EP1015632A1 - Caracterisation d'acide nucleique par spectrometrie de masse - Google Patents

Caracterisation d'acide nucleique par spectrometrie de masse

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Publication number
EP1015632A1
EP1015632A1 EP98942924A EP98942924A EP1015632A1 EP 1015632 A1 EP1015632 A1 EP 1015632A1 EP 98942924 A EP98942924 A EP 98942924A EP 98942924 A EP98942924 A EP 98942924A EP 1015632 A1 EP1015632 A1 EP 1015632A1
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European Patent Office
Prior art keywords
mass
nucleic acid
population
fragments
labels
Prior art date
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EP98942924A
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German (de)
English (en)
Inventor
Günter Schmidt
Andrew Hugin Thompson
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Xzillion GmbH and Co KG
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Brax Group Ltd
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Priority claimed from GBGB9719638.0A external-priority patent/GB9719638D0/en
Priority claimed from GBGB9725630.9A external-priority patent/GB9725630D0/en
Application filed by Brax Group Ltd filed Critical Brax Group Ltd
Publication of EP1015632A1 publication Critical patent/EP1015632A1/fr
Withdrawn legal-status Critical Current

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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
    • 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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6823—Release of bound markers

Definitions

  • This invention concerns a method for analysing nucleic acid.
  • the method is advantageous, since it allows a population of differing nucleic acid fragments to be analysed simultaneously.
  • a further problem of great significance is accurate mass measurement of moderately large biomolecules. This resolution problem limits read lengths of DNA sequences achievable to a significant degree. At present the absolute limit on direct mass analysis of Sanger ladders is determination of sequences of about 100 bases in length and is nearer 30 to 40 bases for practical purposes .
  • GB 9719284.3 describes the use of nucleic acid hybridisation probes cleavably linked to mass labels for the analysis of nucleic acids.
  • GB 9719284.3 describes a method of sequencing nucleic acids exploiting mass labelled sequencing primers or nucleotides to generate Sanger ladders. This sequencing method uses capillary electrophoresis mass spectrometry as the mass spectrometry method to analyse the mass labelled Sanger ladders generated. These methods require a two-stage analysis; a sizing step which determines the lengths of each nucleic acid in a population, i.e. the number of nucleotides that comprise its linear sequence, followed by identification of the mass label each nucleic acid carries.
  • the present invention provides a method for analysing a population of nucleic acid fragments each labelled with a mass label, which method comprises:
  • the population of nucleic acid fragments may be ionised by any suitable method. Electrospray ionisation is particularly useful because it enables direct ionisation from a solution of labelled nucleic acid fragments.
  • the subsequent steps of sorting the ionised population, cleaving each sub-population and determining the mass of each released mass label may be performed in specified zones of a mass spectrometer.
  • mass spectrometer configurations such as those found in ion trap mass spectrometers or Fourier Transform ion cyclotron resonance spectrometers
  • the steps of sorting, cleaving and determining the mass of each released mass label are separated temporally but take place in the same "zone”.
  • the step of sorting the ionised population may be effected by the application of a magnetic field, preferably an electromagnetic field such as from a quadrupole, hexapole or dodecapole.
  • the step of sorting the ionised population may be effected by an ion trap or an ion cyclotron device. It is possible to combine electric and magnetic fields in order to perform the sorting step.
  • the step of cleaving each sub- population may be performed in a cleavage zone by collision or by photo-cleavage, for example using a laser. A choice of how to perform the cleaving steps depends to some extent on how the mass label is linked to its associated fragment. The mass label would typically be linked to its associated fragment by a cleavable linker, which could be photo-cleavable or simply designed to cleave automatically upon collision with a concentration of gas phase or with a solid surface in the mass spectrometer.
  • any suitable mass analyser configuration may be used. This step typically involves separation of the released mass labels from one another followed by detection.
  • the separation may be achieved by any means used in a mass analyser such as a magnetic field, preferably an electromagnetic field including a quadrupole, hexapole or dodecapole.
  • a time of flight configuration it is possible to use a time of flight configuration to separate the released mass labels from one another. Detection may be effected by any suitable means.
