flussgeschwindig- ness of the molecules involved, so that individual molecules can be identified in the measuring volume at a correspondingly high dilution . on details of the process implementation and instrumental details on the materials used for the detection devices is made to the disclosure of European patent No. 0,679,251. the confocal single molecule determination continues (at Rigler and Mets Soc. Photo-Opt.lnstrum. Eng. 1921 (1993), 239 ff.) And Mets and Rigler (J. Fluoresc. 4 (1994), 259-264) described.

Alternatively or additionally, the detection can also be a time-resolved decay, a so-called time gating take place, such as from Rigler et al., "Picosecond Photon Fluorescence Single

. Spetroscopy of Nucleic Acids ", in:" Ultrafast Phenomena ", DH Auston, Ed, Springer 1984., This is done, the excitation of the fluorescent molecules within a measurement volume and subsequently - preferably after a time interval of> 100 ps - the opening of a detection interval. the photo detector. in this way, background signals produced can be kept sufficiently low to permit substantially interference-free detection by Raman effects.

In . a preferred embodiment of the method, the

Provision also include the measurement of a kreuzkorrellierten signal of at least one 2 different labels, in particular

Fluorescent labels, containing nucleic acid molecule or nucleic acid molecule complex originates, wherein a plurality of labeled

Nucleotides, primers and / or nucleic acid templates may be used, each with different markers. This cross-correlation determination is et for example in humidity. al.

(Biophys.J. 72 (1997), 1878-1886) and Rigler et. al. (J. Biotechnol. 63

(1998), 97-109) described.

The detection of incorporated nucleotides preferably comprises a separatism tion of the elongated starting primer from unincorporated nucleotides.

The separation can be carried out, for example, as described in Patent Application DE 100 23 423.2 describes due to the different migration velocity built and non-incorporated nucleotides in elec- trical field. In this way, enrichment by three orders of magnitude or more can be achieved typically.

If the primer or the nucleic acid template is immobilisisert to a carrier particle, this particle can be captured for example with the aid of a home frarotlasers. a washing step can be carried out subsequently then in a directed flow, which can be electroosmotic or hydrodynamic. Because of the favorable flow profile and the higher flow rates hydrodynamic flow is preferred.

It is possible to consider in addition to the detection of whether actually built- in nucleotides are observed or whether contaminating free nucleotides are still present. This is possible, for example by fluorescence correlation spectroscopy. In this method takes advantage of diffusing the extended starting primer much slower than the free chain terminating molecules un therefore longer stays in the illuminated by a confocal microscope range, so that fluorescent light emitted from the extended starting primer an essential longer correlation time ■.'Has a fluorescent light from a free chain termination molecule. satisfy technically less complex correlators for measuring the diffusion limited correlation time, since the correlation times are in the range of ms to several 100 ms.

Another option for quick differentiation of built-in and not built-chain molecules lies in the utilization of energy transfer processes. So (Acad Edman, L., Mets, Ü. And Rigler, R., Proc. Nat.. Sci. USA 93, 6710-6715 (1996)) was, for example, by Edman et al demonstrated that the lifetime of an excited state of tetramethyl rhodamine is drastically reduced by close physical proximity that only occurs at high dilution of the chain termination molecule when the molecule was indeed covalently bonded to the initiating primer.

but according to a further aspect of the present invention, it is also possible abzudauen the unincorporated nucleotides by, for example, an exonuclease again and individually detected. In this case, an at least partial sequence determination of the elongated starting primer is carried out. In this case methods can be used, as described in Patent Application DE 100 31 840.1 and in the publication Dörre et al., Bioimaging 5, 139-152. For carrying out the sequencing reaction, the nucleic acid matrix, or more preferably the starting primer is coupled to a carrier particle.

The single-molecule sequencing preferably comprises the steps of:

(A) introducing the carrier particle in a sequencing device comprising a microchannel,

(B) adherence of the carrier particle in the sequencer,

(C) progressively removing by cleavage individual nucleotide building blocks from said immobilized nucleic acid molecule,

(D) at least partially determining the base sequence of the nucleic acid molecule due to the sequence of the cleaved nucleotide.

