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WO1998003684A1 - Procede de sequençage d'acides nucleiques utilisant la spectrometrie de masse matricielle des temps de vol d'ionisation ou de desorption par laser - Google Patents

Procede de sequençage d'acides nucleiques utilisant la spectrometrie de masse matricielle des temps de vol d'ionisation ou de desorption par laser Download PDF

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WO1998003684A1
WO1998003684A1 PCT/US1997/012688 US9712688W WO9803684A1 WO 1998003684 A1 WO1998003684 A1 WO 1998003684A1 US 9712688 W US9712688 W US 9712688W WO 9803684 A1 WO9803684 A1 WO 9803684A1
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matrix
mer
sequence
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PCT/US1997/012688
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WO1998003684A9 (fr
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Bing H. Wang
Aharon S. Cohen
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Hybridon, Inc.
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Priority to AU40425/97A priority Critical patent/AU4042597A/en
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Publication of WO1998003684A9 publication Critical patent/WO1998003684A9/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
    • C12Q1/6869—Methods for sequencing
    • C12Q1/6872—Methods for sequencing involving mass spectrometry
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
    • H01J49/00—Particle spectrometers or separator tubes
    • H01J49/02—Details
    • H01J49/10—Ion sources; Ion guns
    • H01J49/16—Ion sources; Ion guns using surface ionisation, e.g. field-, thermionic- or photo-emission
    • H01J49/161—Ion sources; Ion guns using surface ionisation, e.g. field-, thermionic- or photo-emission using photoionisation, e.g. by laser
    • H01J49/164—Laser desorption/ionisation, e.g. matrix-assisted laser desorption/ionisation [MALDI]

Definitions

  • the invention relates to the determination of nucleotide sequences for nucleic acids and their analogs. Brief Summary of the Related Art
  • Each of these methods utilizes four separate reaction mixtures to create a nested set of fragments differing by a single nucleotide in length and representing a complete nucleotide sequence, followed by resolution of the fragments based on their size to determine the order of the fragments and hence the nucleotide sequence. Both of these procedures take from numerous hours to days to perform, and neither is effective for determining the nucleotide sequence for certain analogs of DNA. These popular approaches to nucleotide sequence determination are limited by their time consuming methodologies and by their inapplicability to certain types of nucleic acid analytes.
  • oligonucleotides have recently become of interest as tools in molecular biology experiments, as well as for use in the antisense therapeutic approach to disease treatment. Correct sequences are necessary to the efficacy and safety of such oligonucleotides, and effective and rapid analytical approaches are needed for quality control.
  • This class of compounds presents three special problems for traditional sequence determination approaches. First, quality control procedures are needed which are more rapid than the traditional approaches. Second, the oligonucleotides are generally short, often in the range of from about 15 to about 35 nucleotides in length. As a consequence, traditional sequencing approaches result in the loss of much sequence information and thus provide only incomplete information about the nucleotide sequence of the oligonudeotides.
  • oligonucleotides have either modified internucleoside linkages or substitution at the 2' position of the ribose to improve their properties as potential therapeutic agents.
  • U.S. Patent No. 5,220,007 discloses chimeric oligonucleotides having regions of oligonudeoside phosphodiester or phosphorothioate alongside regions of oligonudeoside alkylphosphonate or phosphoramidate.
  • PCT publication WO94/02498 discloses hybrid oligonudeotides having DNA regions alongside 2' -substituted RNA regions.
  • Uhlman and Peyman, Chemical Reviews 90: 544 (1990) discloses oligonudeotides having a variety of modifications along the internucleoside linkages, sugar residues or nucleoside bases.
  • U.S. Patent No. 5,403,709 disdoses a method for sequencing oligonudeotides using another oligonudeotide as an extension and a third, bridging oligonudeotide to hold the first two together for ligation. Conventional primer extension is then used to create a complement for sequencing. This approach requires some advance knowledge of a portion of the sequence of the analyte oligonudeotide.
