Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6869—Methods for sequencing
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor

Definitions

• This invention relates to DNA sequencing. More particularly, it relates to methods and apparatus for determining the sequence of deoxyribonucleotides within DNA molecules .
• DNA sequencing is an important tool.
• a current goal of the biological community in general is the determination of the complete structure of the DNA of a number of organisms, including man. This information will aid in the understanding, diagnosis, prevention and treatment of disease.
• the DNA to be sequenced is enzymatically copied by the Klenow fragment of DNA polymerase I or by a similar polymerase enzyme such as Taq polymerase or SequenaseTM .
• the enzymatic copying is carried out in quadruplicate.
• a low concentration of a chain terminating dideoxynucleotide is present, a different dideoxynucleotide being present in each of the four reactions (ddATP, ddCTP, ddGTP and ddTTP) .
• the polymerase reaction is terminated, again producing sets of nested fragments. Again, the nested fragments have to be separated from one another by electrophoresis to determine the sequence.
• the present invention provides methods and apparatus for determining the sequence of deoxyribonucleotides in a DNA molecule.
• a key characteristic of this invention is that it determines the DNA sequence without recourse to electrophoresis or other size-based separation techniques.
• the present invention provides a method for determining the deoxyribonucleotide sequence, of a single stranded DNA subject molecule.
• This method involves synthesizing, in the presence of a multitude of identical copies of the subject DNA, the DNA molecule which is complementary to it.
• This synthesis is carried out using deoxyribonucleotide triphosphates (dNTP) in a stepwise serial manner so as to simultaneously build Up numerous copies of the complementary molecule, dNTP by dNTP.
• dNTP deoxyribonucleotide triphosphates
• this invention provides apparatus for carrying out the above-described method.
• this method and apparatus for carrying it out can take many different configurations.
• This invention can be carried out in a single reaction zone with multiple differentiable reporters or in multiple reaction zones with a single reporter in each zone. It can be carried out by detecting the incremental signal change after addition of reporters or by noting each added reporter separately. The various reporters can be measured in the reaction zones while attached to the growing molecule or they can be separated from the molecule and then measured.
• the invention can be practiced to create the growing complementary DNA chain without interruption or it can be practiced in stages wherein a portion of the complementary chain is created and its sequence determined; this portion of the chain is then removed; a sequence corresponding to a region of the removed chain is separately synthesized and used to prime the template chain for subsequent chain growth. The latter method can be repeated as needed to grow out in portions the complete complementary chain.
• Figures 1A and IB are schematic diagrams of the process of this invention on a molecular level.
• Figure 2 is a schematic representation of one form of apparatus for practising the invention.
• the DNA growth takes place in a single reaction zone.
• This embodiment uses separate, distinguishable reporters associated with each of the four nucleotides incorporated into the growing molecule. The four different reporters are measured after each addition to detect which base has just been added to that position of the complementary chain.
• Figure 3 is a schematic representation of another form of apparatus for practising the invention.
• This embodiment employs four reaction zones in which the molecular growth is carried out in quadruplicate. In each of the four zones, a different one of the four nucleotides is associated with a reporter (with the remaining three being unlabeled) so that the identity of the nucleotide incorporated at each stage can be determined.
• Figure 4 is a schematic representation of an adoption of the apparatus for practising the invention particularly adapted for carrying out the invention to grow a series of portions of the complementary molecule as opposed to a single continuous complementary molecule.
• Figures 5 through 8 are pictorial representations of chemical reaction sequences which can be used to synthesize representative labeled nucleotide building blocks for use in the practice of this invention.
• dNTPs of these materials are abbreviated as dATP, dCTP, dGTP and dTTP. When these materials are blocked in their 3'-OH position they are shown as 3 'blockeddATP, 3'blockeddCTP, 3 'blockeddGTP and 3 'bl ⁇ ckeddTTP. Similarly, when they are each tagged or labeled with a common reporter group, such as a single fluorescent group, they are represented as dA'TP, dC'TP, dG'TP and dT'TP.
• a solid support 1 is illustrated with a reactive group A attached to its surface via tether 2. This attachment can be covalent, ionic or the like.
• a second reactive group. X capable of bonding to group A, again via a covalent, ionic or the like bond, is attached to the 5' end of a DNA primer 4.
• This primer has a known DNA sequence. When coupled to the substrate via the A-X bond it forms immobilized primer 5.
