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WO1994016107A1 - SEQUENÇAGE D'ADN A L'AIDE DE Bst POLYMERASE - Google Patents

SEQUENÇAGE D'ADN A L'AIDE DE Bst POLYMERASE Download PDF

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Publication number
WO1994016107A1
WO1994016107A1 PCT/US1994/000562 US9400562W WO9416107A1 WO 1994016107 A1 WO1994016107 A1 WO 1994016107A1 US 9400562 W US9400562 W US 9400562W WO 9416107 A1 WO9416107 A1 WO 9416107A1
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sequencing
dna polymerase
group
dna
reagents
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PCT/US1994/000562
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English (en)
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Darwin Johnson Prockop
James Joseph Earley
Geraradus Cornelus Tromp
Sisko Helena Kuivaniemi
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Thomas Jefferson University
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    • 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

Definitions

  • the present invention is directed to sequencing nucleic acids by the dideoxynucleotide sequencing method.
  • the dideoxynucleotide chain termination method is an efficient process for DNA sequencing. It comprises a set of simple reactions that can be performed in a few minutes and can yield a few hundred nucleotides of DNA sequence. Performing many sequencing reactions, however, very quickly becomes tedious and leads to operator errors since optimal results require consistent timing and precise pipetting of small volumes (0.5 to 10 ⁇ l) .
  • the multichannel format is suitable for increasing throughput, not only with manual pipetting, but also with robotic automation. Nonetheless, it is difficult to manually pipet small volumes precisely and reproducibly with a multichannel pipet, since it is easy for one of the channels to fill partially if the pipet is not aligned perfectly with the wells.
  • the problem is compounded in the sequencing protocols, both for manual as well as robotic pipetting, because the small volumes are frequently pipetted from sources of which the volumes are only fractionally larger than the desired volume.
  • Published European Patent Application 0 298 669 discloses the use of dried down enzymes including restriction enzymes; nucleic acid polymerases DNA polymerase I (Klenow fragment) and Tag DNA polymerase; and nucleic acid modification enzymes.
  • the present invention discloses the use of particular dried down enzymes in DNA sequencing. To overcome the limitation of pipetting small volumes, the present invention provides sequencing reagents which have been dried down in multiwell format. As a result, it is possible to pipet volumes that are double or more the volumes used in the standard, non- dried down protocols.
  • a method of sequencing DNA comprising the addition of a nucleic acid template and primer to an effective amount of a DNA polymerase and of other sequencing reagents, wherein said DNA polymerase is Bst DNA polymerase and the Bst DNA polymerase and some or all of the other sequencing reagents are in a dried-down state.
  • the present invention also provides a method of using DNA polymerase and sequencing reagents in a DNA sequencing method wherein said polymerase is Bst DNA polymerase and the DNA polymerase and sequencing reagents are in a dried- down state.
  • Figure 1 is an autoradiograph of sequences obtained with Sequenase in the dried-down format. Sequencing reactions were performed as described and the fragments were separated by electrophoresis through a 6% polyacrylamide DNA sequencing gel. Sequences from eight sets of sequencing reactions (loaded G, A, T, C) are shown. The reagents were dried down on a microtiter plate. Termination reagents were dried slowly whereas the labeling reagents and Sequenase were freeze-dried. All non- enzyme reagents were identical for all sets of reactions. As indicated, one half (four) of the icrotiter plate wells that received labeling reagents and enzymes, received one unit of enzyme whereas the other four wells received two units of enzyme. The lanes with less enzyme show a marked lack of signal intensity as well as almost total premature termination (bands in all lanes) about half way up the gel. The lanes with more (two units) enzyme show satisfactory signal but still show signs of premature termination.
  • Figure 2 is an autoradiograph of sequences obtained with Tag DNA polymerase in the dried-down format. Four sets of reactions (loaded G, A, T, C) are shown. All reagents included non-ionic detergents and were dried down according to the protocol that included applying vacuum in two steps. Sequences were of acceptable quality.
  • Figure 3 is an autoradiograph of sequences obtained with Tag DNA polymerase in the dried-down format demonstrating the effect of additional DTT on the quality of sequences. All reagents included non-ionic detergents and were dried down according to the protocol that applied vacuum in two stages. Six sets of reactions (loaded G, A, T, C) are shown and the presence of additional DTT to 3 mM is shown. There is no apparent difference due to the presence or absence of additional DTT and sequences were of acceptable quality.
  • Figure 4 is an autoradiograph of sequences obtained with Bst DNA polymerase in the dried-down format. Reagents for sequencing with Bst DNA polymerase were dried down according to the protocol that included applying vacuum in two stages. All reagents were identical except that the numbers of units of enzyme dried down were as indicated. Eleven sets of reactions (loaded G, A, T, C) are shown. Bst DNA polymerase generated sequences of good quality with as few as 0.25 U of enzyme per set of reactions.
  • Figure 5 is an autoradiograph of sequences obtained with Tag and Bst DNA polymerases in the dried-down format. Reagents were dried down according to the protocol that included applying vacuum in two stages. The reagents for both enzymes were dried simultaneously under identical conditions.
  • FIG. 6 is an autoradiograph demonstrating the effect of additional DTT as well as storage conditions on Bst DNA polymerase in the dried-down format. Plates with Bst DNA polymerase were prepared as described except that DTT to 3 Mm was added to some wells as indicated. Plates were stored overnight at -20°C(A) or at room temperature (B) .