  • VS. Patent No. 5,525,470 discloses a similar approach which avoids the need for such advance knowledge by utilizing RNA ligase to couple the analyte and extension oligonucleotides.
  • the invention provides a universal analytical method for determining the nucleotide sequence of nucleic acid analytes, including any chemically modified oligonucleotides.
  • This new method utilizes matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) with delayed pulsed ion extraction.
  • MALDI-TOFMS matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
  • This method is extremely rapid and acts directly on the nucleic acid analyte, creating in-source fragmentation (ISF) products which are used to determine the sequence of the nucleic add analyte.
  • ISF products have earlier also been known as prompt fragmentation ions, and such terms can be used interchangeably although in-source fragmentation product is the currently preferred term and will be used throughout herein. It is effective for a variety of nucleic add analytes, including any chemically modified oligonudeotides which have not previously been
  • the invention provides a method for determining the nucleotide sequence of nucleic add analytes comprising providing a suitable target for matrix-assisted laser desorption/ionization, such target comprising a nucleic add analyte in a matrix suitable for such matrix-assisted laser desorption/ionization; irradiating the target with laser irradiation suitable for creating ISF products from the nudeic acid analyte; delaying pulsed ion extraction for a time suffident to reduce formation of PSD ion fragments; ordering the ISF products according to molecular weight by time-of-flight mass spectrometry; and deterrnining the nudeotide sequence of the nudeic add analyte by comparing the molecular weights of the ordered ISF products.
  • the suitable target will indude a solid support, sudi as the commerdally available solid supports provided by Bruker Daltonics, formerly
  • the target is prepared by applying to the solid support a solution comprising the nudeic acid analyte and a matrix suitable for matrix-assisted laser desorption/ionization (MALDI), then allowing the solution on the solid support to evaporate to dryness.
  • the nucleic add analyte can be a naturally occurring or synthetic polynucleotide or oligonudeotide, including oligonucleotides having chemically modified internudeoside linkages, sugar bad ⁇ bones or nudeoside bases.
  • the suitable matrix can be selected from the matrices known in the art to be suitable for MALDI, and includes a cation exchange resin, preferably in ammonium form.
  • Preferred matrices indude, without limitation, 3-hydroxypicolinic acid, 3- hydroxypicolinic acid/picolinic acid, 3-hydroxypicolinic acid/sucrose, 2,5- dihydroxybenzoic acid, sinapinic acid, alpha-cyano-4-hydroxy-cinnamic acid, anthranilic acid, 6-aza-2-thiothymine, nicotinic acid, 2,4,6-trihydroxybenzoic acids, 2,4,6-trihydroxyacetophenone and combinations of the same.
  • Other suitable matrices are identified in Fitzgerald et al, Anal. Chem. 65: 3204 (1993).
  • the matrix is 3-hydroxypicolinic acid/N-(3- indolylacetyl)-l-leucine (3-HPA/IAL).
  • the target can be irradiated with any of a variety of laser irradiation sources.
  • Preferred laser irradiation sources include, without limitation, UV lasers, visible lasers and IR lasers.
  • Particularly preferred UV lasers include a 337 run N 2 laser, a
  • the pulsed ion extraction is delayed for a time sufficient to increase mass resolution of the ISF products and to reduce PSD ion formation sufficiently to improve the profile of the time-of-flight mass spec (TOFMS) by providing a voltage pulse to defied low mass ions and avoid suppression of signals of the larger ions, thus resulting in discrete peaks for each spedes of ISF product.
  • the appropriate time for the voltage pulse depends on the size of the largest ion spedes of interest and can conveniently be adjusted to maximize the mass resolution for such ion spedes.
  • the ISF products are readily separated according to molecular weight by
  • Figure 1 shows TOFMS spectrum for nucleotides N to N-18 for a 20-mer oligonudeotide sequenced using a preferred embodiment of the method according to the invention.