  • nucleic acid fragments and/or mass labels are fragmentation resistant.
  • the population of nucleic acid fragments is produced from a method of DNA sequencing such as disclosed in GB 9719284.3.
  • a template strand of DNA typically a primed template
  • the population of nucleic acid fragments for analysis comprises the series of fragments.
  • each fragment is terminated with a nucleotide which is cleavably attached to a corresponding mass label uniquely resolvable in mass spectrometry for identifying the nucleotide.
  • a further embodiment of this invention employs a modification of the conventional Sanger sequencing strategy that involves degradation of a phosphorothioate containing DNA fragment.
  • This sequencing method utilises alpha-thio dNTPs instead of the ddNTPs used in a conventional Sanger sequencing reaction. These are included with the normal dNTPs in a primer extension reaction mediated by a DNA polymerase.
  • the four sets of base terminating ladders is obtained by including one of the 4 alpha-thio dNTPs in 4 amplification reactions followed by limited digestion with exonuclease III or snake venom phosphodiesterase. (Labeit et al .
  • This method of sequencing is advantageous as it favours the formation of the higher molecular weight termination species.
  • the conventional Sanger sequencing methodology in contrast, generates exponentially less of each termination fragment as the length of the fragment increases.
  • Mass spectrometers are less sensitive to the higher molecular weight species, thus a sequencing method that increases their concentration will improve the sensitivity of the mass spectrometry analysis of these fragments .
  • the population of nucleic acid fragments is provided on a chip, typically a glass chip, whereby each member of the population is present at a discrete location on the chip.
  • the chip may be treated with a MALDI matrix material .
  • the fragments may be desorbed by applying laser light so as to ionise the population.
  • fragments, or groups of fragments, located at discrete regions on the chip may be selectively desorbed from the chip by appropriate spatial addressing of the laser light.
  • Laser desorption of fragments may typically be effected in an evacuated chamber which may be integral with the mass spectrometer.
  • This invention describes the use of Tandem Mass Spectrometry techniques as a detection method for nucleic acid sequencing and for other nucleic acid sizing assays that use cleavable mass labels.
  • Capillary electrophoresis mass spectrometry uses a capillary electrophoresis separation to determine the lengths of nucleic acids in a population followed by ionisation of the eluent from the capillary electrophoresis separation and cleavage of the mass labels from the nucleic acids which are then analysed by mass spectrometry.
  • the same size separation, label cleavage and label analysis steps can be performed in a tandem mass spectrometer.
  • Tandem Mass spectrometry describes a variety of techniques where the components of an ion stream pass through more than one mass analysis step.
  • multiple mass labelled nucleic acids can be separated by length in the first mass analyser of a tandem configuration. This is followed by cleavage of mass labels from their associated nucleic acid between the first and second mass analyser. The cleaved mass labels are finally analysed in the second mass analysis stage of the instrument.
  • the tandem mass spectrometry approach is very desirable as such separations can take place in fractions of seconds rather than in the order of tens of minutes to an hour for a capillary electrophoresis mass spectrometry separation.
  • Capillary electrophoresis based methods face the same problems as gel electrophoresis based separation systems for sizing of nucleic acids although the problems are much more controllable in a capillary system. These problems include band- broadening due to temperature effects, compressions due to secondary structure in the template nucleic acids and inhomogeneities in the separation gels. Determination of the mass of a nucleic acid molecule, even at a low resolution to determine its length will avoid these problems.
  • Mass labels can be chosen to take a different charge to DNA in the mass spectrometer. This means that after cleavage of labels from their corresponding DNA molecule, labels can be exclusively selected for analysis in the second mass analyser by using the appropriate mode of analysis. DNA tends to form ions with a net positive charge, so negative ion mode is generally more effective. Further selectivity is possible if scanning mass analysers, such as quadrupoles, are used for the second mass analysis component as these can filter out any fragment noise.