  • Figure 7 is a layout of a Master and Target microtiter plate during dispensing of reagents.
  • Reagent stocks were placed in five wells (12B through 12F) of the master plate. Seven microliters were transferred successively to rows of wells (five wells at a time) on the target plate.
  • the reagents were dispensed to the target plate such that the wells 2A through 2F contained the labeling reagents and enzyme, 3A through 3H contained the guanosine termination mix, 4A through 4H contained the adenosine termination mix, 5A through 5H contained the thymidine termination mix and wells 6A through 6H contained the cytosine termination mix.
  • the present invention is directed to a method of DNA sequencing using dried-down polymerase and reagents.
  • the present invention is also directed to a method of using dried- down reagents, particularly sequencing reagents, in DNA sequencing.
  • the methods of the present invention may be especially useful for sequencing in a multiwell format.
  • DNA sequencing includes, and is not limited to, the use of one or more radiolabeled deoxynucleotide(s) .
  • radiolabeled primers fluorescently-labeled primers or dideoxynucleotides, biotin- labeled primers or deoxynucleotides may be used.
  • Reactions may also be performed in the absence of label and detection achieved by hybridization of short oligonucleotide primers that may be labeled with radioisotopes or other components that allow for use of signal-producing enzymes such as luciferase.
  • polymerases for use in DNA sequencing include and are not limited to, DNA polymerases such as Bst DNA polymerase.
  • the present invention is directed to the method of DNA sequencing wherein the DNA polymerase is dried-down Bst DNA polymerase; and the use of dried-down Bst DNA polymerase in DNA sequencing.
  • sequencing reagents include, and are not limited to, pH buffers, EDTA for example; non-ionic detergents, one or more of Brij-35, NP-40, Triton X-100®, Tween® or a mixture of equal parts of these detergents for example; a dye such as xylene cyanol, Fuchsin S, brilliant green, bro ophenol blue, methyl blue, methylene blue or toluidine blue; deoxynucleotides; dideoxynucleotides; a primer, such as M13 universal primers, forward and reverse, for 3-galactosidase-containing vectors, and primers for promoters of SP6, T3, and T7 RNA polymerases; and control DNA.
  • non-ionic detergents one or more of Brij-35, NP-40, Triton X-100®, Tween® or a mixture of equal parts of these detergents for example
  • a dye such as xylene cyanol, Fuch
  • the dideoxynucleotide method of sequencing DNA is an enzymatic chain termination method which is detailed in Sa brook et al. , Molecular Cloning: A Laboratory Manual , second edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. Briefly, a short oligonucleotide primer is annealed to template DNA and DNA is synthesized enzymatically by extending the annealed primer with a DNA polymerase, usually Sequenase®, Tag DNA polymerase or Bst DNA polymerase.
  • a DNA polymerase usually Sequenase®, Tag DNA polymerase or Bst DNA polymerase.
  • a collection of randomly terminated fragments is generated by specific incorporation, four separate reactions of the four dideoxynucleotides (ddA, ddC, ddG and ddT) that prevent further extension of the DNA chains.
  • Conventional templates may be used in accordance with the present invention.
  • the templates include and are not limited to bacteriophage M13 vectors such as M13mpl8 or M13mpl9, phagemid vector which may be single or double stranded, plasmid vectors, and polymerase chain reaction (PCR) products which may be single or double stranded.
  • Primers, which anneal to vector sequences flanking the target DNA may be about 15 to about 29 nucleotides in length.
  • the length of the primer is determined by the nature of its DNA sequence as well as the template used.
  • the collection of randomly terminated DNA fragments are then separated according to size (length) by electrophoresis on a denaturing polyacrylamide gel and the sequence is determined by the ladder of fragments differing by a single base in length that are produced from the four dideoxynucleotide reactions.
  • “dried-down” refers to desiccation (removal of water) of DNA sequencing reagents and is not limited to a two stage vacuum drying such that in the first stage, the reagents are exposed to a pressure of about 125 Pascals (Pa) to about 375 Pa for about 10 minutes to about 15 minutes; preferably a pressure of about 125 Pa for about 10 minutes. In the second stage, the reagents are exposed to a pressure of about 25 Pa to about 50 Pa for about 20 minutes to about 30 minutes; preferably a pressure of about 25 Pa for about 30 minutes.
  • Pa Pascals
  • second stage the reagents are exposed to a pressure of about 25 Pa to about 50 Pa for about 20 minutes to about 30 minutes; preferably a pressure of about 25 Pa for about 30 minutes.
  • the preferred enzyme for the purposes of the present invention is Bst DNA polymerase, a thermostable DNA polymerase from Bacillus stearothermophilus .
  • the Bst DNA polymerase is dried down prior to use in the present invention.
  • About 0.7 units to about 1.3 units, preferably about 1.0 unit of the Bst DNA polymerase is used in a method of DNA sequencing.
  • the advantages of using a thermostable DNA polymerase for sequencing are that a microtiter plate with all the reagents can be heated during the denaturation step without loss of enzyme activity and that the extension and termination reactions occur at elevated temperatures so that -GC- rich templates can be sequenced with greater resolution.