  • the "w” ions are marked and the molecular weight assignments are shown over each peak.
  • Figure 2 shows TOFMS spectrum for nucleotide N-19 for the same oligonudeotide shown in Figure 1.
  • Figure 3 shows TOFMS spectrum for a 15-mer PS all DNA oligonudeotide. The "w” ions are marked and the molecular weight assignments are shown over each peak.
  • Figure 8 RP-HPLC chromatograms of a neat 25-mer PS ODN (upper trace) and a mixture of the 25-mer PS ODN and a 17-mer PS ODN (lower trace). The mixture was separated into 5 fractions as indicated.
  • FIG. 9 MALDI-TOF mass spectra of the 4 of the 5 fractions separated by RP- HPLC.
  • Figure 10. Proposed fragmentation pathway for modified oligonucleotides (MONs) containing negatively charged backbone linkage.
  • MONs modified oligonucleotides
  • Figure 11 Proposed fragmentation pathway for modified oligonucleotides containing neutral backbone linkage.
  • the invention relates to the determination of nudeotide sequences for nucleic acids and their analogs.
  • the patents and publications cited herein are known to those skilled in this field and are hereby incorporated by reference in their entirety.
  • the invention provides an analytical method for determining the nucleotide sequence of nucleic acid analytes, including any chemically modified oligonucleotides.
  • This new method utilizes matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) with delayed pulsed ion extraction. This method is extremely rapid and acts directly on the nucleic add analyte. It is effective for a variety of nudeic acid analytes, induding any chemically modified oligonudeotides which have not previously been successfully sequenced.
  • MALDI-TOFMS matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
  • the invention provides a method for determining the nucleotide sequence of nucleic acid analytes comprising providing a suitable target for matrix-assisted laser desorption/ionization, such target comprising a nudeic acid analyte in a matrix suitable for such matrix-assisted laser desorption/ionization; irradiating the target with laser irradiation suitable for creating ISF products from the nudeic acid analyte; delaying pulsed ion extraction for a time suffident to reduce formation of PSD ion fragments; ordering the ISF products according to molecular weight by time-of-flight mass spectrometry; and determining the nudeotide sequence of the nucleic add analyte by comparing the molecular weights of the ordered ISF products.
  • the suitable target will indude a solid support, such as the commerdally available solid supports provided by Bruker Analytical Systems
  • the target is prepared by applying to the solid support a solution comprising the nudeic acid analyte and a matrix suitable for matrix-assisted laser desorption/ionization (MALDI), then allowing the solution on the solid support to evaporate to dryness.
  • the nucleic acid analyte can be a naturally occurring or synthetic polynucleotide or oligonudeotide, including oligonudeotides having diemically modified internucleoside linkages, sugar backbones or nucleoside bases.
  • oligonudeotide includes polymers of two or more deoxyribonucleoside, ribonucleoside or 2'-0-substituted ribonucleoside monomers, or any combination thereof.
  • oligonucleotides will have from about 2 to about 100 monomers, and most preferably from about 13 to about 40.
  • Such monomers may be coupled to each other by any of the numerous known internucleoside linkages.
  • these internucleoside linkages may be phosphodiester (PO), phosphotriester, phosphorothioate (PS), or phosphoramidate linkages, or combinations thereof.
  • oligonudeotide also encompasses such polymers having chemically modified bases or sugars and/ or having additional substituents, induding without limitation lipophilic groups, intercalating agents, diamines and adamantane.
  • the term "2'-0-substituted” means substitution of the 2' position of the pentose moiety with an -O-lower alkyl group containing 1-6 saturated or unsaturated carbon atoms, or with an -O-aryl or allyl group having 2-6 carbon atoms, wherein such alkyl, aryl or allyl group may be unsubstituted or may be substituted, e.g., with halo, hydroxy, trifluoromethyl, cyano, nitro, acyl, acyloxy, alkoxy, carboxyl, carbalkoxyl, or amino groups; or such 2' substitution may be with a hydroxy group (to produce a ribonucleoside), an amino or
  • the suitable matrix can be selected from the matrices known in the art to be suitable for MALDI, and indudes a cation exdiange resin, preferably in ammonium form.
