WO2002010446A2 - Procede permettant de determiner une sequence nucleotidique partielle d'une molecule d'acide nucleique - Google Patents

Procede permettant de determiner une sequence nucleotidique partielle d'une molecule d'acide nucleique Download PDF

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WO2002010446A2
WO2002010446A2 PCT/EP2001/008671 EP0108671W WO0210446A2 WO 2002010446 A2 WO2002010446 A2 WO 2002010446A2 EP 0108671 W EP0108671 W EP 0108671W WO 0210446 A2 WO0210446 A2 WO 0210446A2
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nucleic acid
primer
acid molecule
nucleotide sequence
polymerase
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PCT/EP2001/008671
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German (de)
English (en)
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WO2002010446A3 (fr
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Eckhard Nordhoff
Christine Luebbert
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MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V.
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Priority to AU2001282031A priority Critical patent/AU2001282031A1/en
Priority to EP01960566A priority patent/EP1356098A2/fr
Publication of WO2002010446A2 publication Critical patent/WO2002010446A2/fr
Publication of WO2002010446A3 publication Critical patent/WO2002010446A3/fr

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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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6853—Nucleic acid amplification reactions using modified primers or templates
    • 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
    • C12Q1/6872—Methods for sequencing involving mass spectrometry

Definitions

  • the present invention relates to a method for determining a partial nucleotide sequence of a nucleic acid molecule, comprising the steps: (a) incubation of a primer which hybridizes to the nucleic acid molecule with multiple copies of the nucleic acid molecule in the presence of a polymerase and a mixture of deoxyribonucleotides at an annealing temperature which is between 5 ° C and 40 ° C above the theoretical melting temperature of the primer; (b) determination of the molecular masses of successive nucleic acid fragments, which were generated by elongation of the primer in step (a), by means of mass spectrometry; and (c) determining the nucleotide sequence by determining the molecular weight increases between the successive size nucleic acid fragments.
  • the present invention further relates to a method for determining a partial nucleotide sequence of a nucleic acid molecule, comprising the steps of: (a) incubating a primer which hybridizes to the nucleic acid molecule with multiple copies of the nucleic acid molecule in the presence of a polymerase and a mixture of deoxyribonucleotides; (b) heat denaturation of the complexes consisting of nucleic acid molecule and extended primer and / or nucleic acid molecule, extended primer and polymerase; (c) optionally repeating steps (a) and (b) one or more times; (d) determination of the molecular masses of successive nucleic acid fragments, which were generated by elongation of the primer in step (a), by means of mass spectrometry; and (e) determining the nucleotide sequence by determining a molecular weight increase between the successive size nucleic acid fragments.
  • the invention also relates to a method for determining a partial nucleotide sequence of a nucleic acid molecule, comprising the steps of: (a) incubating a primer that hybridizes to the nucleic acid molecule with multiple copies of the nucleic acid molecule in the presence of a polymerase and a mixture of deoxyribonucleotides at an annealing temperature that is between 5 ° C and 40 ° C above the theoretical melting temperature of the primer, with each deoxyribonucleotide labeled with a different dye; (b) electrophoretic or chromatographic separation of the
  • the invention relates to a method for determining a partial nucleotide sequence of a nucleic acid molecule, comprising the steps: (a) incubation of a primer which hybridizes to the nucleic acid molecule with multiple copies of the nucleic acid molecule in the presence of a polymerase and a mixture of deoxyribonucleotides, each deoxyribonucleotide with a different dye is marked; (b) heat denaturation of the complexes consisting of nucleic acid molecule and extended primer and / or nucleic acid molecule extended primer and polymerase; (c) optionally repeating steps (a) and (b) one or more times; (d) electrophoretic or chromatographic separation of the nucleic acid fragments generated by elongation of the primer in step (a); and (e) determination of the nucleotide sequence by determination of the dye (s) with which nucleic acid fragments successively in size were marked by incorporation of the corresponding deoxyribonucleo
  • ddNTPs dideoxynucleoside triphosphates
  • populations of DNA molecules are formed whose elongation terminates statistically at positions that correspond to each complementary C, T, A or G of the template strand. If the populations of DNA molecules are marked differentially (for example, by using ddNTP-specific fluorescent dyes), the enzymatic reaction can be carried out in a reaction mixture which contains all four different ddNTPs. If the populations of DNA molecules are indistinguishably marked, the enzymatic reaction must be carried out in four different reaction batches, each containing one of the four different ddNTPs. The populations of labeled DNA molecules can finally be separated using polyacrylamide gel electrophoresis, for example, and the sequence of the DNA template can be determined.