  • the standard microtiter plates with the dried down sequencing reagents for sequencing with Bst DNA polymerase can be used with standard laboratory robots to automate DNA sequencing. All the components are present on one plate which may be manipulated in its entirety for all steps. Because the enzyme is thermally stable, the entire plate with all 96 wells can be heated as a single entity, and therefore it can easily be used with laboratory automation equipment designed to manipulate whole, intact 96-well plates.
  • the volumes that need to be pipetted are in the precise range provided by standard laboratory pipets and robots.
  • Bst DNA polymerase is more stable after drying down, in that the sequencing results generated with it are more consistent from batch to batch of plates prepared.
  • Experiments with plates containing Tag DNA polymerase which produced poor results demonstrated that the Tag enzyme could be replaced with new enzyme to produce good results.
  • Replacement of Tag DNA polymerase in half the wells of a plate generated good results with the new Tag DNA polymerase and poor results with the dried-down Tag DNA polymerase and therefore confirmed the variability of the Tag DNA polymerase.
  • the initial reaction volume during the denaturation (at about 65°C for about 5 minutes) and annealing (at about room temperature for about 5 minutes) steps of DNA sequencing may be about 45 ⁇ l (microliters) to about 50 ⁇ l; preferably about 47 ⁇ l. There is a loss of about 6 ⁇ l due to evaporation at the elevated temperature during denaturation and annealing.
  • the entire volume is successively transferred to the wells containing dNTPs (that may include a radiolabeled dNTP) and to the wells containing DNA polymerase.
  • the reaction is incubated at about 63°C to about 67°C, preferably about 65°C for about 4.5 minutes to about 5.5 minutes; preferably about 5 minutes. Due to the approximate 6 ⁇ l lost to evaporation, about 35 ⁇ l remains.
  • the reaction is then divided into 4 termination reactions by transferring about 6 ⁇ l to about 8 ⁇ l; preferably about 7 ⁇ l to each of the columns of wells containing the dideoxynucleotide reagent mixes.
  • the reactions are incubated for about 4.5 minutes to about 5.5 minutes, preferably about 5 minutes at about 63°C to about 67°C, preferably about 65°C, to incorporate the dideoxynucleotides.
  • Sequenase® a T7 bacteriophage DNA polymerase conventionally used in DNA sequencing, modified such that 3' - 5' exonuclease activity is eliminated. Sequenase® was dried down on microtiter plates and sequences were obtained ( Figure 1) . Sequenase®, however, is known to be labile and the results obtained with the dried enzyme demonstrated a detectable loss of activity in terms of decreased incorporation (as detected by decreased band intensity) or inability to continue extension. The loss of activity was compensated for by increasing the number of units of enzyme dried down per well ( Figure 1) . The loss of enzyme activity was attributed to denaturation of some of the enzyme molecules during the drying process.
  • Results from using plates with dried reagents together with non-dried enzyme were comparable to those of conventional sequencing reactions.
  • An inherent limitation of using Sequenase® for the dried down format is that the enzyme cannot be heated to the temperature used to denature the template. Therefore, an additional set of steps to denature the template is required and the plate cannot be heated and manipulated in its entirety. While these limitations make dried down Sequenase® unsuitable for automation, DNA sequencing using dried down Sequenase® by manual operation remains possible.
  • Tag DNA polymerase Another candidate enzyme for the drying method was Tag DNA polymerase from Thermus aguaticus .
  • Tag DNA polymerase is thermally stable and is also conventionally used for DNA sequencing. Initial experiments demonstrated that the well-known stability of Tag DNA polymerase appeared to produce results equal to and better than those obtained with Sequenase®. Tag DNA polymerase survived the drying down better than Sequenase® and generated sequences of good quality. Further experimentation revealed that there was variability in incorporation and extension achieved after drying. The instability in the Tag DNA polymerase was often the cause of variability.
  • Tag DNA polymerase is supplied in a buffer that contains dithiothreitol (DTT) . Therefore, the possibility that the enzyme was adversely affected by oxidation during the drying was investigated. Inclusion of additional DTT in the enzyme dilution buffer had no adverse effect. Enzyme lots stored (at -20°C) for about 7 days to about two months in the laboratory were more likely to yield poor results after drying. However, Tag DNA polymerase from the same tube still functioned well in the polymerase chain reaction (PCR) .
  • PCR polymerase chain reaction
  • DNA polymerase Bst from Bacillus stearothermophilus has properties that make it extremely suitable for DNA sequencing. It is extremely stable in solution as it may be incubated for 14 days at room temperature without loss of activity. Bst DNA polymerase generated excellent sequences after drying and yielded reproducible results.
  • Bst DNA polymerase was remarkably well suited to the dry-down method. Few experiments were necessary to define conditions for drying and sequencing that yielded reproducible results of excellent quality. In contrast to Tag DNA polymerase which appeared to be unaffected by additional DTT, Bst DNA polymerase was inhibited such that the reactions did not extend as far as reactions in the absence of additional DTT. The enzyme generated good quality sequences after drying, even when using as few as about 0.25 units of enzyme per reaction. It was remarkably robust as exemplified by generation of good quality sequences over a range of Mg 2+ concentrations as well as when plates were left out at room temperature overnight.
  • Drying reagents in microtiter plate wells allows a larger volume to be transferred to the well containing the dried down reagents or enzymes. Therefore, the dry-down method circumvents the volume limitation of robots and workstations and also reduces the probability of volume errors when multichannel pipets are used manually.