The detection and manipulation of loaded carrier particles, for example, according to Holm et al. (Analytical Methods and Instrumentation, Special Issue TAS 96, 85-87), Eigen and Rigler (Acad Sci Proc. Natl... USA 91 (1994), 5740-5747) (or Rigler J. Biotech. 41 (1995) carried out by methods described 177-186) that include a detection with a confocal microscope. The manipulation of loaded carrier particles in microchannel structures is preferably effected by means of a trapping laser, for example an infrared laser. Suitable methods are for example Ashkin et al. (Nature 330 (198_7), 24-31) and Chu (Science 253 (1991), 861-866) described.

Preferably, the holding of the carrier particle is carried out by an automated process. For this, the carrier particles are passed in the hydrodynamic flow through the microchannel, where they pass through a detection element. The detector in the detection window is adjusted so that it detects a marked due to the ball thereon fluorescently labeled DNA and / or an additional fluorescent-labeled probe, and then automatically causes the activation of the trapping laser in the measuring space.

In order to cleave single nucleotides from the extended Startprimer- an exonuclease molecule is used, for example, T7 DNA polymethyl rase as exonuclease, E. coli exonuclease I or E. coli exonuclease III.

In the simplest case, only one starting primer for the extension reaction is used. but it is also possible to have multiple use binding starting primer at various locations on the die and extend. The start primers are then encoded preferably different, for example • • by different fluorescent labels or by different combinations of fluorescent labels. In particular, fluorescently labeled dNTPs in the start can be installed primer for identification of the starting primer. If a different fluorescent label is used for each nucleotide can be distinguished with n fluorescently labeled positions 4 π different starting primer. An even larger number arises advertising when used at different positions for the same nucleotide different fluorescent analogue to.

According to a first embodiment, the extension reaction is carried out by adding a single, fluorescence-labeled Kettenabbruchmole- CRWT to or the initiating primer (see Figure 1 a for example). As capping molecules dideoxynucleotides are preferably used. but it is also possible to use different modified deoxyribonucleic acids, if they are not recognized by the enzymes used. It is conceivable, for example, to modify the 3'-position of the Desox- yribosemoleküls by a halogen atom or an alkyl or alkoxy. According to a second embodiment of the present invention, several consecutive nucleotides can be characterized. In this case, the termination of the extension reaction is not through the incorporation of a suitable chain termination molecule, but by a block primer enforced (see Figure 1 b for an example). The block primer is bound downstream of the to be investigated polymorphism at the nucleic acid and itself is protected against extension at its 3 'end by suitable chemical modification. For example, the location furthest downstream nucleotide of the primer may be a block chain termination molecule. In this embodiment, it is possible to use several different coded start / block primer pairs that can bind to different locations on the die (see Figure 1 c for an example).

The blocking of the block primer may, optionally with the exception of blocking of the most downstream block binding primer be reversible. For reversibly blocking a removable protective group, for example, a photo-labile protecting group can be used. Particularly preferred are the block primers carry a phosphate group at the 3'-position of the sugar on the 3 'end. This phosphate group at the 3'-end to prevent elongation by the polymerase and can be cleaved for deblocking readily with a 3 'phosphatase.

After the extension reaction of the starting primer is no covalent bond to the immediately downstream block primer. But this bond can be linked, for example enzymatically with a ligase. The ligation takes place much more easily when the block primers bear at their 5'-end of a phosphate group.

According to a third embodiment / the gap (s) between pairs of an elongated by fluorescent nucleotides starting primer and the downstream each block primer can be filled by deoxyribonucleotides after removal of the 3 'blocking the block primer and körinen covalent bonds between the extended Blockprimem and the immediately downstream Startprimem be closed (see Figure 1 d is an example). To carry the block primer preferably a 5'-phosphate. In this embodiment, it is not absolutely necessary to provide the different start / block primer pairs with encodings.

Another object of the invention is the combination of the chain termination with a marker detection in completely or partly transparent microwells (see patent application DE 100 23 421 .6). This - method comprises the steps of:

(A) providing a carrier particle having immobilized thereon a nucleic acid molecule consisting of a single-stranded nucleic säurematrize and an initiating primer,

(B) elongating the starting primer by a fluorescently labeled chain termination molecule,

(C) optionally washing the wells to remove non-incorporated labels, and (d) detecting the incorporated into the starting primer fluorescent label.