  • ddNTPs ddGTP, ddATP, ddTTP, ddCTP
  • PCR polymerase chain reaction
  • mass spectrometry mass spectrometry
  • the base-specific termination of the elongation reaction through the incorporation of modified stop nucleotides, usually ddNTPs, as practiced with the Sanger sequencing method and all related sequencing techniques, has a number of disadvantages. For example, for optimal results it is necessary to carefully match the ratio of dNTPs and stop nucleotides. If the DNA template to be sequenced was amplified in a previous PCR reaction, it is therefore necessary, as far as possible, to remove dNTPs that were not used in the PCR reaction before sequencing. With a large number of samples to be sequenced, this step is time consuming and costly. Another disadvantage is that only DNA polymerases can be used, the stop nucleotides as the substrate accept and install correctly. Finally, the commercially available stop nucleotides are significantly more expensive than unmodified nucleotides.
  • the technical problem underlying the present invention was to provide a simpler, faster and less expensive method for sequencing nucleic acid molecules.
  • the present invention thus relates to a method for determining a partial nucleotide sequence of a nucleic acid molecule, comprising the steps: Temperature that is between 5 ° C and 40 ° C above the theoretical melting temperature of the primer; (b) determination of the molecular masses of successive nucleic acid fragments, which were generated by elongation of the primer in step (a), by means of mass spectrometry; and (c) determining the nucleotide sequence by determining the molecular weight increases between the successive size nucleic acid fragments.
  • partial nucleotide sequence means that only that part of the sequence of a nucleic acid molecule that does not hybridize to the primer is determined. If the nucleic acid molecule has an excess of length of preferably up to 25 nucleotides that does not hybridize with the primer and the primer hybridizes flush at the 3 'end of the nucleic acid molecule, the sequence of the entire part that does not hybridize with the primer can be determined.
  • nucleic acid molecules which have a total length of preferably less than 50 nucleotides and the primers have a total length of preferably not more than 25 nucleotides
  • the sequence hybridizing to the primer (which was either already known and served as the basis for the synthesis of the primer sequence) can be added has or the complete Hybridization of the primer can be derived from the primer sequence as a complementary sequence), the total sequence of the nucleic acid molecule can be determined.
  • these embodiments also belong to the invention.
  • the primer is preferably incubated under stringent hybridization conditions which permit hybridization of the primer only at the region of the nucleic acid molecule which is complementary in its sequence to the sequence of the primer.
  • stringent hybridization conditions which permit hybridization of the primer only at the region of the nucleic acid molecule which is complementary in its sequence to the sequence of the primer.
  • non-stringent hybridization conditions which allow hybridization of the primer to a region of the nucleic acid molecule whose sequence at one or more positions is not complementary to the sequence of the primer. This area preferably has no more than three “mismatches”.
  • Stringent or non-stringent hybridization conditions are known to the person skilled in the art and can easily be determined depending on, for example, the length of the primer to be used (see, for example, Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd edition, CSH Press, Cold Spring Harbor, New York (1989); Ausubel et al., Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, New York (1989); or Higgins and Harnes, Nucleic Acid Hybridization, A Practical Approach, EAL Press Oxford, Washington DC (1985)).
  • Stringent hybridization conditions are, for example, hybridization in 6xSSC and 0.1% SDS at 65 ° C or in 50% formamide and 4xSSC at 42 ° C and subsequent washing in 0.1xSSC and 0.1% SDS at 65 ° C.
  • Non-stringent hybridization conditions are, for example, hybridization and subsequent washing in 4xSSC and 1% SDS at 50 ° C.