  • the DNA polymerase In order for the dry-down sequencing method to be successful, the DNA polymerase should withstand drying and reproducibly generate sequences of good quality.
  • Bst DNA polymerase meets the criteria and appears to be extremely well suited to the dry- down method; it may be dried down on microtiter plates and generates sequences of good quality. It is robust in the dried form; it is stable for more than a week at -20°C and at least overnight at room temperature.
  • the dried down Bst is useful in DNA sequencing methods; particularly the dideoxynucleotide methods; more particularly a multiwell format.
  • DNA sequencing reactions currently comprise two major steps: a single extension reaction in the absence of dideoxynucleotides that extends the primer and usually incorporates labeled DNTP, and four separate termination reactions during which dideoxynucleotides (one per reaction) are incorporated causing the termination of DNA synthesis.
  • the volume of the extension reaction is at least four times the volume of the termination reactions.
  • the template Prior to extension, the template is heat-denatured and annealed to the primer.
  • the dried down Bst DNA polymerase reaction can be divided into the following steps: Heat denaturation of the template, annealing of primer to the template, extension of the primer and four termination reactions.
  • primer, template DNA, water and buffer are added to each of the wells in a column of wells.
  • the starting volume is in the range of about 45 ⁇ l to about 50 ⁇ l, preferably about 47 ⁇ l.
  • concentrations of the components are as follows: control DNA template (M13mpl8) , 17 ng/ml; primer, 21.3 nM (21.3 fmol/ ⁇ l) ; MgCl 2 , 10.2 mM; Tris-HCl pH 8.8 at room temperature, 34.0 mM.
  • the plate is heated to between about 63°C to about 67°C, preferably to about 65°C, for about 4.5 minutes to about 5.5 minutes, preferably about 5 minutes.
  • the plate is transferred to about room temperature, about 20°C to about 23°C, preferably about 21°C.
  • concentrations of the reagents are about: control DNA, 19.5 ng/ ⁇ l; primer, 24.3 nM (24.3 fmol/ ⁇ l) ; MgCl 2 , 11.7 mM; Tris-HCl pH 8.0 at room temperature, 39.0 mM.
  • the entire volume is transferred to one or more wells to reconstitute dried down reagents that include Bst DNA polymerase.
  • the plate is incubated at about 63°C to about 67°C, preferably about 65°C, for about 4.5 minutes to about 5.5 minutes, preferably about 5 minutes. At the elevated temperature there is again loss of about 6 ⁇ l due to evaporation and as a result the volume is about 35 ⁇ l at the end of the 5 minute extension reaction.
  • concentrations of the reagent at the after extension are about as follows: MgCl 2 , 13.7 mM; EDTA, 0.029 mM; BSA, 0.029 ⁇ g/ml; xylene cyanol, 0.001%; non-ionic detergents, 0.009%, Bst DNA polymerase, 0.029 U/ ⁇ l; and Tris-HCl pH 8.8 at room temperature, 39.25 mM. Due to the nature of the reaction, the concentrations of dNTPS, primer and free template cannot be determined after extension.
  • the extension reaction volume is transferred to teach of four wells containing the dideoxynucleotide termination reagents.
  • the deoxy- and dideoxy- nucleotides are dried down in a buffer that, when resuspended in 7 ⁇ l, contributes: EDTA, 0.125 mM; bovine serum albumin, 0.125 ⁇ g/ml; xylene cyanol, 0.043%; non-ionic detergents, 0.37% and Tris-HCl pH 7.0 at room temperature, 1.25 mM.
  • dATP deoxynucleotide
  • dCTP deoxynucleotide
  • dGTP dGTP
  • dTTP deoxynucleotide
  • concentration of ddATP is about 200 ⁇ M to about 400 ⁇ M, preferably about 314 ⁇ M.
  • concentration of ddCTP is about 450 ⁇ M to about 850 ⁇ M, preferably about 650 ⁇ M.
  • the concentration of ddGTP is about 340 ⁇ M to about 630 ⁇ M, preferably about 485 ⁇ M.
  • the concentration of ddTTP is about 80 ⁇ M to about 150 ⁇ M, preferably about 114 ⁇ M.
  • the overall concentration of buffer reagents is about as follows: MgCl 2 , 13.7 ⁇ M; EDTA, 0.154 mM; bovine serum albumin, 0.154 ⁇ g/ml; xylene cyanol, 0.005%; non-ionic detergents, 0.046%; Tris-HCl pH 8.8 at room temperature, 47.25 mM.
  • the termination reactions are incubated at about 63°C to about 67°C, preferably about 65°C, for about
  • Reactions are terminated by the addition of about 6 ⁇ l to about 8 ⁇ l, preferably about 7 ⁇ l of STOP buffer consisting of 95% forma ide, 20 mM EDTA pH 8.0, 0.05% bromophenol blue and 0.05% xylene cyanol.
  • the final volume, with the addition of 7 ⁇ l of STOP buffer, is about 8 to 9 ⁇ l due to evaporation.
  • Reactions may also be terminated by cooling the plate, e.g., by placing the plate on ice or at 4°C, prior to the addition of STOP buffer.
  • EXAMPLE 1 General dry down procedure Several parameters were investigated to expedite the drying process while minimizing damage to the enzymes. These parameters included the effect of the magnitude of vacuum used to dry the reagents, the interval necessary to dry the reagents, and the effect of surface tension (adherence of reagents to the plates) .