  • the cation exchange resin is a 200-400 mesh in ammonium form.
  • Preferred matrices include, without limitation, 3-hydroxypicolinic add, 3-hydroxypicolinic add/picolinic add, 3-hydroxypicolinic add/sucrose, 2,5- dihydroxybenzoic add, sinapinic add, alpria-cyancH-hydroxy-c namic add, anthranilic acid, 6-aza-2-thiothymine, nicotinic add, 2,4,6-trihydroxybenzoic adds, 2,4,6-trihydroxyacetophenone and combinations of the same.
  • Other suitable matrices are identified in Fitzgerald et al, Anal. Chem. 65: 3204 (1993).
  • the matrix is 3-hydroxypicolinic acid/N-(3- indolylacetyl)-l-leucine (3-HPA/IAL).
  • the target can be irradiated with any of a variety of laser irradiation sources.
  • Preferred laser irradiation sources include, without limitation, UV lasers, visible lasers and IR lasers.
  • Particularly preferred UV lasers include a 337 nm N 2 laser, a 266 nm laser and a 193 nm laser.
  • One particularly preferred laser irradiation source is the 337 nm N 2 laser provided with the MALDI unit produced by Bruker Analytical Systems. The laser power is sufficient to efficiently produce ISF products without saturating the detector system.
  • a laser attenuation level of from about 40 to about 50 is used to give an irradiance of 1-2 107W/cm2.
  • attenuation levels of from about 10 to about 110 may be advantageously used.
  • the laser attenuation level can be measured using a power meter (Laser Probe, Inc., Utica, NY, USA).
  • the ion source of the mass spectrometer is opened so that the energy detector could be placed in the laser beam path in the ion source region.
  • the energy output of the laser is recorded at a series of the neutral density attenuator settings.
  • the voltage on the target will be from about +/- 3-30 kV (for positive or negative polarity).
  • such acceleration voltage will be about +25 kV.
  • such acceleration voltage will be about +20 kV.
  • the voltage on the auxiliary plate after switching (P2) is preferably from about +/- 2-27 kV.
  • P2 voltage is about +22.5 kV.
  • the lens voltage is preferably from about +/- 0- 15 kV. In one particularly preferred embodiment, such lens voltage is about +9.2 kV.
  • the voltage on the dual-microchannel-plates detector is preferably from about +/- 1.3-2.0 kV. In one particularly preferred embodiment, such detector voltage is about +1.5 kV.
  • the time elapsed between laser firing and voltage switching on the auxiliary plate is preferably from about 100 to about 400 ns. In one particularly preferred embodiment, such elapsed time is about 250 ns.
  • the number of spectra summed may preferably range from about 1 to about 1000. In one particularly preferred embodiment, such number will be about 80.
  • the vacuum in the sample chamber region is preferably at or below 5 x 10 "6 torr. In one preferred embodiment, such vacuum is 5 x 10 "7 torr.
  • the pulsed ion extraction is delayed for a time sufficient to increase mass resolution of the ISF products, and to reduce PSD ion formation sufficiently to improve the profile of the time-of-flight mass spec (TOFMS) by providing a voltage pulse to deflect low mass ions and avoid suppression of signals of the larger ions, thus resulting in discrete peaks for each of the ISF products.
  • TOFMS time-of-flight mass spec
  • the MALDI-TOFMS unit produced by Bruker is available with the MS modified as to allow delay of the pulsed ion extraction following laser irradiation.
  • the appropriate voltage and time for the ion deflection voltage pulse depends on the size of the largest ion species of interest and can conveniently be adjusted to maximize the mass resolution for such ion species.