• Primer 5 is then hybridized to template DNA strand 6 which is made up of an unknown region 7 inserted between regions 8 and 8 ' . Regions 8 and 8' are located at the 5' and 3' ends of the unknown region and have known sequences.
• the 8' region's known sequence is complementary to the sequence of primer 4 so that those regions hybridize to form immobilized template DNA 9. Therefore the individual dNTPs are serially added to form the DNA sequence complementary to the unknown region of the template. 11 and 12 represent the first two such dNTPs incorporated into the growing molecule. These in turn provide the identity of their complements 11' and 12' respectively. This growth continues until the entire complementary DNA molecule has been constructed. Completion can be noted by identifying the sequence corresponding to the 8 region of template 6. Turning to Figure IB, a variation of this chemistry is shown in that the template 6* carries the reactive group X which bonds to the substrate via the A-X bond to form an immobilized template 5*.
• Device 13 for carrying out the invention is shown schematically.
• Device 13 includes a reaction zone 14 which carries inside it a surface 15.
• a plurality of copies of a subject primed single stranded DNA are immobilized on this surface 15. This is the strand of DNA for which the sequence is desired.
• the immobilized DNA is depicted fancifully on surface 15 as if it were present as a series of separately visible attached strands. As will be appreciated, this is not in fact the case and is only done to guide the reader as to the location of the DNA strands .
• the reaction zone 14 may be configured to permit direct reading of reporter signals emanating from within. Examples of this configuration include equipping the reaction zone to permit measuring fluorescence or luminescence through one or more transparent walls or detecting radionuclide decay.
• Reaction zone 14 is fitted with inlet 16 for the addition of polymerase or another suitable enzyme capable of moderating the templat ⁇ e-directing coupling of nucleotides to one another.
• the reaction zone is ⁇ also accessed by inlet lines, 18a-18d for four differently labeled blocked dNTPs, that is 3'blockeddA'TP, 3 'blockeddC' 'TP, 3 'blockeddG' ' 'TP, and 3'blockeddT' ' ' 'TP. These materials can be added in four separate lines, as shown, or can be premixed, if desired, and added via a single line. Buffer and other suitable reaction medium components are added via line 20.
• the polymerase and the four labeled dNTPs are added to the reaction zone 14 under conditions adequate to permit the enzyme to bring about addition of the one, and only the one, of the four labeled blocked dNTPs which is complementary to the first available template nucleotide following the primer.
• the blocking group present on the 3 '-hydroxyl position of the added dNTP prevents inadvertent multiple additions .
• the liquid in reaction zone 14 is drained through line 22 either to waste, or if desired to storage for reuse.
• the reaction zone and the surface 15 are rinsed as appropriate to remove unreacted, uncoupled labeled blocked dNTPs.
• the first member of the complementary chain is now in place associated with the subject chain attached to surface 15.
• the identity of this first nucleotide can be determined by detecting and identifying the label attached to it.
• This detection and identification can be carried out in the case of a fluorescent label by irradiating the surface with a fluorescence-exciting beam from light source 24 and detecting the resulting fluorescence with detector 26.
• the detected florescence is then correlated to the fluorescence properties of the four different labels present on the four different deoxynucleotide triphosphates to identify exactly which one of the four materials was incorporated at the first position of the complementary chain. This identity is then noted.
• a reaction is carried out to remove the blocking group and label from the 3' position on the first deoxynucleotide triphosphate.
• This reaction is carried out in reaction zone 14.
• a deblocking solution is added via line 28 to remove the 3' hydroxyl labeled blocking group. This then generates an active 3' hydroxyl position on the first nucleotide present in the complementary chain and makes it available for coupling to the 5' position of the second nucleotide.
• removal of the deblocking solution via line 22 and rinsing as needed the four blocked, labeled deoxynucleotide triphosphates, buffer and polymerase are again added and the appropriate second member is then coupled into the growing complementary chain.
• the second member of the chain can be identified based on its label. This process is then repeated as needed until the complementary chain has been completed.
• the sequence of incorporated deoxynucleotides is known, and therefore so is the sequence of the complement which is the subject chain. It will be appreciated that this process is easily automated. It is a series of fluid additions and removals from a reaction zone. This can be easily accomplished by a series of timer-controlled valves and the like. This technology has been well developed in the area of oligonucleotide synthesizers, peptide synthesizers, and the like. In such an automated system, the timing can be controlled by a microprocessor or, in most cases, by a simple programmable timer. The rate and " extent of reaction can be monitored by measurement of the reporter concentration at various stages .