Depending on the arrangement of the fluorescence exciting light source and detector, the use of completely or partially transparent microwells is required. The excitation and / or detection of the fluorescence can be carried out for example by a built-in micro-well semiconductor laser and / or semiconductor detector (see Figure 2 for an example). but the excitation light source and / or the detector can be outside of the microstructure. The method is ideally suited for automation, since a plurality of reactions on a microwell plate can be performed in parallel or sequentially. If the amount of starting primer and the amount of added labeled nucleotide low (nM) is held, the distinction between native and non-incorporated chain terminator molecules may be effected for example by FCS (fluorescence correlation spectroscopy) as explained above. Alternatively, energy transfer processes can also be explained above, be utilized.

Alternatively, and preferably higher, for example μWΛ concentrations of primers and chain terminating molecules used because the incubation period may then be kept lower. However, at least the chain termination molecules must then after the primer extension reaction by - be a wash step removed. By micro-wells can be used with one or more small holes or a Größenausschlussmem- bran which the labeled, restrain to a carrier particle bound DNA dene and unlabelled chain terminating molecules to pass through (see, for. Example, Figure 2).

Various combinations of Startprimem and chain terminating molecules are conceivable. In the simplest case, for the characterization of SNPs, loaded two or more (up to four) wells with in each case only one fluorescently labeled chain terminating molecule and the starting primer is hybridized whose 3 'end immediately before the nucleotide to be investigated. Only in one of the wells there is an elongation reaction. Since it is known that corrugated containing chain termination molecule, for all chain terminating molecules have the same fluorescent label can be used. Since the extension reaction stops, unless the correct nucleotide is present for the extension, even Desoxynukleo- tide can be used in this case. but a chain terminating molecule is preferably as described above, for example, selected from the group consisting of ddATP, ddUTP, ddTTP, ddCTP and ddGTP provided. a solid phase with a plurality of wells such as, for example, in Patent Application DE 100 23 421.6 is preferable described, is used. In one approach, a plurality of SNPs can be assayed in parallel in this manner. Preferably takes place here a parallel detection of 4 wells.

but it is also possible to use a starting primer along with several, preferably four different chain terminating molecules corresponding to the four nucleobases. However, the chain termination molecules must then carry different labeling groups. A distinction of the marker groups is about the wavelength of the exciting and / or emitted light, or over the lifetime of the excited

State possible. The measurement of the lifetime of the excited state is effected by measurement of fluorescence decay time (FD, fluorescence decay).

In this measurement method, the to be assayed molecule is excited by a pulsed laser (such as a mode locked laser). The detection of the emitted fluorescent photons takes place as a function of time since the decay of the laser pulse whose duration must be small compared to the time of life to be examined excited state.

In special cases it is possible to work with several Startprimem and more chain terminating molecules in one well. For example, by a SNP is known that only one of the bases A or T is to be expected and is known by another SNP that only either G or C occur, the two polymorphisms can be studied in parallel. Other situations where several nucleotide positions can be examined simultaneously due to additional information about the polymorphisms are readily apparent to those skilled in the art.

With yet another embodiment of the present invention, several SNPs can be analyzed simultaneously, even if is to be expected at the polymorphism with the occurrence of all four nucleotides. For this, a start is used primer whose 3 'end is immediately upstream of the respectively to be characterized for each nucleotide polymorphism site. Then the extension reaction with terminators molecules occurs. Start complementary primers are then in a further step selected restriction sites added so that digestion of the nucleic acid template can be carried out in fragments characteristic length. By examining the diffusion behavior of the fragments by means of FCS, the fluorescence signals can then be assigned to the individual polymorphic nucleic acid positions.

A principle similar procedure is possible if a sequence-specific ligase is used instead of the restrictase • •. Sequence-specific league tion can, for example, by "reverse" reach-powered restrictases. Since the hydrolysis reaction consumes one molecule of water and the ligation reaction releases a molecule of water, the equilibrium in the direction of the ligation can be shifted by using an anhydrous reaction medium as possible. In the analogous case of proteases "Reverse operation" of the enzyme was successfully realized by the addition of large amounts of polyethylene glycol or organ Sichen solvents to the reaction buffer.