  • such ion deflection voltage will range from about 0 to about 10 kV. In one particularly preferred embodiment, such ion deflection pulse will be about +2 kV. Preferably, such time will be from about 0-10 microseconds. In one particularly preferred embodiment, such time is about 1-2 microseconds.
  • the ISF products are readily separated according to molecular weight by TOFMS, which orders the products according to their molecular weight.
  • the observed ISF products include the "w” and “d” ions, and also the "a” and “z” ions.
  • the w and z ions are generated by fragmentation of the sequence from the 3' end and the a and d ions are generated by fragmentation of the sequence from the 5' end. Since the mass of each nudeotide is known, this allows determination of the nudeotide sequence of the nucleic add analyte by simple comparison of the molecular weights of the consecutive spedes in the ordered array.
  • the w ions together with the protonated molecular ions often allows the determination of the complete nudeotide sequence.
  • the w ions alone are insufficient to determine the entire sequence, and in these cases the d, a and z ions provide supplemental information which allows the construction of the complete nucleotide sequence.
  • the presence of the d, a, and z ions also fadlitates the determination of the backbone linkage.
  • the sequence and backbone linkage of an unknown sequence which includes modified nucleotides can be determined using a single technique.
  • PS/MP methylphosphosphonate
  • Example 1 Preparation of a matrix suitable for MALDI 3-hydroxypicolinic acid (3-HPA; Aldrid Chemical Co., Milwaukee, WI; 10.0 +/- 0.5 mg) was weighed and put into a 1.5 ml Eppendorf tube. To the Eppendorf tube was added 75 microliters HPLC grade water (J.T. Baker) and 75 microliters HPLC grade acetonitrile (EM Sdence, Gibbstown, NJ). The tube was capped and fixed onto the platform of a Vortex Gene 2 (Sdentific Industries, Inc. Bohemia, NY) vortexer, which was operated at vibration level 6 for 5 minutes. N-(3-indolylacetyl)- 1-leudne (IAL; Aldrich; 2.0 +/- 0.4 mg) was weighed and put into a 1.5 ml
  • Example 2 Preparation of an exemplary MALDI target An 20-mer oligonudeotide phosphorothioate having two 5' terminal 2'-0- methyl nucleosides and four 3 * terminal 2'-0-methyl nucleosides was prepared as a solution in HPLC grade water (Baker) at a concentration of 5000 ppm. Four microliters of the matrix prepared according to Example 1 was transferred to an
  • Eppendorf tube to which two microliters of the oligonudeotide solution was added. To the tube was then added 3 mg of cation exchange resin in ammonium form (200- 400 mesh), and the tube was then vortexed for 10 seconds. The Eppendorf tube was allowed to stand briefly, then 1 microliter of the clear solution was withdrawn and deposited on a stainless steel target (Bruker). The solvent was then allowed to evaporate at ambient conditions.
  • the MALDI target prepared according to Example 2 was placed in the sample chamber of a Bruker Matrix-assisted Laser Desorption/ionization Time-of- Flight Mass Spedrometer equipped to allow delay of pulsed ion extraction following laser irradiation.
  • Additional fragment ion series include "d” and "z” (see Tables 1 and 2). Because the modification occurs to the nucleosides, each residue mass used to assign terminal nudeosides is increased by 30 ⁇ into 238 (C), 239 (T), 263 (A), and 278 (G), respectively. Similarly, the residue mass for each nucleotide containing a 2"-0-methyl group is 335 (C), 350 (T), 359 (A), and 375 (G). The terminal nudeotides containing a 2'-0-methyl group should have the following m/z in the positive ion mode, 354 (C), 369 (T), 378 (A), and 394 (G).
  • Example 4 Sequence determination of a 17-mer PS/PO chimeric nucleic acid analyte using MALDI-TOFMS with delayed pulsed ion extraction
  • a 17-mer PS/PO chimeric oligodeoxynucleotide MALDI target was placed in the sample chamber of a Bruker Matrix-assisted Laser Desorption/ionization Time- of-Flight Mass Spectrometer equipped to allow delay of pulsed ion extraction following laser irradiation.