  • the stringency can also be regulated exclusively via the temperature. In this case, stringent hybridization conditions are usually achieved by selecting the hybridization temperature higher than the melting temperature of the nucleic acid molecules to be hybridized.
  • At least 6 x 10 8 copies, more preferably at least 3 x 10 10 copies and most preferably at least 3 x 10 11 copies of the nucleic acid molecule to be sequenced are preferably used to carry out the method according to the invention.
  • the copies of the to be sequenced Nucleic acid molecules are identical at least in the area of the nucleic acid sequence to be sequenced and the primer binding site. They are preferably identical copies of the nucleic acid molecule to be sequenced.
  • the term “mixture of deoxyribonucleotides” means that only nucleotides are used in step (a), the incorporation of which allows a further elongation of the primer in the 5'-3 'direction by the polymerase activity. In other words, this mixture contains no nucleotides, such as, for example, dideoxyribonucleotides, the incorporation of which would prevent further elongation of the primer due to the missing 3'-OH group.
  • Suitable nucleotides are thus, for example, the deoxyribonucleotides dGTP, dATP, dTTP and dCTP or derivatives thereof.
  • Suitable dNTP derivatives are, in particular, those which have improved ion stability, such as, for example, 7-deazadATP and -dGTP (F. Kirpekar et al., Rapid Commun. Mass Spectrom. 9 (1995) 525. K. Schneider and BT Chait , Nucleic Acids Res. 23 (1995) 1570) or 2'-F-dNTP (W. Tang, L. Zhu, LM. Smith, Anal. Chem. 69 (1997) 302).
  • the theoretical melting temperature of any primer can be readily determined by the person skilled in the art (see, for example, Sambrook et al., Loc. Cit.).
  • the molecular masses of the nucleic acid fragments generated in step (a) are determined by mass spectrometry and then the molecular mass increases between the primer used and the nucleic acid fragment that corresponds to the primer extended by one nucleotide, the latter nucleic acid fragment and the nucleic acid fragment that corresponds to the one extended by two nucleotides etc. determined.
  • nucleotides can be determined by determining the increase in molecular weight which differ in size from two nucleic acid fragments, can be determined simply and unambiguously, and thus the associated sequence of the nucleic acid molecule due to the complementarity of double-stranded nucleic acid molecules.
  • the increases in molecular mass can be derived in a simple manner from the molecular masses of the generated nucleic acid fragments determined by mass spectrometry.
  • the specific molecular masses of successive nucleic acid fragments that is to say nucleic acid fragments which differ from one another only by the incorporation of an additional nucleotide, are subtracted from one another. These values are then compared with the molecular mass increases expected for the incorporation of nucleotides A, C, G or T.
  • the assignment of the built-in nucleotide takes place within maximum deviation tolerances, for example, +/- 4 Da.
  • the smallest difference between the four possible molecular mass increases (+ A, + C, + G or + T) is nominally 9 Da and concerns the incorporation of A (+313 Da) versus T (304 Da). This is sufficiently taken into account by a maximum deviation tolerance of +/- 4 Da.
  • nucleic acid fragments resulting from the incorporation of one of the four possible nucleotides by addition of the associated known molecular mass increases (nominal: A: +313 Da, C: +289 Da, T: +304 Da, G: +329 Da ) be calculated.
  • the four possible molecular masses calculated in this way are then compared with the actually determined value.
  • nucleotide The assignment of which nucleotide was incorporated is carried out within predefined deviation tolerances, eg +/- 4 Da.
  • the four possible molecular masses of the nucleic acid fragments extended by a further nucleotide are then calculated by means of addition and compared with the associated experimental value.
  • the molecular masses of nucleic acid molecules can be precisely calculated in a simple manner known to the person skilled in the art from the known atomic masses of the various chemical elements by means of addition. This calculation takes into account the chemical composition of the nucleotide monomers (i.e.
  • the monoisotopic or average atomic masses can be used for these calculations.
  • the monoisotopic molecular mass is calculated, which is used for comparison if the isotope of the nucleic acid molecules to be analyzed can be separated. The high instrumental resolution required is provided by modern mass spectrometers.