  • the first attempts at drying the reagents simulated freeze-drying. Microtiter plates containing reagents were placed on dry ice. After freezing, the plates were placed in a vacuum chamber and vacuum of about 25 Pa for about 30 minutes was applied. The dried plates contained tiny flakes in the wells. The flakes were easily disturbed by air currents as well as electrostatic charges. To overcome these problems, drying was attempted without first freezing the reagents. The applied vacuum, of about 25 Pa to about 50 Pa, for about 20 to about 30 minutes continued to result in freezing with similar consequences. A two-stage drying procedure that avoided freezing of the reagents by ensuring that the initial vacuum was in the range of from about 60 to about 90 Pa (500 to 750 millitorr) for about 10 to about 15 minutes was adopted.
  • the concentration was raised to 0.075%.
  • Inclusion of non-ionic detergents produced a more uniform distribution of the reagents at the bottoms of the wells. Additionally, the reagents adhered tightly to the plate such that the flakes were difficult to disturb by air currents or electrostatic charge.
  • One consequence of including surfactants was the need to modify the two-stage dry-down such that the first stage served to de-gas the reagents to prevent bubbles rising above the wells.
  • the first- stage vacuum was held between about 125 Pa and about 375 Pa (1,000 to 3,000 millitorr) for about 10 minutes to about 15 minutes; more preferably about 125 Pa for about 10 minutes.
  • the second stage remained at about 25 Pa for about 20 minutes.
  • a second consequence of including surfactants was that bubbles were generated during the sequencing reactions when mixing and dissolving the dried reagents by pipetting the solution up and down rapidly. The bubbles had no detectable effect on the sequences. The inclusion of the surfactants, however, resulted in more rapid resuspension and dissolution of the reagents.
  • the buffer provided in the kit was diluted four-fold and xylene cyanol was added to give final concentrations of: 2.5 mM Tris-HCl pH 7.5, 1.25 mM DTT, 125 ⁇ g/ml BSA and 0.005% xylene cyanol.
  • Triton X-100®, and Tween-20® were mixed to prepare the 10% NID.
  • the 10X concentration buffer consisted of: 15 mM
  • the buffer was identical in composition to E-DD buffer except that the pH was 7.0.
  • the labeling solution was prepared.
  • the solution contained the non-radioactive deoxynucleotide triphosphates for the extension and labeling reaction.
  • the stock solutions of dCTP, dITP (Sequenase® and Tag DNA polymerase) , 7-deaza dGTP ⁇ Bst DNA polymerase) , and dTTP were diluted to 75 ⁇ M each in 0.1 mM EDTA and 10 mM Tris HCl (pH
  • Sequencing Templates Any template suitable for DNA sequencing may potentially be used. M13mpl8 was used as a control template to develop the system. Single-stranded DNA produced by asymmetrical PCR with primers specific for the particular PCR products has also been used. Termination Mixes
  • Labeling deoxynucleotide solution was used as a stock such that the concentration each of the deoxynucleotides was 13.7 ⁇ M for all four termination mixes.
  • the optimal ratio of i the dideoxynucleotide/deoxynucleotide (ddNTP/dNTP) for each base was determined empirically for Tag and Bst DNA polymerases.
  • the final concentrations of the deoxynucleotides and dideoxynucleotides used with Bst DNA polymerase were as shown in Table 1.
  • ddA( ⁇ M) 314.0 ddC 651.0 ddG 485.0 ddT 114.0 dNTPS ( ⁇ M) 13.5 13.5 13.5 13.5 13.5 dNTPS equal concentrations of dATP, dCTP, dGTP, dTTP. The concentration of each dNTP is equal to the amounts indicated in Table 1.
  • Typical plates were prepared in batches of six or multiples thereof.
  • a total of six solutions (enzyme in appropriate dilution buffer, Sequenase® in SED (Sequenase® dilution buffer) and Bst or Tag DNA polymerase in E-DD (Enzyme dry down buffer) , LDN and the four termination mixes AT, CT, GT, TT) were prepared and kept on ice.
  • the enzyme was diluted in IX E-DD buffer. To simplify the pipetting, all reagents were made up so that it was possible to use a constant volume of 7 ⁇ l to dispense the six different solutions.
  • the master mixes were dispensed into a single master plate that was kept on ice.
  • FIGURE 7 A well in a column of wells (see FIGURE 7) on the master plate received 200 ⁇ l of the appropriate solution.
  • the reagents in a column of wells were sufficient for three target plates. Empty target plates were placed on the bench, six at a time, at room temperature and the solutions were rapidly dispensed. After a batch of six plates were completed, the plates were stored at 4°C until the solutions were dispensed to all batches of plates.
  • Sequencing kits for Sequenase® Modified T7 DNA polymerase; Sequenase® Version 2.0
  • M13 universal primer (-40) and M13mpl8 template DNA were obtained from United States Biochemical Corporation, USB (Cleveland, OH) .
  • Acetylated BSA was obtained from Promega (Madison, WI) .
  • Glycerol and xylene cyanol were obtained from Sigma (St. Louis, MI) .
  • Falcon® flexible U-bottom PVC microtiter plates and lids were obtained from Thomas Scientific (Swedesboro, NJ) . Heating blocks with sculpted surfaces that match the U-bottom microtiter plates were obtained from USA Scientific Plastics (Ocala, FL) .