  • labels are well-characterised molecules, picking up a signal from these is greatly simplified in a tandem analysis. Since ionisation is essentially a statistical process, there will be a small background noise of labels from DNA fragmentation products carrying labels though. However by modifying the energy imparted to ions, one can potentially favour the formation of neutral labelled fragments which will not appear in any spectrum. Alternatively one can simply choose mass labels that adopt the same charge as their corresponding DNA molecule but whose peaks in the mass spectrum do not coincide with DNA fragmentation products.
  • the mass resolution problem is particularly acute for sequencing by single stage mass spectrometry as the length of a DNA ladder and its terminating base are determined by accurate measurement of the mass of the molecule, which requires mass accuracy approaching a single dalton.
  • This invention proposes a tandem scheme where the first mass analyser determines the length of the DNA ladder, which has a mass resolution requirement of the order of 300 daltons followed by cleavage of a label identifying the terminating base in a collision chamber, or another induced fragmentation step. The cleaved label is identified subsequently in the second mass analyser. Labels can be small molecules and can be analysed at high resolution in the second mass spectrometer.
  • An advantageous embodiment of this technology is the use of fluorinated mass labels when high resolution mass analysis of labels is employed after cleavage from their nucleic acid.
  • a hydrogenated molecule whose integral mass is 100 will have a fractionally higher real mass when measured at very high resolution.
  • a fluorinated molecule whose integral mass is 100 will tend to have a fractionally lower real mass.
  • fluorinated molecules are not common in living systems, this means that a fluorinated mass label will be distinguishable in the mass spectrum even in the presence of contaminating peaks due to fragmentation or buffers as long as the nucleic acids and reagents used are not fluorinated.
  • An important feature of the invention is the mechanism of cleavage of the labels from a mass labelled nucleic acid which occurs after the first mass analysis step. Collision induced dissociation of labels from their corresponding DNA is one method of cleavage currently used for peptide sequencing. An alternative method would be photon induced cleavage of the mass label from its DNA.
  • tandem mass spectrometers typically have a linear configuration in which a separate component performs each step of the process and the ion stream is directed from one component to the next. Multiple configurations of linear instruments are possible as discussed later. Certain instruments, however, such as ion trap instruments and fourier transform ion cyclotron mass spectrometers, permit all these steps to occur in a single component.
  • DNA sizing assays that are compatible with capillary electrophoresis mass spectrometry as discussed in PCT/US97/01046 are equally applicable to Tandem Mass Spectrometry applications. These include but are not limited to differential display, restriction fragment length polymorphism analysis, and linkage analysis.
  • This invention is highly advantageous for high throughput analysis of mass labelled DNA molecules as it permits very rapid analysis of those molecules. Furthermore, this invention permits multiplexing of a number of labelled nucleic acids. The degree of multiplexing is limited only by the number of mass labels available and resolvable in the mass spectrometer.
  • Each sequencing reaction would be performed separately and then all the templates would be combined at the end of the sequencing reactions.
  • the Sanger ladders generated are then all separated together in a tandem mass spectrometer, using one of the soft ionisation techniques described below.
  • Each set of 4 mass labels then correlates to a single source template.
  • RNA polymerases are used in conjunction with ribonucleotides or their analogues since most RNA polymerases use promoter sequences rather than primers and so incorporation of labels would have to be effected via labelled nucleotides .
  • labelled nucleotides are a favourable embodiment in that it avoids certain potential problems associated with primer labelled sequencing.
  • Polymerase reactions often terminate prematurely, without the intervention of blocked nucleotides. This is a problem with primer labelled sequencing because the premature termination generates a background of labelled fragments that are terminated incorrectly. Labelling the blocking nucleotides ensures only correctly terminated fragments are labelled so only these are detected by the mass spectrometer. This then permits cycle sequencing where multiple rounds of primer are add to the template.
  • the sequencing reaction is performed using a thermostable polymerase. After each reaction the mixture is heat denatured and more primer is allowed to anneal with the template. The polymerase reaction is repeated when primer template complexes reform. Multiple repetition of this process gives a linear amplification of the signal, enhancing the reliability and quality of the sequence generated. This an advantage over direct mass analysis techniques which must deal with prematurely terminated products which will appear in the mass spectrum and may result in incorrect base calls.