  • 3a demonstrates this using an example of a set of nucleic acid fragments generated according to the invention.
  • the molecular mass increases for the nucleic acid fragments were determined to within 0.1 Da. If the mass spectrometer does not succeed in separating the isotopy of nucleic acid molecules, for example because they are too large, average atomic masses are used for the calculation and thus average molecular masses are calculated.
  • the method according to the invention advantageously allows nucleic acid molecules to be sequenced without using stop nucleotides, which lead to base-specific termination of the elongation reaction.
  • the method according to the invention is based on the surprising finding that oligonucleotides hybridize at complementary sequences of larger nucleic acid molecules at a temperature which is well above their melting temperature for a sufficient time to be extended by one or more nucleotides in the presence of a polymerase and dNTPs the double strand formed is thermally denatured again.
  • the nucleic acid fragments arise in the process according to the invention in that the attachment of the primer is short-lived at the high annealing temperature and the complex consisting of nucleic acid molecule (template), primer and enzyme is thermally unstable.
  • formed synthesis complexes disintegrate after a short time and leave a primer that has only been extended by one or a few nucleotide (s).
  • nucleic acid molecules no longer have to be purified before sequencing in order to remove nucleotide contamination, for example by a previous PCR amplification.
  • the method according to the invention permits automation of the method steps, which further accelerates the implementation. It is also conceivable to use the method according to the invention for "multiplexing", ie for the simultaneous determination of different partial nucleotide sequences within a nucleic acid molecule.
  • the specific primers are marked differently, so that the associated nucleic acid fragments resulting from the elongation of the primers can be separated, for example, by reversible affinity binding (solid phase purification). These can then be analyzed separately from one another by mass spectrometry.
  • primers can be used whose molecular mass is changed by an additional chemical group (mass tag, eg half the mass of one of the four nucleotides) so that the sequencing ladder in the mass spectrum can be clearly separated for the incorporation of one or a few nucleotides.
  • mass tag eg half the mass of one of the four nucleotides
  • Such techniques or the specific "mass tagging" of nucleic acids are known in the prior art (for example P. Ross et al., High level of multiplex genotyping by MALDI-TOF mass spectrometry, Nature Biotechnology 16 (1998) 1347, WO 99/29898 , or WO 99/29897).
  • the method according to the invention is particularly suitable for the sequencing of short nucleic acid sections of up to 25 nucleotides, preferably of up to 15 nucleotides, more preferably of up to 10 nucleotides and most preferably of up to 7 or 5 nucleotides.
  • the method according to the invention is therefore particularly suitable for the detection of single nucleotide polymorphisms (single nucleotide polymorphisms, SNPs). Examples 2 and 3 and the associated Figures 2 and 3 also document possible applications and characteristics.
  • the nucleic acid molecule to be sequenced can of course also be, for example, a cDNA, a vector, a plasmid, a cosmid, an artificial bacterial chromosome (BAC), artificial yeast chromosome (BAC) Chromosomes, YAC) or natural genomic DNA or act as a fragment thereof.
  • BAC bacterial chromosome
  • BAC yeast chromosome
  • YAC natural genomic DNA or act as a fragment thereof.
  • foreign DNA segments for example cDNA copies of isolated messenger RNA (English: messenger RNA, mRNA) or fragments of genomic DNA, are routinely used in cloning techniques
  • Vectors, plasmids or cosmids installed and then introduced into guest cells, for example E. coli or yeast cells.
  • the complete sequence of the externally introduced nucleic acid can thus be determined by determining the partial nucleotide sequence, for example of a vector.
  • the sequencing technique according to the invention enables partial and rapid sequence information (eg terminal partial nucleotide sequences) of the DNA sections incorporated in the different clones to be generated quickly and efficiently. This information can then be used to identify clones by means of sequence comparison or to make statements about their quality.
  • sequence information comprising a few nucleotides is already sufficient to answer this question.
  • the method according to the invention comprises the following steps between step (a) and (b): (aa) heat denaturation of the complex consisting of nucleic acid molecule and extended primer; and (ab) optionally repeating steps (a) and (aa) one or more times.