  • a Titertek multichannel pipet for the range from 5 to 50 ⁇ l was obtained from Flow Laboratories, ICN (Costa Mesa, CA) .
  • the 35 S-dATP (500 Ci/mmol) used as label was obtained from Du Pont NEN Research Products (Boston MA) .
  • the components of the Sequenase® kit (USB) were dried down on a PVC microtiter plate. Sequenase® for several reaction wells - about 0.25 ⁇ l (about 3.25 U) for each reaction well - was diluted with 1.75 ⁇ l SED (Sequenase enzyme dilution buffer) as recommended by the supplier.
  • the enzyme was further diluted by adding 5 ⁇ l water for each reaction well. Therefore, a volume of 7 ⁇ l of diluted Sequenase® was pipetted to the third column of a PVC microtiter plate.
  • a solution of the labeling mix (dNTPs) from the kit, DTT from the kit and S-dATP was prepared such that 1 ⁇ l of the labeling mix, 0.5 ⁇ l of the 35 S-dATP (20 nM, 500 Ci/mmol) and 1 ⁇ l of 0.1 M DTT per well was diluted with water to 7 ⁇ l per well. Seven ⁇ l of the labeling solution was pipetted to each well in column two of the PVC microtiter plate.
  • the four termination mixes from the kit were diluted with water to a volume of 7 ⁇ l per well.
  • 3.5 ⁇ l of the kit reagents were diluted with 3.5 ⁇ l of water.
  • the four termination mixes were dispensed to columns 4 - 7 of the microtiter plate. The plate was kept on ice during the time that the reagents were dispensed.
  • the reagents, labeling, solution, diluted enzyme, and termination mixes were dried down by the application of vacuum. As described, in initial experiment, the plate with reagents was frozen on dry ice prior to applying vacuum. In subsequent experiments, the plate was exposed to vacuum without freezing.
  • enzyme was dried down in four of the eight wells in a column.
  • enzyme in the conventional format (in solution) was added to the wells that did not have any enzyme dried down.
  • Sequenase® appeared to be more stable when the drying was done in a manner similar to freeze-drying, i.e., rapid application of a vacuum with a pressure less than 25 Pa (about 200 millitorr) .
  • a vacuum with a pressure less than 25 Pa about 200 millitorr
  • the reagents dried to the flaky deposits described above. Drying under a vacuum with a pressure greater than 25 Pa was detrimental to the enzyme and yielded more variable sequences.
  • the labeling and termination mixes were dried down in the absence of the enzyme and the enzyme was dried subsequently under its optimal conditions, the results were still variable.
  • a buffering component that stabilizes the enzyme against loss of activity, such as bovine serum albumin (BSA) appeared to stabilize the enzyme although variability was not completely eliminated.
  • BSA bovine serum albumin
  • the template was denatured at about 65°C for 5 minutes by placing the U-bottom microtiter plate on a sculpted heat block. The plate was transferred to a block at 45°C and the primer was allowed to anneal for 5 minutes. The entire remaining volume of about 35 ⁇ l per well was successively transferred with a multichannel pipet to the wells containing the dried labeling mix and the wells contained the dried Tag DNA polymerase. Reagents were resuspended by rapid aspiration and dispensing with the multichannel pipet. The plate was incubated for 5 minutes at 45°C for the labeling/extension reaction. For each reaction, 7 ⁇ l of the reaction was pipetted to teach of the wells with the dried termination mixes using a multichannel pipet.
  • M13 universal primer (-40) and M13mpl8 template DNA were obtained from United States Biochemical Corporation, USB (Cleveland, OH) .
  • Bst DNA polymerase was obtained from Bio-Rad Laboratories (New York, NY) .
  • Deoxynucleotides and dideoxynucleotides were obtained as solutions from Boehringer Mannheim Biochemicals (Indianapolis, IN) .
  • Acetylated BSA was obtained from Promega (Madison, WI) .
  • Tris buffer, DTT, EDTA, MgCl 2 , gelatin and xylene cyanol were obtained from Sigma (St.
  • Heating blocks with sculpted surfaces that match the U-bottom microtiter plates were obtained from USA Scientific Plastics (Ocala, FL) .
  • a Titertek multichannel pipet for the range from 5 to 50 ⁇ l was obtained from Flow Laboratories, ICN (Costa Mesa, CA) .
  • the 35 S-dATP (500 Ci/mmol) used as label was obtained from Du Pont NEN Research Products (Boston, MA) .
  • the 41 ⁇ l that remained per well after evaporation was successively transferred with a multichannel pipet to the wells containing labeling mix and dried down enzyme.
  • Reagents were resuspended by repetitive, rapid aspiration and dispensing.
  • the plate was moved to a sculpted heat block at 65°C and incubated for 5 minutes.
  • 7 ⁇ l of the remaining 35 ⁇ l was transferred to the wells containing the dried termination reagents.
  • the termination reactions were carried out at 65°C for 5 minutes. Reactions were stopped by the addition of 7 ⁇ l of STOP buffer.
  • the enzyme generated good quality sequences after drying, even when as few as 0.25 units of the enzyme were used per reaction ( Figure 4) .