  • each template In order to permit simultaneous sequencing reactions with mass labels one requires that the Sanger ladder generated for each template be distinguishable from those generated from other templates. This can be achieved using uniquely labelled sequencing primers for each template. In order to ensure that each template bears a unique sequencing primer site one could conceivably engineer a family cloning vectors that bear different primer sequences flanking the integration site for the exogenous DNA to be sequenced. Each sequencing reaction would be performed on a group of templates where only one template derived from each vector type is present so that all the templates in a reaction bear unique primers.
  • Adapters to Introduce primers to restriction fragments One can, however, exploit the ability to sequence numerous templates simultaneously to cut out sub-cloning steps in a sequencing project.
  • a large DNA fragment such as a mitochondrial genome or a cosmid.
  • the majority of properly restricted fragments should as a result bear an adapter at each of their termini. This permits amplification of the adaptered restriction fragments at this stage if that is desired.
  • the capture primer could be biotinylated and presented to the adaptered fragments free in solution, after which captured fragments can be immobilised onto a solid phase support derivitised with avidin. Alternatively the primer could be immobilised onto a solid phase support prior to exposure to the adaptered restriction fragments.
  • the captured fragments are made double stranded at this stage by reaction with a polymerase. This means that immobilised copies of all sequences should be present.
  • the hybridised captured strand can be melted off at this stage and be disposed of if that is desired.
  • primers bear the primer sequence in the adapter and the restriction site by which the adapters were originally ligated to the DNA and an additional overlap of a predetermined number of bases. If one has 64 labels available the overlap can be 3 bases. Each of the possible 3 base overlaps can be identified by a unique mass label. Given a population of the order of 50 to 60 templates one would expect the majority to have a different 3-mer adjacent to the ligated primer. Thus the majority of templates will be expected to hybridise to a distinct primer. Any template that bears a 3-mer immediately adjacent to the adapter that is the same as that on another template would only be resolvable if one is able to determine by the quantity of each template which template to assign a base call to.
  • the Sanger ladders from each of the four sequencing reactions are then preferably pooled and analysed together by ES tandem mass spectrometry so as to avoid any ambiguities in assigning bases due to experimental differences.
  • Each pool of templates would thus have to have its primers labelled with a unique set of mass labels. Thus a total of 256 mass labels would be required.
  • Each primer thus has four labels, one four each terminator reaction.
  • the labels assigned to each primer should be close in mass and size to minimise differences in migration between each termination reaction.
  • a further embodiment of this invention is generating multiple template ladders simultaneously in the same reactions with labelled nucleotides.
  • N 4 there would be 256 discrete locations on the array. It is expected that a group of templates would in most cases have distinct sequences immediately adjacent to the primer. This would be an expensive exercise for sorting templates from just one reaction vessel. With a large number of mass labels, however, one can have distinct sets of 4 mass labels identifying blocking nucleotides in a large number of reactions.
  • multiple templates can be added to different reaction vessels, preferably different templates to each reaction vessel. After generating Sanger ladders in each vessel, the reactions can be pooled and the templates from each reaction can be sorted simultaneously. One would expect the majority of ladders of each template from each reaction to segregate to discrete locations on an array and that each location on the array would receive template ladders from a number of distinct reactions.
  • different primers can be linked to a 'sorting sequence', a length of oligonucleotide that could be used to sort ladders with different primers onto a hybridisation chip.
  • sorting sequences would ideally be non-complementary to each other to prevent cross hybridisation with each other and should minimally cross-hybridise with the complementary sequences of all other sorting sequences.
  • a full discussion of minimally cross- hybridising sets of oligonucleotides is discussed in PCT/US95/12678.
  • a series of sequencing templates identified by different primers linked to distinct sorting sequences can be used to generate Sanger ladders in the same reaction with the same labelled nucleotide terminators.
  • the resultant Sanger ladders can then be sorted onto a hybridisation array comprising the sequences complementary to the sorting sequences so that each Sanger ladder identified by a particular primer can be sorted to a discrete location on the array.
  • a hybridisation array could comprise an array of wells on microtitre plates, for example, such that each well contains a single immobilised oligonucleotide that is a member of the array.