  • the present invention also relates to a method for determining a partial nucleotide sequence of a nucleic acid molecule, comprising the steps of: (a) incubating a primer which hybridizes to the nucleic acid molecule with multiple copies of the nucleic acid molecule in the presence of a polymerase and a mixture of deoxyribonucleotides; (b) heat denaturation of the complexes consisting of nucleic acid molecule and extended primer and / or nucleic acid molecule, extended primer and polymerase; (c) optionally repeating steps (a) and (b) one or more times; (d) determining the molecular masses of successive nucleic acid fragments, generated by elongation of the primer in step (a) by means of mass spectrometry; and (e) determining the nucleotide sequence by determining the molecular weight increases between the successive size nucleic acid fragments.
  • nucleic acid fragments can also be generated by elongation of the primer of different lengths in that after the primer has hybridized to the nucleic acid molecule to be sequenced, the annealing temperature is increased rapidly and without interruption so that it the complexes consisting of nucleic acid molecule and extended primer and / or nucleic acid molecule, extended primer and polymerase are denatured.
  • the temperature is raised to above 80 ° C, more preferably to above 90 ° C and most preferably to above 94 ° C.
  • the annealing temperature and duration in step (a) is of minor importance in this embodiment.
  • the annealing temperature is preferably below the optimum temperature of the polymerase used, so that there is negligible elongation of the primer during step (a).
  • Hybridization preferably takes place for 1 to 10 seconds, more preferably for 1 to 5 seconds and most preferably for 1 second.
  • One skilled in the art can readily determine the optimal annealing temperature and duration for a given primer.
  • the nucleic acid fragments are purified / isolated before the mass spectrometric analysis.
  • sequencing products can be cleaned and concentrated very quickly and cost-effectively for mass spectrometric analysis by the use of ZipTips (P10 pipette tips with a small reverse phase column integrated at the end) sold by Millipore Corporation, Bedford, MA, USA, and the associated purification protocols.
  • ZipTips P10 pipette tips with a small reverse phase column integrated at the end
  • mass spectrometry is matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS) or electrospray ionization mass spectrometry (ESI-MS).
  • MALDI-TOF-MS matrix-assisted laser desorption / ionization time-of-flight mass spectrometry
  • ESI-MS electrospray ionization mass spectrometry
  • the present invention relates to a method for determining a partial nucleotide sequence of a nucleic acid molecule, comprising the steps: (a) incubation of a primer which hybridizes to the nucleic acid molecule with multiple copies of the nucleic acid molecule in the presence of a polymerase and a mixture of deoxyribonucleotides an annealing temperature which is between 5 ° C and 40 ° C above the theoretical melting temperature of the primer, each deoxyribonucleotide being labeled with a different dye; (b) electrophoretic or chromatographic separation of the primer to form a primer.
  • Electrophoretic or chromatographic methods for the separation of nucleic acid fragments are known to the person skilled in the art and include, for example, gel electrophoretic, capillary electrophoretic, ion exchange chromatographic or reverse phase chromatographic methods.
  • the present invention also relates to a method for determining a partial nucleotide sequence of a nucleic acid molecule, comprising the steps: (a) incubating a primer that hybridizes to the nucleic acid molecule with multiple copies of the nucleic acid molecule in the presence of a polymerase and a mixture of deoxyribonucleotides, each deoxyribonucleotide being labeled with a different dye; (b) heat denaturation of the complexes consisting of nucleic acid molecule and extended primer and / or nucleic acid molecule, extended primer and polymerase; (c) optionally repeating steps (a) and (b) one or more times; (d) electrophoretic or chromatographic separation of the nucleic acid fragments generated by elongation of the primer in step (a); and (e) determination of the nucleotide sequence by determination of the dye (s) with which nucleic acid fragments successively in size were marked by incorporation of the corresponding deoxyribon
  • nucleic acid sections mentioned above apply.
  • 1 or 2 nucleotides are preferably identified from the sequence or their sequence is established.
  • the dyes are fluorescent dyes, the fluorescence being excited by means of a laser.