  • sequencing reagents for use in the present invention include and are not limited to pH buffers, such as 1.5 M Tris-HCl, 0.15 mM EDTA; stabilizers such as 0.15 mg/ml bovine serum albumin or acetylated bovine serum albumin; 0.075% non-ionic detergents (NID)/surfactants including one or more of Brij-35, NP-40, Triton X-100®, Tween 20® or a mixture of equal parts of these detergents in 1.5 mM Tris-HCl pH 8.0 at room temperature; 0.005% of a dye such as xylene cyanol, Fuchsin S, brilliant green, bromophenol blue, methyl blue, methylene blue or toluidine blue; deoxynucleotides in a buffer suitable for drying down, such as 0.15 mM EDTA, 0.15 mg/ml acetylated BSA, 0.075% non-ionic detergents such as those indicated above in 1.5 mM Tris
  • reagents include and are not limited to, radiolabeled deoxynucleotides, primers for DNA sequencing (labeled or not labeled) , and fluorescently-labeled dideoxynucleotides template DNA for control reactions.

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Abstract

Procédé de séquençage d'ADN consistant à ajouter une amorce et une matrice d'acide nucléique à une dose efficace d'une polymérase, cette dernière étant la Bst polymérase à l'état asséché. La présente invention se rapporte également à un procédé d'utilisation de la polymérase dans le cadre d'un procédé de séquençage d'ADN, ladite polymérase étant la Bst polymérase à l'état asséché.
PCT/US1994/000562 1993-01-12 1994-01-12 SEQUENÇAGE D'ADN A L'AIDE DE Bst POLYMERASE WO1994016107A1 (fr)

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Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995027067A1 (fr) * 1994-04-01 1995-10-12 Gen-Probe Incorporated POLYMERASE PURIFIEE D'ADN DE $i(BACILLUS STEAROTHERMOPHILUS)
EP0726310A1 (fr) * 1995-02-10 1996-08-14 Gen-Probe Incorporated Compositions d'enzyme stabilisées pour l'amplification d'acides nucléiques
EP0757100A1 (fr) * 1995-08-02 1997-02-05 New England Biolabs, Inc. Surexpression et purification d'ADN polymérase thermostabile en forme de tronque par une protéine de fusion
WO1997011197A1 (fr) * 1995-09-20 1997-03-27 E.I. Du Pont De Nemours And Company Controles de la pcr
US5876992A (en) * 1996-07-03 1999-03-02 Molecular Biology Resources, Inc. Method and formulation for stabilization of enzymes
US6291164B1 (en) 1996-11-22 2001-09-18 Invitrogen Corporation Methods for preventing inhibition of nucleic acid synthesis by pyrophosphate
US6391622B1 (en) 1997-04-04 2002-05-21 Caliper Technologies Corp. Closed-loop biochemical analyzers
US6403338B1 (en) 1997-04-04 2002-06-11 Mountain View Microfluidic systems and methods of genotyping
US6436677B1 (en) 2000-03-02 2002-08-20 Promega Corporation Method of reverse transcription
EP1115848A4 (fr) * 1998-09-21 2002-08-21 Shanghai Inst Biochemistry Adn polymerase pouvant reduire la discrimination selective de l'innate vis a vis de didesoxynucleotides marques par un colorant fluorescent
US6632645B1 (en) 2000-03-02 2003-10-14 Promega Corporation Thermophilic DNA polymerases from Thermoactinomyces vulgaris
EP2021508A4 (fr) * 2006-05-23 2009-09-30 Molecular Detection Inc Kits stables à la température ambiante pour des diagnostics moléculaires
US7611871B2 (en) 2000-09-05 2009-11-03 Biochip Technologies Gmbh Method for the specific determination of DNA sequences by means of parallel amplification
WO2011106629A3 (fr) * 2010-02-26 2012-01-19 Life Technologies Corporation Protéines modifiées et leurs procédés de fabrication et d'utilisation
US9714915B2 (en) 2010-02-26 2017-07-25 Life Technologies Corporation Modified proteins and methods of making and using same
US20190309796A1 (en) * 2016-06-27 2019-10-10 Ks Gleitlager Gmbh Plain Bearing Bush
US11866740B2 (en) 2015-10-01 2024-01-09 Life Technologies Corporation Polymerase compositions and kits, and methods of using and making the same

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Non-Patent Citations (2)

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Title
BIOTECHNIQUES, Volume 11, No. 1, issued 1991, MEAD et al., "Bst DNA Polymerase Permits Rapid Sequence Analysis from Nanogram Amounts of Template", pages 76-87. *
BIOTECHNIQUES, Volume 12, No. 2, issued 1992, DALE, "Direct Microtiter Plate Sequencing of PCR-Amplified M13 Clones from Plaques Using Dried Reagents", pages 195-197. *

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WO1995027067A1 (fr) * 1994-04-01 1995-10-12 Gen-Probe Incorporated POLYMERASE PURIFIEE D'ADN DE $i(BACILLUS STEAROTHERMOPHILUS)
EP0699760A1 (fr) * 1994-04-01 1996-03-06 Gen-Probe Incorporated ADN polymérase de bacillus stearothermophilus purifiée
US6100078A (en) * 1994-04-01 2000-08-08 Gen-Probe Incorporated Purified DNA polymerase from bacillus stearothermophilus ATCC 12980