For all described embodiments, the carrier particles preferably has a size in the range of 0.5 to 10 microns, and most preferably from 1 to 3 // m. Examples of suitable materials of carrier particles are plastic materials such as polystyrene, glass, quartz, metals or semimetals such as silicon, metal oxides such as silicon dioxide or compound materials containing several of the aforementioned components. optically transparent carrier particles, for example of plastics or particles are particularly preferred core with a plastic and used a silicon dioxide shell. The immobilization to a carrier particle can be done either through the die or the initiating primer. For the process, it does not matter at what time of immobilization occurs. This step is possible i) prior to the hybridization step, ii) after the hybridization step, but prior to extension of the starting primer by the chain termination molecule, and preferably, iii) after the extension reaction. The advantage of the late immobilization is that a potentially disruptive influence of the carrier is avoided in the hybridization and elongation reaction.

The binding of the starting primer or the nucleic acid template to the carrier

•; can be done by covalent or non-covalent interactions.

For example, the binding of the polynucleotides to the substrate by high-affinity interactions between the partners of a specific binding pair, for example biotin / streptavidin or avidin, hapten / anti-hapten. Antibody, sugar / lectin, etc., can be imparted. Thus, biotinylated nucleic acid molecules may be coupled to streptavidin-coated support. Alternatively, the nucleic acid molecules can also be adsorptively bound to the support. Thus, a binding by incorporation of alkanethiol modified nucleic acid molecules to metallic substrates, such as gold carriers, done. Yet another alternative is the covalent immobilization, whereby the binding of the polynucleotides by reactive silane groups on a silica Oberf pool can be conveyed. If a mixture of two or more is present at the location of Einzelnukleotidpoly- morphism different DNA molecules as a template, it is low as in the single-molecule sequencing to bind only a maximum of one molecule of the template or the starting primer to a single carrier particle. This can easily be achieved by a sufficiently high molar surplus of carrier particles against the die or the initiating primer.

If the DNA molecules used as template, however, all are uniform, it is even low wells in particular for the embodiment of the invention in micro- to bind more molecules of a template or starting primer to a carrier particle. The exonuclease digestion then leads to elimination of several identical fluorescent-labeled chain termination molecules, so that the fluorescence signal and thus the signal to noise ratio is improved.

When using several fluorescent-labeled components in the inventive Polymorphismuscharakterisierungen entstehtdas problem to separate the different markings effective. can be carried out as described above, this inter alia, by the use of different wavelengths at the excitation and emission of fluorescent light. The spectral splitting is carried out according to the prior art with dichroic mirrors. Disadvantage of this approach is the relatively high losses, particularly in the spektalen splitting of the light emitted by the fluorophore photons. Surprisingly, it was found that the losses can be reduced when the spectral splitting instead of a dichroic mirror having a dispersion element such as a grid, for example a holographic or scratched grating or a prism is performed (see Figure 3). It is favorable, the reflections at the entrance of the light in the dispersion element and / or at the exit of the light from the dispersion element, for example, be as completely as possible suppressed by suitable coating of the glass surfaces with a prism. The use of a dispersion element instead of a dichroic mirror is not limited to use in the characterization of nucleotide polymorphisms. It is also possible in the direct detection of single molecules (see, for example, application DE 100 23 423.2), at the single-molecule sequencing methods (see for example Application DE 100 31 840.1), in methods for the selection of particles (see, eg, application DE 100 31 842.8), in methods for detecting polynucleotides (see, for example, application DE 100 23 421 .6), in processes for the separation of labeled biopolymers (see for example application DE 100 23 422.4) and Multiplexsequenzierverfahren (see for example application DE 100 31 842.8)

Fig. 1 shows various embodiments of the polymorphism characterization. In (a) the extension of the initiating primer to a single fluorescently labeled chain terminating molecule is carried out. In (b) the initiating primer is extended by different fluorescently labeled deoxynucleotides to the 3'-end of a downstream block binding primer. The block primer itself is blocked at its 3 'end so that it is not extended. In (c) a plurality of start / block primer pairs are used. In this case, it is necessary to encode the starting primer by fluorescent markers. multiple start / block primer pairs are also used in (d), in addition, the blocking of the block primers (except for the blocking of the block primer furthest downstream) reversibly at the 3 'end, thus for example kierung a 3'-PhosphatblocK-. In a first step, in the presence of the 3 'blocking fluorescent nucleotides are incorporated. After a washing step to remove non-incorporated nucleotides is then filled by unlabeled deoxynucleotides in a second step after removal of the 3'-blocking of the gap between the block and the following primers initiating primer. The missing covalent bonds consecutive nucleotides are linked by ligase. Shown is the

Result of this procedure.