  • a sample of the pooled reactions is added to each well and allowed to hybridise to the immobilised oligonucleotide present in the well.
  • the unhybridised DNA is washed away.
  • the hybridised DNA can then be melted of the capture oligonucleotide and injected into an electrospray interface to a tandem mass spectrometer.
  • the array could be synthesised combinatorially on a glass 'chip' according to the methodology of Southern or that of Affymetrix, Santa Clara, California, or using related ink-jet technologies such that discrete locations on the glass chip are derivitised with one member of the hybridisation array.
  • the chip can then be treated with a MALDI matrix material such as 3-hydroxypicolinic acid. Having prepared the chip in this way it can be loaded into a MALDI based tandem mass spectrometer and Sanger ladders from discrete locations on the array can be desorbed by application of laser light to the desired location on the array. Direct desorption of DNA from a hybridisation matrix has been demonstrated by Koster et al. (Nature Biotech. 14, 1123 - 1128). The length of the fragments can be analysed in the first mass analyser followed by cleavage of labels and analysis of these labels in the second mass analyser.
  • multiplexing and sorting templates is the ability to avoid a number of sub-cloning steps in a large scale sequencing project.
  • Each sequencing reaction would be performed on a group of templates where only one template derived from each vector type is present so that all the templates in a reaction bear unique primers.
  • fragmentation of DNA applies particularly to the use of MALDI techniques in that the protonation mechanism that leads to cleavage is thought to be exacerbated by the matrices used to ionise the nucleic acid, since many of these are moderately acidic compounds such as cinnamic acid derivatives, 2,5- dihydroxybenzoic acid, etc.
  • the matrix 3-hydroxypicolinic acid has been shown to produce less fragmentation than most which improves the potential of MALDI based approach.
  • the mass labelling technology is however also highly compatible with ESI based approaches where buffering agents and control over ionisation conditions might allow reduction of the protonation problem.
  • Mass labels can be chosen to take a different charge to DNA in the mass spectrometer. This means that after cleavage of labels from their corresponding DNA molecule, labels can be exclusively selected for analysis in the second mass analyser. Since only labels are analysed in the second mass analyser, most DNA fragments will not appear in the spectrum, or if the labels bear the same charge as the DNA they can be chosen to have masses that are discrete from DNA fragmentation products allowing them to be easily identified. There will still however be a small background from DNA fragments carrying labels which can also be dealt with to some extent by this invention. Fragmentation of singly charged species, generated by the 'mild' ionisation techniques such as Electrospray, MALDI and FAB, generally results in the formation of a charged fragment and an uncharged fragment.
  • the DNA fragments without labels, whether charged or not, will not be seen in the second mass analysis phase or should be resolvable from mass label peaks depending on the label used. Uncharged species with labels will also not be seen in the final spectrum. If the fragmentation paths in (1) and (2) are equally likely then clearly, one would expect half the fragmentation noise when compared with the noise seen in direct mass spectrometry of Sanger ladders but the formation of the ions is not equally likely but is determined by the heats of formation of the species involved. Generally the stability of a bond is analysed by comparing the heat of formation of the ion species on the left in the equations above with the heat of formation of the neutral species on the right, as discussed below.
  • the fragmentation of molecular ions can to some extent be controlled by determining the energy imparted to the ions in the ionisation process. This is not easy to control in MALDI based techniques which is intrinsically a relatively high energy process, but in electrospray, APCI (Atmospheric Pressure Chemical Ionisation) and FAB based techniques it is relatively easy to control the energy imparted to ions through control of the accelerating potential used.
  • RNA is chemically less stable than DNA but is more resistant to fragmentation in the mass spectrometer. Generally RNA is disliked as a material to work with as it is so easy to contaminate with degrading enzymes in manual experiments . However for automated high throughput sequencing this may not be a significant problem as contamination by RNAses, etc. can be much more rigorously controlled. For use in sequencing one would require terminating ribonucleotides or analogues that are accepted by an RNA polymerase. Such terminators could be generated by synthesising ribonucleotides with the 3' hydroxyl blocked. The blocking group could be a linker to a cleavable mass label identifying the nucleotide.