US6066483A (en) * 1994-04-01 2000-05-23 Gen-Probe Incorporated Purified DNA polymerase from Bacillus stearothermophilus
US5874282A (en) * 1994-04-01 1999-02-23 Gen-Probe Incorporated Purified DNA polymerase from Bacillus stearothermophilus ATTC 12980
US5834254A (en) * 1995-02-10 1998-11-10 Gen-Probe Incorporated Stabilized enzyme compositions for nucleic acid amplification
US5614387A (en) * 1995-02-10 1997-03-25 Gen-Probe Incorporated Method of making stabilized enzyme compositions for nucleic acid amplification
WO1996024664A1 (fr) * 1995-02-10 1996-08-15 Gen-Probe Incorporated Compositions d'enzymes stabilisees pour amplification d'acide nucleique
EP0726310A1 (fr) * 1995-02-10 1996-08-14 Gen-Probe Incorporated Compositions d'enzyme stabilisées pour l'amplification d'acides nucléiques
US5814506A (en) * 1995-08-02 1998-09-29 New England Biolabs, Inc. Over-expression and purification of a truncated thermostable DNA polymerase by protein fusion
EP0757100A1 (fr) * 1995-08-02 1997-02-05 New England Biolabs, Inc. Surexpression et purification d'ADN polymérase thermostabile en forme de tronque par une protéine de fusion
WO1997011197A1 (fr) * 1995-09-20 1997-03-27 E.I. Du Pont De Nemours And Company Controles de la pcr
US5876992A (en) * 1996-07-03 1999-03-02 Molecular Biology Resources, Inc. Method and formulation for stabilization of enzymes
US6294365B1 (en) 1996-07-03 2001-09-25 Molecular Biology Resources, Inc. Method and formulation for stabilization of enzymes
US6291164B1 (en) 1996-11-22 2001-09-18 Invitrogen Corporation Methods for preventing inhibition of nucleic acid synthesis by pyrophosphate
US7344835B2 (en) 1996-11-22 2008-03-18 Invitrogen Corporation Methods for preventing inhibition of nucleic acid synthesis by pyrophosphate
US6764839B2 (en) 1996-11-22 2004-07-20 Invitrogen Corporation Methods for preventing inhibition of nucleic acid synthesis by pyrophosphate
US6440722B1 (en) 1997-04-04 2002-08-27 Caliper Technologies Corp. Microfluidic devices and methods for optimizing reactions
US7238323B2 (en) 1997-04-04 2007-07-03 Caliper Life Sciences, Inc. Microfluidic sequencing systems
US6391622B1 (en) 1997-04-04 2002-05-21 Caliper Technologies Corp. Closed-loop biochemical analyzers
US6406893B1 (en) 1997-04-04 2002-06-18 Caliper Technologies Corp. Microfluidic methods for non-thermal nucleic acid manipulations
US6444461B1 (en) 1997-04-04 2002-09-03 Caliper Technologies Corp. Microfluidic devices and methods for separation
US6849411B2 (en) 1997-04-04 2005-02-01 Caliper Life Sciences, Inc. Microfluidic sequencing methods
US6670133B2 (en) 1997-04-04 2003-12-30 Caliper Technologies Corp. Microfluidic device for sequencing by hybridization
US6403338B1 (en) 1997-04-04 2002-06-11 Mountain View Microfluidic systems and methods of genotyping
EP1115848A4 (fr) * 1998-09-21 2002-08-21 Shanghai Inst Biochemistry Adn polymerase pouvant reduire la discrimination selective de l'innate vis a vis de didesoxynucleotides marques par un colorant fluorescent
US7094539B2 (en) 2000-03-02 2006-08-22 Promega Corporation Bacillus stearothermophilus reverse transcription compositions and kits
US7214522B2 (en) 2000-03-02 2007-05-08 Promega Corporation Thermophilic DNA polymerases from Thermoactinomyces vulgaris
US6436677B1 (en) 2000-03-02 2002-08-20 Promega Corporation Method of reverse transcription
US7504220B2 (en) 2000-03-02 2009-03-17 Promega Corporation Method of reverse transcription
US6632645B1 (en) 2000-03-02 2003-10-14 Promega Corporation Thermophilic DNA polymerases from Thermoactinomyces vulgaris
US7611871B2 (en) 2000-09-05 2009-11-03 Biochip Technologies Gmbh Method for the specific determination of DNA sequences by means of parallel amplification
EP2021508A4 (fr) * 2006-05-23 2009-09-30 Molecular Detection Inc Kits stables à la température ambiante pour des diagnostics moléculaires
WO2011106629A3 (fr) * 2010-02-26 2012-01-19 Life Technologies Corporation Protéines modifiées et leurs procédés de fabrication et d'utilisation
AU2011220536B2 (en) * 2010-02-26 2012-05-24 Life Technologies Corporation Modified proteins and methods of making and using same
US9714915B2 (en) 2010-02-26 2017-07-25 Life Technologies Corporation Modified proteins and methods of making and using same
US10059987B2 (en) 2010-02-26 2018-08-28 Life Technologies Corporation Modified proteins and methods of making and using same
US11866740B2 (en) 2015-10-01 2024-01-09 Life Technologies Corporation Polymerase compositions and kits, and methods of using and making the same
US20190309796A1 (en) * 2016-06-27 2019-10-10 Ks Gleitlager Gmbh Plain Bearing Bush
US10641326B2 (en) * 2016-06-27 2020-05-05 Ks Gleitlager Gmbh Plain bearing bush

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