• the labels present in the blocked dNTPs can be incorporated in one of several manners. For one, they can be incorporated directly and irremovably in the deoxynucleotide triphosphate unit itself. Thus, as the complementary chain grows there is a summing of signals and one identifies each added nucleotide by noting the change in signal observed after each nucleotide is added.
• the label is incorporated within the blocking group or is otherwise incorporated in a way which allows it to be removed between each addition. This permits the detection to be substantially simpler in that one is noting the presence of one of the four reporter groups after each addition rather than a change in the sum of a group of reporter groups .
• the presence of reporter signal is noted directly in the reaction zone 14 by the analytical system noted as source 24 and detector 26. It will be appreciated, however, that in embodiments where the reporter group is removed during each cycle, it is possible to read or detect the reporter at a remote site after it has been carried out of the reaction zone 14.
• drain line 22 could be valved to a sample collector (not shown) which would isolate and store the individual delabeling product solutions for subsequent reading.
• the various removed labels could be read as they flowed out of the reaction zone by equipping line 22 with an in-line measurement cell such as source 24' and detector 26' or the like.
• a second embodiment of this invention employs four separate parallel reaction zones. This method has the advantage of requiring only one type of labeling and being able to use it with all four dNTPs.
• Figure 3 shows a schematic representation of a device 30 which has the four reaction zone configuration. In this configuration there are four reaction zones 32a through 32d, each of which resembles the reaction zone 14 in Figure 2. In these cases each of the four reaction zones contains a surface 34a-d to which is immobilized numerous copies of a primed subject single stranded DNA. Each reaction zone is supplied with polymerase via lines 36a-d. Each zone is supplied with suitable reaction medium via lines 38a-38d. The four dNTPs are supplied in blocked form to each zone, as well.
• zone 32a one of the blocked dNTPs is labeled, for example "A'"; in zone 32b a second dNTP is labeled, for example "C”; in zone 32c a third dNTP is labeled, for example "G'”; and in 32d the fourth labeled dNTP "T' M is present.
• These labeled materials are supplied via lines 40a through 40d respectively.
• Unlabeled blocked dNTPs are supplied via lines 42a-d so that each of the four reaction zones contains three unlabeled blocked dNTPs and one labeled blocked dNTP.
• the various labeled and unlabeled dNTP ' s can be premixed. These premixed materials can be added to the various reaction zones via single addition lines.
• the single stranded DNA hybridized to a primer and attached to each of surfaces 34a-34d is contacted with polymerase (supplied via lines 36a-36d), buffer (supplied via lines 38a-38d) and the four bases in each of the four reaction zones .
• the blocked dNTP which complements the first base on the subject chain couples.
• this base is labeled.
• this label is incorporated into the growing chain, one can determine the identity of the dNTP which is incorporated at the first position. This determination of the identity of the first unit of the chain can be carried out using signal sources and detectors such as 44a-44d and 46a-46d, respectively.
• Deblocking is carried out by adding deblocking solution to the reaction zone through lines 48a-48d.
• Lines 50a-50d are drain lines for removing material from the reaction zones following each step.
• all of the variations noted with reference to the device described in Figure 2 can also be used including cumulating reporter signals and generating reporter signals away from the reaction zone by removing the reporter groups as part of each of the sequential couplings.
• this embodiment can be readily automated, as well.
• One obvious potential shortcoming of the present invention is that it employs a long sequence of serial reactions. Even if the efficiency and yield of each of these reactions are relatively high, the overall yield becomes the product of a large number of numbers, each of which is somewhat less than 1.00, and thus can become unacceptably low. For example if the yield of a given addition step is 98% and the deblocking is 98% as well, the overall yield after 15 additions is 48 , after 30 additions it is 23% and after 60 additions it is 5.3%.
• This limitation can be alleviated by periodically halting the DNA molecule growth and using the sequence data obtained prior to halting the growth to externally recreate a portion of the molecule which can then be used as a primer for renewed DNA fabrication. This process is illustrated in Figure 4.
• FIG 4 shows a schematic of an automated sequencer 52 employing the present invention.
• Sequencer 52 has a single reaction zone 14 combining the subject primed DNA, immobilized therein such as on surface 15.
• the four 3-blocked DNTP's are fed to the reaction zone through line 18.