Fig. 2 (a) shows a plan view, (b) a side view of microwells, suitable for use in the present invention. Fig. 3 (a) shows the formula used to determine single molecule previously

Optics, (b) shows the optical system according to the invention using a dispersion element for separating the different wavelengths.

The determination can be made about the fluorescence intensities (Aλ) at different wavelengths and / or via fluorescence decay times (r) at various wavelengths using a plurality of detectors.

Claims

claims
1. A method for characterizing nucleic acid polymorphisms, uu mf sse a nd the steps of:
(A) providing a nucleic acid template to be examined,
(B) annealing at least one starting primer to the nucleic acid template, wherein the 3'-end of the primer is located upstream of a start to be examined Nukleinsäurepolymorphismus,
(C) elongating the starting primer by at least one fluorescently labeled nucleotide, and
(D) detecting built into the starting primer by nucleotides single molecule determination.
2. The method of claim 1, d u rc h ad n ge ken zeic et hn, that the detection of incorporated nucleotides includes a separation of the elongated starting primer from unincorporated nucleotides.
3. The method of claim 1 or 2, d ad u rc h., ge ken nzeic et hn, that the detection of the incorporated nucleotide comprises a sequence at least partially determining the elongated starting primer.
4. The method according to any one of the preceding claims, d ad u rch ge ken n zeic hn et that is coupled to conduct the single molecule determination of the starting primer to a carrier particle.
5. The method of claim 3 or 4, d ad u rch geken nzeich net, the single-molecule sequencing comprising the steps of:
(A) introducing the carrier particle in a sequencing device comprising a microchannel,
(B) adherence of the carrier particle in the sequencer,
(C) progressively removing by cleavage individual nucleotide building blocks from said immobilized nucleic acid molecule,
(D) at least partially determining the base sequence of the nucleic acid molecule due to the sequence of the cleaved nucleotide.
6. The method according to any one of the preceding claims, ABy d in that an enzymatic cleavage of appended to the starting primer nucleotides by an exonuclease, in particular by T7 DNA polymerase as exonuclease, E. coli exonuclease I or E. coli exonuclease III is carried out.
7. The method according to any one of the preceding claims, d ad u rch labeled in zeic HNET that a single initiating primer is used.
8. A method according to any one of claims 1 to 6, d ad u rch ge ken I nze et n that a plurality of initiating primer are used.
9. A method according to any one of claims 7 or 8, d ad u rch ge ken n net drawing that the extension reaction involves the attachment of a single fluorescently labeled chain terminating molecule.
10. The method of claim 9, d ad u rch geken nzeic h net, that it is in the chain termination molecule is a dideoxynucleotide.
11. The method according to any one of claims 7 or 8, d ad u rc h ge ken nzeichnet that the extension reaction comprises attaching a plurality of fluorescently labeled nucleic acid building blocks.
12. The method according to claim 11, d ad u rch et ekennzeichn g, that one or more pairs of start and block primers are used, wherein the 5 'end of each primer block at a predetermined distance downstream of the 3' end of the associated
Starting primer binds to the nucleic acid template, wherein the 3'-end of the primer is blocked block.
13. The method according to claim 12, d ad u rch gekennzeich net that several pairs of start and block primers are used, and the start / block primer pairs can be identified by different codes.
14. The method of claim 12 or 13, d ad u rch ge ken nzeic HNET that blocking the 3 'end of the primer block, optionally is reversible, with the exception of the most downstream block binding primer.
15. The method according to any one of claims 12 to 14, that block primers are used which carry a 3'-phosphate group d adu rch g eken n n et drawing.
16. The method according to any one of claims 12 to 15, d ad u rch ge characterizing et n, that a covalent bond between the extended by fluorescent nucleotides starting primer and the block primer is closed.
17. The method according to any one of claims 12 to 16, d ad u rch ge ken nzeich et n that the covalent bond is enzymatically, closed, for example using a ligase.