  • the flight path is 1.6 m.
  • the desorption and ionization was effected with a 337 nm N 2 laser.
  • the deflection duration was set at 1000 ns, while for those with m/z above 1500 the deflection duration was set at 2000 ns.
  • a mass spectrum was calibrated in two segments. In the lower mass region, a matrix ion peak and the doubly protonated molecular ion were used as the calibrants. In the higher mass region, the singly and doubly protonated molecular ions were used as the calibrants.
  • Figure 4 shows a mass spectrum of a 17-mer oligodeoxynudeotide consisting of both PS and PO nucleotides.
  • Prominent ion series observable in the mass spectrum include “d” and “w” ion series.
  • Also prominently present in the lower m/z region of the mass spectrum are the “a” ions (Tabled- Because the measurement was taken in positive ion mode, all ions should have two extra protons compared to their negative ion analogs, assuming they are all even electron species. For simplidty the same notation for the same ion type is used regardless the ion polarity.
  • the presence of a complete set of w ions (w,-w 16 ) provides for ready sequence determination. Confirmation of the 17-mer sequence can be made by a simple comparison between the expected and experimentally determined m/z of "w” ions (Tabled ).
  • a sequence can be constructed from the "w” ions together with (M+H) + ions. The sequence is then compared with segments of sequences constructed from other ion series for verification. In general, this is a more useful strategy because it can be used to determine an unknown sequence.
  • the first step of the sequence determination is to find the w k-1 peak (k being the total number of the nucleotides in a given sequence).
  • the difference in m/z between (M+H) + and w k _ corresponds to the mass of a 5'-terminal nucleoside residue, it should match one of the five possible values for unmodified deoxynudeoside, 209 (C), 210 (U), 224 (T), 223 (A), and 249 (G).
  • the m/z difference between two adjacent "w" ions should correspond to the mass of a nucleotide residue.
  • the w k-2 is located by finding the peak having a m/z smaller by one nudeotides the residues mass are 289 (C), 304 (T), 313 (A), 329 (G).
  • PS w can have the following masses: 324 (C), 339 (T), 348 (A), and 364 (G); PO w ⁇ has smaller masses by 16 ⁇ .
  • sequence determination could be achieved with the "w" ion series alone, other type of ions provide useful confirmation.
  • the w 13 ion peak at m/z 4212 is not well separated from the peak at m/z 4205.
  • the w 12 peak at m/z 3910 is also weak. This affects the assignment of nucleotides at position 4, 5, and 6 (counting from the 5' end). However, information can be unambiguously obtained from d3 (m/z 979), d4 (m/z 1308), d5 (m/z 1613), and d6 (m/z 1942). As shown in Table3 ⁇ r ⁇ e partial sequence constructed from these ions match the corresponding sequence constructed from "w" ions.
  • FIG. 5 shows a mass spectrum of a 25-mer PS ODN.
  • Table 4 A complete set of “w” ions can be identified (Table 4). Accompanying the "w” ions are the “a” ions giving a distinct pattern of paired peaks in the mass spectrum (Table 5). Other ion series with generally lower abundances, such as “b”, “c", “d”, and “z” ions have also been found (Table 6). As shown, the replacement of the non-bridging oxygen atom by a sulfur atom enhances the generation of "a” ions.
  • a 18-mer PS/MP chimeric oligodeoxynucleotide was sequenced by MALDI- TOFMS using the instrument settings described in Example 4 above.
  • methylphosphonate another popular modified backbone structure is methylphosphonate.
  • the residue masses for methylphosphonate deoxynucleotide are 287 (C), 302 (T), 311 (A), and 327 (G).