• Polymerase and buffers are added via lines 16 and 20, respectively.
• the dNTP 's, polymerase and buffer can be recycled from step to step via lines 54 and 56 and holding vessel 58. All of the valves admitting and removing fluids from reaction zone 14 can be controlled by central computer 60 which functions as a valve control clock.
• This computer 60 can also control the addition of deblocker from line 28, deblocking eluent with cleaved labels (as obtained when the label is present in the blocking group) is removed via line 22 and detected via detector system 24/26 reading label values in detector vessel 62.
• This embodiment illustrates the use of a fluorescent label system and shows the addition of fluorescent sensitizer (flooder) via line 64 to the fluorescent detection zone 62.
• the deblocking solution and detected label are discarded via line 66.
• the signal presented by the label identified by detector 26 is passed to analog/digital converter 68 and therein to a memory in central computer 60 where it is stored.
• the memory in computer 60 contains the sequence of an initial portion of the complementary DNA molecule which has been constructed in association with the subject or target DNA molecule contained within reactor 14. After some number of units have been assembled - typically 25 to 300, or more; preferably 50 to 300, or more; and more preferably 100 to 300, or more - the growing complementary DNA molecule is stripped from the immobilized subject DNA molecule and discarded. This stripping (denaturing) can be done by art-known methods such as by warming the reaction zone to 75°C or higher (preferably 90-95°C) for a few (1-15) minutes.
• the sequence information stored in computer 60 is used to drive DNA synthesizer 70 to externally create a new DNA primer corresponding to at least a portion of the discarded DNA molecule. (The sequence can also be read on printer 72, if desired.)
• This newly constructed DNA primer molecule is fed through line 74 to reaction zone 14 under hybridization conditions so as to join to the complementary region of the subject DNA molecule as a new primer.
• the length of the primer must be adequate to. unambiguously and strongly hybridize with a single region of the subject DNA. As is known in the hybridization art, this can depend upon factors such as the sequence, environmental conditions, and the length of the subject DNA. For efficiency of operation, the primer should ideally be as short as possible.
• Primer lengths typically range from about 10 bases to about 30 bases, although shorter primers would certainly be attractive if they met the above criteria, and longer primers could be used albeit with an increase in cost and time. Good results generally are achieved with primers from 12 to 20 bases long. This gives the molecular growth reaction a "new start" with a large number of properly primed identical molecules. This allows a strong signal to be generated when the next dNTP is coupled.
• the coupling process employed in this invention to incorporate each of the blocked deoxynucleotide triphosphates into the growing complementary chain is an enzyme moderated process.
• Each member of the complementary DNA chain is added using a suitable template-dependent enzyme.
• One enzyme which can be used is Sequenase TM enzyme (an enzyme derived from bacteriophage 7 DNA polymerase that is modified to improve its sequencing properties - see Tabor and
• Sequenase TM examples include but are not limited to
• the coupling conditions which are employed are those known in the art for these enzymes.
• these include temperatures in the range of from about room temperature to about 45 C; a buffer of pH 7 to 8 and preferably pH 7.3 to 7.7; an enzyme concentration of from about 0.01 units per microliter to about 1 unit per microliter and a reaction time of from about 1 to about 20 minutes and preferable 1 to 5 minutes.
• a typical buffer for use with Sequenase TM is made up of
• these typical conditions include temperatures in the range of from about 10 C to about 45 C and preferably from about 15°C to about 40°C; a buffer of pH 6.8 to 7.4 and preferably pH 7.0 to 7.4; an enzyme concentration of from about 0.01 units per microliter to about 1 unit per microliter and preferably from about 0.02 to about 0.15 units per microliter and a reaction time of from about 1 to about 40 minutes.
• a typical buffer for use with Klenow fragment of DNA polymerase I is made up of
• 3 '-blocking groups include: (l) the ability of a polymerase enzyme to accurately and efficiently incorporate the dNTPs carrying the 3 '-blocking groups into the cDNA chain,
• the 3 '-blocking group carries a reporter group, it is desirable that the reporter permit sensitive detection either when part of the cDNA chain before deblocking or subsequent to deblocking in the reaction eluant.
• 3 '-blocked dNTPs are used that can be incorporated in a template-dependent fashion and easily deblocked to yield a viable 3 ' -OH terminus.
• the most common 3 '-hydroxyl blocking groups are esters and ethers.