18. The method of claim 16 or 17, characterized in that at least one primer carries a block δ'-phosphate group.
19. A method according to any one of claims 14 to 18, d ad u rch geken n characterized in that the gap (s) between the extended by fluorescent nucleotides start primers and the block primers downstream in each case filled by deoxyribonucleotides after removal of the 3 'blocking the block Primer and are covalent bonds between the extended primers and the start block immediately downstream primers are closed.
20. A method for characterizing nucleic acid polymorphisms in a microwave, and mf assend the steps of:
(A) providing a carrier particle with a subsequent real-stabilized nucleic acid molecule consisting of a nucleic acid template and a first-class start primer
(B) elongating the starting primer by a fluorescently labeled chain termination molecule,
(C) optionally washing the wells to remove non-incorporated labels, and
(D) detecting the incorporated into the starting primer fluorescent label.
21. The method according to claim 20, d ad u rc h ge characterizing net that is used in the microwave integrated semiconductor laser and / or semiconductor detector for the excitation and / or for the detection of fluorescence.
22. The method of claim 20 or 21, d ad u rch et gekennzeich n that a plurality of reactions on a microwell plate is carried out in parallel or sequentially.
23. The method according to any one of claims 20 to 22, characterized labeled in ad d urch that is available for the extension of the initiating primer only one type of labeled nucleotide selected from the group consisting of ddATP, ddUTP, ddTTP, ddCTP, ddGTP.
24. The method according to any one of claims 20 to 22, dadu rc HGE ke nn ze et ichn that there are several, distinguishable by their fluorescence labeling chain terminating molecules available for the extension of the initiating primer.
25. The method according to any one of the preceding claims, dadu rc HGE ke nn zei et chn that one uses a carrier particle made of plastic, glass, quartz, metals, semimetals, metal oxides or of a composite material.
26. The method according to any one of the preceding claims, dadu rc HGE ke n nze ic et hn that the carrier particle has a diameter of 1 nm to 10 microns.
27. The method according to any one of the preceding claims, dadu rc HGE ken nzei et chn that the nucleic acid template on the carrier particles over their
5'-terminus or the starting primer is immobilized via its 3'-terminus by means of bioaffinity interactions.
28. The method of claim 1, dadurchgekennzeichn et that a biotinylated nucleic acid molecule is immobilized on an avidin or streptavidin beschich.tetes carrier particles.
29. The method according to any one of the preceding claims, d ad u rch geken nzeich net, that the distinction of different fluorescent labels due to the wavelength, the excited state lifetime, or a combination thereof is carried out.
30. The method according to any one of the preceding claims, d ad u rch geken nzeich net that the determination is carried out by confocal single molecule detection and / or by time-resolved decay.
31. A method according to any one of the preceding claims, d ad u rch in that the determination comprises measurement of a cross-correlated signal from a different, at least 2
comes markings, in particular fluorescent labels, containing nucleic acid molecule or nucleic acid molecule complex.
32. A method for Steigerug the detection efficiency in the detection of fluorescence from single molecules, d ad u rc HGE ke n nzeich et n that for the separation of light of different wavelengths
Dispersion element is used.
33. The method of claim 32, dadu rc h geken nzeich net that a prism or grating is used as the dispersing element.
EP20010993709 2000-11-13 2001-11-13 Detection of nucleic acid polymorphisms Withdrawn EP1409721A2 (en)

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DE10056226 2000-11-13
DE10056226 2000-11-13
DE10065631 2000-12-29
DE10065631A DE10065631A1 (en) 2000-11-13 2000-12-29 Characterizing nucleic acid polymorphisms, useful e.g. for detecting inherited disease, by extension of starter primer then single-molecule detection of incorporated nucleotide
PCT/EP2001/013120 WO2002038806A2 (en) 2000-11-13 2001-11-13 Detection of nucleic acid polymorphisms

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