  • the annealing temperature is between 10 ° C., preferably 15 ° C. and 20 ° C., preferably 30 ° C., above the theoretical melting temperature of the primer.
  • steps (a) and (b) are repeated between 2 and 40 times.
  • steps (a) and (b) are repeated between 5 and 20 times.
  • step (b) is carried out by means of microwaves or infrared light irradiation or in a thermal cycler.
  • an ultrasound treatment is carried out between steps (a) and (b).
  • the polymerase is a thermostable polymerase.
  • Thermostable polymerases are known to the person skilled in the art and include, for example, the thermostable Taq DNA polymerase which has been isolated from the thermophilic microorganism Thermus aquaticus.
  • Other examples are the naturally occurring thermostable DNA polymerases Hot Tub TM, Pyrostase TM, Pfu, Pwo, Tbr, Tfl and the genetically engineered thermostable polymerases Tli, Amplitaq®, Thermo Sequenase TM, Vent TM, Deep Vent TM, Tth and UlTma TM ,
  • the nucleic acid molecule is double-stranded and is denatured before step (a).
  • nucleic acid molecule is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N
  • DNA polymerase is used.
  • the primer is 10 to 15 nucleotides long.
  • Another advantage of the method according to the invention is that short primers can be used for sequencing nucleic acid molecules. This not only enables very stringent conditions in the hybridization of the primer, which leads to incorrect attachment of the primer can be excluded, but also reduces due to the lower synthesis costs additionally the costs incurred in carrying out the method according to the invention.
  • the short length of the primers used has a positive effect on the mass spectrometric analysis. This is due to the fact that shorter oligonucleotides are detected in mass spectrometry with higher sensitivity and mass resolution than longer ones.
  • the primer is 10 to 12 nucleotides long.
  • Figure 1 MALDI-TOF mass spectrum of a Sanger sequencing reaction (a) and a sequencing reaction according to the invention (b).
  • Example 1 The spectra of the sequence reaction products generated in Example 1 were recorded in the linear mode of a Bruker Scout MTP Reflex III mass spectrometer. The selected section shows the signals of the generated first four conductor oligonucleotides (primer + 1, 2,3,4 nucleotides). The incorporation of the corresponding dideoxy and deoxyribonucleotides is indicated in each case.
  • Figure 2 MALDI-TOF mass spectra of the sequencing conductors generated with different values T on neai.
  • the spectra of the sequencing reaction products generated in Example 2 were recorded in the linear mode of a Bruker Scout MTP Reflex III mass spectrometer.
  • the individual values for T ann eai are given in the corresponding partial illustrations. It can clearly be seen how the yield of the sequencing products increases from 41 ° C. to 63 ° C. with increasing T a nn ⁇ ai.
  • the expected (calculated) melting temperature for the primer used is 45 ° C.
  • Nucleotide internals are shown for the first three nucleotides in the upper part of the illustration. The insertion of G in one but not the other sequencing strand results in a signal doublet for the first nucleotide (incorporation of complementary C versus A).
  • the signals coincide due to the same resulting empirical formula (incorporation of C followed by A versus incorporation of A followed by C). The result is a signal but two mass differences from the previous doublet, on the basis of which the incorporation of A versus C was determined.
  • incorporation of G versus C which confirms the insertion of G two nucleotides beforehand.
  • P indicates the signal of the primer used.
  • Figure 3 MALDI-TOF mass spectra of the sequencing products generated in Example 3 for DNA templates (a-d).
  • the spectra were recorded in the high-resolution reflector mode of a Bruker Scout MTP Reflex III mass spectrometer.
  • the selected section shows the signal of the primer and the signals of the generated first four conductor oligonucleotides.
  • the inserts in FIG. 3a show the associated signal greatly enlarged in the X direction.
  • the resolution of the isotopy on which the corresponding oligonucleotides are based can be clearly recognized therein.
  • the nucleotide incorporation on which the different sequencing products are based, assigned on the basis of the determined mass differences, is indicated.
  • the assignments substitution (b), insertion (c) and deletion (d) are obvious.