• ester blocking groups such as lower (1-4 carbon) alkanoic acid and substituted lower alkanoic acid esters, for example formyl, acetyl, isopropanoyl, alpha fluoro- and alpha chloroacetyl esters and the like; ether blocking groups such as alkyl ethers; phosphate blocking groups; carbonate blocking groups such as 2-nitrobenzyl; 2,4-dinitrobenzene-sulfenyl and tetrahydrothiofuranyl ether blocking groups.
• Blocking groups can be modified to incorporate reporter moieties, if desired, including radiolabels (tritium, C 14 or F ⁇ ** 2 , for example), enzymes, fluorophores and chromophores .
• selectively-removable amine protection groups include carbamate ⁇ cleavable by acid hydrolysis [t-butyl, 2-(biphenyl)isopropyl] and certain amides susceptible to acid cleavage (formamide, trichloroacetamide) (Greene, 1981) .
• nucleotide derivatives protection of the primary amino groups is performed prior to phosphonation.
• standard amino protecting groups cleavable by ammonolysis may be used.
• the sequencing scheme After successfully incorporating a 3 '-blocked nucleotide into the DNA chain, the sequencing scheme requires the blocking group to be removed to yield a viable 3 '-OH site for continued chain synthesis.
• the deblocking method should:
• the exact deblocking chemistry selected will, of course, depend to a large extent upon the blocking group employed. For example, removal of ester blocking groups from the 3 'hydroxyl function is usually achieved by base hydrolysis. The ease of removal varies widely; generally, the greater the electro-negativity of substituents on the carbonyl carbon, the greater the ease of removal. For example, the highly electronegative group trifluoroacetate is cleaved rapidly from 3' hydroxyls in methanol at pH 7 (Cramer et al. , 1963) and thus would not be stable during coupling at that pH.
• Phenoxyacetate groups are cleaved in less than one minute but require substantially higher pH such as is achieved with NH-/ methanol (Reese and Steward, 1968).
• the ester deblocking rate is advantageously selected so as to exhibit a deblocking rate of less than 10 -3s-1 during the incorporation, and at least 10 ⁇ s during the deblocking stage. Ideally, this rate change is achieved by changing the buffer pH from 7 to about 10, but care must be taken not to denature the DNA.
• hydroxyl blocking groups are cleaved selectively using chemical procedures other than base hydrolysis.
• 2,4-Dinitrobenzenesulfenyl groups are cleaved rapidly by treatment with nucleophiles such as thiophenol and thiosulfate (Letsinger et al., 1964).
• Allyl ethers are cleaved by treatment with Hg(II) in acetone/water (Gigg and Warren, 1968) .
• Tetrahydrothiofuranyl ethers are removed under neutral conditions using Ag(I) or Hg(II) (Cohen and Steele, 1966; Cruse et al . , 1978).
• These protecting groups which are stable to the conditions used in the synthesis of dNTP analogues and in the sequence incorporation steps, have some advantages over groups cleavable by base hydrolysis - deblocking occurs only when the specific deblocking reagent is present and premature deblocking during incorporation is minimized.
• Photochemical deblocking can be used with photochemically-cleavable blocking groups.
• Several blocking groups are available for such an approach.
• the use of o-nitrobenzylethers as protecting groups for 2 '-hydroxyl functions of ribonucleosides is known and demonstrated (Ohtsuka et al. , 1978); removal occurs by irradiation at 260 nm.
• Alkyl o-nitrobenzyl carbonate protecting groups are also cleaved by irradiation at pH 7 (Cama and Christensen, 1978).
• Enzymatic deblocking of 3 '-OH blocking groups is also possible. It has been demonstrated that T4 polynucleotide kinase can convert 3 '-phosphate termini to 3 '-hydroxyl termini that can then serve as primers for DNA polymerase I (Henner et al . , 1983). This 3 '-phosphatase activity is used to remove the 3 '-blocking group of those dNTP analogues that contain a phosphate as the blocking group; the radioactive label enables the incorporation of the nucleotide analogue and the removal of the phosphate group to be followed easily. . If the use of radioisotopes represents too great a drawback, it is possible to use unlabeled phosphate monoesters with a cleavable fluorescent label (see below).
• each dNTP into the complementary chain is noted by detecting a label or reporter group present in or associated with the incorporated dNTP.
• the labels or markers are "innocuous".