  • Example ' To investigate the origin of the ISF Products the 25-mer PS ODN was mixed with a 17-mer PS-ODN of the same sequence nested on the 3'-end in a molar ratio of 100:1. When analyzed by RP-HPLC, the two PS ODN appear as two separate components ( Figure 8). Fractionation of the mixture at time points indicated in the chromatogram gave 5 fractions. Analysis by MALDI-TOFMS revealed that Fraction A consisted mostly of the 17-mer. Due to low the concentration, the signal of the 17-mer is low (data not shown). The MALDI-TOFMS mass spectra of the other fractions are shown in Figure 9. In Fraction B besides the 17-mer no other accompanying fragments are present.
  • Example 2 The influence of experimental conditions No qualitative difference has been observed when the 25-mer PS ODN, shown in Figure 5, was analyzed in either the positive or negative polarity, using 3- HPA/IAL as the matrix. As in the positive polarity, the dominant ion series are "w” and “a” ions. Additional ion series include “d” and "z” ion, the latter appear only in the lower m/z region. The tendency to arc in the ion source at high laser irradiance makes the positive polarity a better choice for routine analysis.
  • the signal to noise ratio (S/N) of the fast fragment ions can be improved as the irradiance increases from the threshold irradiance, which is defined as the irradiance necessary to give a S/N of 2 for a given fragment ion, the abundance of the fragment ions relative to that of the (M+H) + ion of the 17-mer PS ODN did not scale linearly with the irradiance applied at the sample.
  • the threshold irradiance which is defined as the irradiance necessary to give a S/N of 2 for a given fragment ion

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Abstract

La présente invention concerne un procédé analytique permettant de déterminer la séquence des analytes acides nucléiques des nucléotides, y compris les oligonucléotides chimiquement modifiés. Ce nouveau procédé utilise la spectrométrie de masse matricielle des temps de vol d'ionisation ou de désorption par laser ou 'MALDI-TOFMS' (pour 'Matrix-Assisted Laser Desorption/Ionization - Time-Of-Flight Mass Spectrometry') à extraction à retard d'ions pulsés.
PCT/US1997/012688 1996-07-19 1997-07-19 Procede de sequençage d'acides nucleiques utilisant la spectrometrie de masse matricielle des temps de vol d'ionisation ou de desorption par laser WO1998003684A1 (fr)

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Cited By (56)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998040520A1 (fr) * 1997-03-14 1998-09-17 Hybridon, Inc. Methode de sequençage d'acides nucleiques modifies au moyen d'une spectrometrie de masse a transformee de fourier a ionisation par electropulverisation
WO1998045700A3 (fr) * 1997-04-09 1999-03-11 Joachim W Engels Procede de sequençage de biopolymeres par spectrometrie de masse
WO1999057318A3 (fr) * 1998-05-07 2000-03-23 Sequenom Inc Analyse de molecules par spectrometrie de masse a desorption/ionisation par laser, assistee par matrice absorbant l'infrarouge
US6218118B1 (en) 1998-07-09 2001-04-17 Agilent Technologies, Inc. Method and mixture reagents for analyzing the nucleotide sequence of nucleic acids by mass spectrometry
GB2399218A (en) * 2003-01-17 2004-09-08 Bruker Daltonik Gmbh Tandem mass spectrometry utilising in-source decay (ISD)
US6994969B1 (en) 1999-04-30 2006-02-07 Methexis Genomics, N.V. Diagnostic sequencing by a combination of specific cleavage and mass spectrometry
US7169560B2 (en) 2003-11-12 2007-01-30 Helicos Biosciences Corporation Short cycle methods for sequencing polynucleotides
US7220549B2 (en) 2004-12-30 2007-05-22 Helicos Biosciences Corporation Stabilizing a nucleic acid for nucleic acid sequencing
US7397546B2 (en) 2006-03-08 2008-07-08 Helicos Biosciences Corporation Systems and methods for reducing detected intensity non-uniformity in a laser beam
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