  • P indicates the signal of the primer used.
  • Example 1 Sanger versus sequencing reaction according to the invention, implementation: thermal denaturation of the active synthesis complex consisting of DNA template, primer and DNA polymerase.
  • the reaction temperature is increased rapidly, so that the synthesis complex consisting of DNA template, primer and DNA polymerase is thermally denatured during the ongoing extension of the primer. This process is statistically distributed, decreasing in frequency with the length of the synthesis products, and results in a sequencing ladder according to the invention. Both sequencing reactions were carried out in 10 ⁇ ⁇ reaction volume.
  • Sanger sequencing reaction 54 ° C / 20 s, 72 ° C / 30 s, 94 ° C / 20 s
  • sequencing reaction according to the invention 54 ° C / 1s, 94 ° C / 5s.
  • a comparison of the data shown in FIG. 1 shows that the reaction products according to the invention can be obtained as disruptive by-products in Sanger sequencing reactions.
  • the stop with dA in addition to ddA is particularly troublesome for the detection of SNPs, since the molecular masses of dA and ddG are identical and the side reaction in question can thus simulate the exchange of A for G in an allele. This problem is avoided in the sequencing reaction according to the invention.
  • the selected annealing temperature T an n ea i significantly exceeds the melting temperature of the primer T m , its attachment and thus also the lifespan of the synthesis complex consisting of DNA template, primer and DNA polymerase is short-lived. As a result, the primer becomes only a few nucleotides extended before the synthesis stops. This process is statistically distributed, decreasing in frequency with the length of the synthesis products, and results in a sequencing ladder according to the invention.
  • DNA double strand template 47/48 bp PCR product.
  • Human genomic DNA served as the starting material.
  • the amplified regions (alleles) differ by inserting G at position 25 of the sequencing strand in an allele.
  • a primer that was only 12 nucleotides long was chosen, which attaches to the sequencing strand immediately before the expected mutation. This mutation was clearly detected by the detected sequencing products (see Figure 2). All sequencing reactions were carried out in 10 ⁇ ⁇ reaction volume.
  • reaction buffer 20 mM (NH 4 ) 2 SO 4 , 2 mM MgCl 2 , 75 mM Tris-HCl, pH 9.5 (reaction buffer). All reaction mixtures were subjected to 20 reaction cycles to increase the yield of sequencing products. For this purpose, the reaction mixtures were first heated to 94 ° C for 2 minutes in a thermal cycler (Eppendorf mastercycler gradient), followed by 20 temperature cycles: Tanneai / 1s, 94 ° C. The values used for T an neai ranged from 41 ° C. to 80 ° C. within a selected gradient (see FIG. 2).
  • Example 3 Use of the sequencing reaction according to the invention, implementation: Tanneai »T m , for the detection of SNPs (single nucleotide polymorphisms).
  • CGC AAC CTT CGC 1 unit thermosequenase
  • dNTP deoxyribonucleoside triphosphate
  • reaction buffer 20 mM (NH 4 ) 2 SO 4

Abstract

La présente invention concerne un procédé permettant de déterminer une séquence nucléotidique partielle d'une molécule d'acide nucélique. Ce procédé consiste: (a) à incuber une amorce qui s'hybride à la molécule d'acide nucléique, avec des copies multiples de la molécule d'acide nucléique en présence d'une polymérase et avec un mélange de désoxyribonucléotides; (b) à déterminer la masse moléculaire de fragments d'acide nucléique qui se succèdent en taille et qui sont générés par élongation de l'amorce en (a) par spectrométrie de masse; et (c) à déterminer la séquence nucléotidique par détermination de la croissance de la masse moléculaire entre les fragments d'acide nucléique qui se succèdent en taille.
PCT/EP2001/008671 2000-07-27 2001-07-26 Procede permettant de determiner une sequence nucleotidique partielle d'une molecule d'acide nucleique WO2002010446A2 (fr)

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DE10036743A DE10036743A1 (de) 2000-07-27 2000-07-27 Verfahren zur Bestimmung einer partiellen Nukleotidsequenz eines Nukleinsäuremoleküls

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