• An "innocuous marker or label or reporter” refers to a radioactive, fluorescent, or the like marker or reporter which has physical and chemical properties which do not interfere with either the enzymatic addition of the marked nucleotide to the cDNA, or the subsequent deblocking to yield a viable 3 '-OH terminus .
• One simple labeling approach is to incorporate a radioactive species within the blocking group or in some other location of the dNTP units. This can be done easily by C 14 labeling or P32 labeling.
• Another labeling approach employs fluorescent labels. These can be attached to the dNTP's via the 3 '0H- blocking groups or attached in other positions. There are two general routes available using fluorescent tags:
• the first route is fairly straightforward and can employ a range of known fluorophores such as rhodamines, fluoresceins and the like, typically including those fluorophores known as useful in labeling dNTP's and the like.
• fluorophores such as rhodamines, fluoresceins and the like, typically including those fluorophores known as useful in labeling dNTP's and the like.
• the second route can employ a fluorophore where only a fragment is attached to the dNTP. This can reduce size and minimize steric interference. In the second route, rapid reaction of a normally nonfluorescent probe or molecule with specific functional group(s) found only on the label fragment leads to the formation of a fluorescent addition product. This leads to a signal only when the particular label
• Blocking groups or other label fragment groups containing free thiol functions can be used for this approach.
• the blocking group or other label fragment can contain a metal-binding ligand, e.g. a carboxylic acid group which will react with added rare earth metal ions such as europium or terbium ions to yield a fluorescent species.
• This dNTP can be incorporated and the fluorescence measured and removed according to the methods described below.
• One method involves the use of a fluorescent tag attached to the base moiety.
• the tag may be chemically cleaved (either separately from or simultaneously with the deblocking step) and measured either in the reaction zone before deblocking or in the reaction eluant after cleavage.
• the fluorescent moiety or other innocuous label can be attached to the dNTP through a spacer or tether.
• the tether can be cleavable if desired to release the fluorophore or other label on demand.
• Typical tethers are from about 2 to about 20, and preferably from about 3 to about 10 atoms in length.
• the C-8 position of the purine structure presents an ideal position for attachment of a label.
• Sarfati et al . (1987) describes a derivatization of deoxyadenosine at C-8 of the purine to prepare, ultimately, an 8-substituted biotin aldylamino dATP.
• the Sarfati et al . (1987) approach can be used to prepare the appropriate fluorescent, rather than biotinylated, analogues.
• a number of approaches are possible to produce fluorescent derivatives of thymidine and deoxycytidine.
• One quite versatile scheme is based on an approach used by Prober et al . (1987) to prepare ddNTPs with fluorescent tags. Structures A, B, C and D below illustrate the type of fluorescent dNTPs that result from these synthetic approaches.
• the synthetic routes have a great flexibility in that the linker can be varied with respect to length or functionality.
• the terminal fluorescent moiety can also be varied according to need.
• the labels so incorporated in the growing cDNA chain are detected by conventional analytical methods .
• increased detection sensitivity is a major advantage of the present method.
• the signal is based on a low level of fluorophores and is superimposed on a background of scatter from the gel and glass plates. This decreases sensitivity and often constrains current methods to the use of laser illumination to maximize sensitivity (Smith et al., 1986; Prober et al . , 1987; Ansorge et al . , 1986) .
• Detection of fluorophores is readily achievable in commercial non-excited spectrofluorometers , such as are sold by Perkin-Elmer.
• LED light- emitting diodes
• Typical LEDs include:
• Red LED emitting at approximately 650 nm
• Green LED emitting at approximately 540 nm
• Blue LED emitting at approximately 450 nm
• the solution containing cleaved blocking groups or nucleotides is directly injected into a field ionization mass spectrometer. Identification of the particular nucleotide incorporated or cleaved is achieved by monitoring the relative abundance of molecular ion peaks corresponding to the specific nucleotides or blocking groups; for example, four distinct acetyl blocking groups differing by one mass unit (replacement of 0 to 3 hydrogens by deuterium) could be detected by monitoring a small “window. "
• Immobilization of Subject DNA In the present invention, single stranded subject DNA or its primer is immobilized.
• One approach to this immobilization is to attach the DNA to a solid substrate.
• DNA and RNA are commonly attached noncovalently through ionic interactions along their length to various types of membranes (Southern, 1975; Maniatis, Fritsch, and Sambrook, 1982; Chuvpilp and Kravchenko, 1984).
• polynucleotides are covalently attached along their length to membranes (Goldberg, et al . , 1979), resins (Seed, 1982; Arndt-Jovin, et al .
• the inner quartz or glass surface can be advantageously functionalized using silanizing reagents such as triethoxysilylpropylamine or dichlorodi ethylsilane. This is followed by covalent attachment of a long-chain alkylamine to these functionalizing groups.
• the single stranded subject DNA is attached to the long chain amine.
• immobilization is carried out by attaching the subject DNA to a plastic surface.
• a thin polypropylene chamber wall designed to pass Cer'enkov radiation from 32P, for example, can serve as a suitable substrate for DNA immobilization.
• a plastic surface it is preferable to use the method of Kremsky et al .
• the reaction zone has one or more openings covered with a membrane such as an ultrafiltration membrane, for example, Amicon's PM-5 or PM-10 membranes which have nominal molecular weight cut offs of 5000 and 10,000 respectively.
• a membrane such as an ultrafiltration membrane, for example, Amicon's PM-5 or PM-10 membranes which have nominal molecular weight cut offs of 5000 and 10,000 respectively.
• the single stranded DNA is suspended in liquid in the reaction zone.
• the labeled and unlabeled dNTPs and other coupling reagents are flowed into the zone. Materials are removed from the zone through such a filter which retains the DNA chains.
• the polymerase or other enzyme which is used to effect coupling is generally of a size to be retained by the membrane. This scheme works for chemical but not enzymatic deblocking, since in enzymatic deblocking the polymerase and phosphatase must be cycled separately through the cell.
• the DNA can be immobilized on particles of resin or polymer microspheres and these particles retained within the chamber.
• the filter material is unimportant as long as the DNA is attached to resin particles which are of a size that cannot penetrate the filter pores .
• oligonucleotides or polynucleotides are linked through their 5' end to cellulose (Gilha , 1968; Clerici et al . 1979), Sephacryl (Langdale and Malcolm, 1985), or latex microspheres (Kremsky et al., 1987).
• the DNA is available for interactions with other nucleic acids or proteins .
• the DNA is coupled covalently to streptavidin-agarose beads by an alkylbiotinylated oligonucleotide (Kremsky et al., 1987).
• the single-stranded DNA is coupled to DBM paper such as a filter in the presence of a protecting strand. After coupling, the protecting strand is released, leaving the immobilized template and priming site free for successive enzymatic reactions (Hansen et al . , 1987) .
• This method and the other single-point methods described above are useful for immobilizing DNA while leaving it free for interactions with enzymes used in DNA sequencing- Examples
• the organic layer is separated and the aqueous layer washed with 2 x 200 ml CH 2 C1 2
• the combined CH-Cl- extracts are dried over magnesium sulfate (MgSO.), filtered and evaporated to dryness under vacuum at room temperature.
• the crude 5 '-dimethoxytrityl-3 'thymidine H-phosphonate II is then treated with 2% benzenesulfonic acid in CH ⁇ Cl- -.methanol (MeOH) (7:3) (200 ml) for one hour.
• the solution is washed with 10% sodium bicarbonate (NaHCO-) and water, dried over magnesium sulfate and evaporated to dryness.
• the crude 3 '-thymidine- H-phosphonate III is recrystallized from ethanol/ether.
• the mixtu-re is stirred for 12 hours at 4 C, neutralized with NaHCO.. solution,- and added to 150 ml water.
• the aqueous solution is washed with benzene (2 x 100 ml) and ether ( 2 x 100 ml), and diluted to 0.8 liters with water and charged on a 2.5 x 50 cm column of DEAE-cellulose.
• the products are eluted using a linear gradient of pH 8.5 ammonium bicarbonate solution (0.05 to 0.25 M) .
• the fractions collected are analyzed by HPLC to determine the desired product-containing fractions, and these are evaporated to dryness under vacuum. The residue is repeatedly re-evaporated with water to remove salts .
• the 5 '-monophosphate IV (16 mmole) is then dissolved in 30 ml of dimethylformamide (DMF) and treated with N,N'-carbonyldiimidazole (30 mmole) at room temperature for one hour.
• the reaction is quenched by addition of 5 ml methanol, and 60 ml of a 0.5M solution of bis (tri-n-butyl-ammonium) pyrophosphate in DMF is added dropwise over 10 minutes.
• the solution is diluted with water to 1 liter and treated with 100 ml of a solution of 0.1 M iodine (I-.) in 5% pyridine/water. After one hour, the solution is deposited on a DEAE-cellulose column from Sigma (5x50cm) or Sephradex from Pharmacia. The column is washed with water and eluted with triethylammonium bicarbonate solution
• the 5 '-triphosphate-3 '-phosphate thymidine product V is obtained by evaporation of the appropriate fractions collected.
• Example 3 Quartz Surface Immobilization of Subject DNA Four 25 microliter volume quartz cuvette reaction chambers are prepared. These chambers are configured like chamber 32 in Figure 3 with the exception that they use their inner walls as the surface to which the DNA is affixed. The inner surfaces are cleaned and dried.
• Triethoxysilylpropylamine (5 microliter in 20 microliter CHC1-.) is added and held at 5°C for 120 minutes under anhydrous conditions . This couples the triethoxysilylpropylamine to the surface and gives an amine character to the surface.
• the subject DNA is then attached to the amine surface.
• This is carried out by first attaching a long chain alkyl amine (n-octylamine) to the base at the 5' end of the subject DNA molecule or to the base at the 5 ' end of a suitable primer, such as an M13 primer for example the 17-mer dGTAAAACGACGGCCAGT, and then joining the alkylamine to the aminopropyl ⁇ ilane surface groups by reaction with glutaraldehyde (1.5 equivalents, 25°C, 120 minutes).
• a suitable primer such as an M13 primer for example the 17-mer dGTAAAACGACGGCCAGT
• Other functional groups pendant to the base moiety or attached to the 5' position can also be used [for example: aldehydes or carboxylic acids (Kremsky et al) ] for covalent immobilization on derivatized quartz or glass surfaces.
• Example 4 Incorporation of Labeled Nucleotide Analogs into DNA
• the 25 microliter reaction zones are charged with a reaction mixture which contains three Units of Sequenase TM enzyme.
• the reaction mixture also contains an appropriate buffer for this enzyme (20 mM Tris-HCl pH 7.5, 10 mM MgCl, 25 mM NaCl, 0.01 M dithiothreitol), the i single-stranded primed subject DNA is present at a concentration of approximately 0.1 M attached to the surface of the reaction chamber at its 5' end, (see Example 3), three unlabeled, 3 '-blocked deoxynucleotide triphosphate (dNTP) analogs at a concentration of 1.5 micromolar each, and one 3 '-blocked, fluorescently labeled dNTP analog of Example 2 at a concentration of 30 micromolar are each present in each of the four reaction zones. In each zone a different one of the four dNTPs is labeled.
• the reaction
• the identity of the added dNTP is determined by exciting the fluorophores present in the one cuvette which incorporated its fluorescently-labeled dNTP.
• the fluorescent group is removed before measurement.
• the 2 ,4-dinitrobenzenesulfenyl fluorescent blocking groups are removed with a deblocking reagent which consists of 0.1 M pyridine/pyridinium chloride buffer (pH 7.8) containing thiourea 0.05 M.
• the deblocking reaction is allowed to proceed for one minute at 40 C-
• the reaction chamber is then drained and washed twice with 100 mM Tris-HCl buffer, pH 6.5.
• the release of the fluorescent blocking group is measured in the initial eluate from the reaction chamber using a flow-through cell. Depending on the cell in which the fluorescent group is present, the identity of the nucleotide which has been added to the DNA chain is determined.
• the blocking group were a dansylcadaverine type ester such as in reaction scheme 4 , it could be removed by treatment with 50% methanol/50% water pH 10.0 for one minute.
• Example 6 Enzymatic Deblocking
• the blocking group can also be removed enzymaticall .
• the deblocker fed into the reaction chamber contains 100 mM Tris-HCl (pH 6.5) 10 mM MgCl-, 5 mM 2-mercaptoethanol, and one Unit T4 polynucleotide kinase.
• the reaction proceeds for one minute at a temperature of 37 C.
• the 3 ' -phosphatase activity of T4 polynucleotide kinase converts 3 '-phosphate termini to 3 '-hydroxyl termini which then serve as primers for further synthesis.
• a simple clock mechanism or microprocessor driven timer circuit can be used to actuate a plurality of electrically controlled valves in. sequence to add the various reagents for adding building blocks, deblocking and the like with the result that the sequence of the target DNA single strand can be obtained with minimum involvement of lab personnel .

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