  • RNA analogues that are resistant to enzymatic degradation and are fragmentation resistant in a mass spectrometer such as 2'-fluoro sugar analogues or 2'-0-methyl sugar analogues. Terminators could be generated as described for ribonucleotides above.
  • DNA carries the appropriate charge If one wishes to use mass labels that take a different charge from DNA, one should ensure that the DNA carries the appropriate charge. To be certain one can tag the DNA with a charge carrier that forms the appropriate ion with a very high probability or is already charged prior to ionisation such as quaternary ammonium ions which could be attached by a fragmentation resistant linkage to a sequencing primer.
  • a charge carrier that forms the appropriate ion with a very high probability or is already charged prior to ionisation such as quaternary ammonium ions which could be attached by a fragmentation resistant linkage to a sequencing primer.
  • a DNA molecule with a mass of the order of 6000 daltons, which is outside the most sensitive range of most instruments, but carrying 3 positive charges will have a mass/charge ratio of about 2000 which falls well into the sensitive range of most mass spectrometers .
  • Tandem separation of mass labelled Sanger Ladders requires that in the first analyser, molecules are separated by length. As mentioned above this has a lower requirement for mass accuracy than conventional approaches. However if a number of labelled templates are to be analysed simultaneously it may be advantageous to normalise base masses, i.e. synthesis nucleotide analogues for adenine, cytosine, guanine and thymine that have the same mass, so that addition of any of the four nucleotides to an oligonucleotide increases its mass by the same amount. This normalisation should allow one to avoid any overlap in masses between labelled molecules of different lengths ensuring that labelled molecules arrive sequentially prior to removal and analysis of the mass label identifying the terminating nucleotide.
  • Mass Spectrometry Techniques Present approaches to direct analysis of Sanger ladders tend to favour the use of MALDI TOF instruments.
  • MALDI approaches generally do not induce fragmentation in ions but the acidic matrices used in much DNA work are believed to be responsible for much fragmentation. Thus unless fragmentation resistant DNA analogues are available or better matrices are found this technique will always face this problem.
  • TOF instruments are limited in the mass accuracy achievable for high molecular weight species. This is exacerbated by the use of MALDI as an ionisation technique as this generates ions with quite a broad kinetic energy distribution, although this problem can be countered to some extent in reflectron instruments.
  • Electrospray ionisation produces ions with a very narrow energy distribution. Furthermore it generally does not induce fragmentation in molecular ions. As DNA is presented to the mass spectrometer in solution one can also avoid acid induced fragmentation by using appropriate buffers. Similarly liquid phase based Fast Atom Bombardment ionisation techniques could be used to generate very restricted ion populations. These techniques may be advantageous to improve mass resolution in higher molecular mass species and in reducing fragmentation.
  • Mass spectrometry is a highly diverse discipline and numerous mass analyser configurations exist and which can often be combined in a variety of geometries to permit analysis of complex organic molecules such as the peptide tags generated with this invention.
  • Tandem mass spectrometry describes a number of techniques in which a ions from a sample are selected by a first mass analyser on the basis of their mass charge ratio for further analysis by induced fragmentation of those selected ions.
  • the fragmentation products are analysed by a second mass analyser.
  • the first mass analyser in a tandem instrument acts as a filter selecting ions to enter the second mass analyser on the basis of their mass charge ratio, such that essentially a species of only a single mass/charge ratio enter the second mass analyser at a time.
  • the selected ion passes through a collision chamber, which results in fragmentation of the molecule.
  • a mass labelled nucleic acid molecule, or group of molecules can be separated from other molecules of different length by a relatively low resolution mass filtering step in the first mass analyser.
  • the mass labels on selected species can then be cleaved from the DNA in a collision induced fragmentation step.
  • the labels can then be analysed in the second mass analyser of the tandem instrument.
  • tandem geometries are possible.
  • Conventional 'sector' instruments can be used where the electric sector provide the first mass analyser stage, the magnetic sector provides the second mass analyser, with a collision cell placed between the two sectors.
  • This geometry is not ideal for peptide sequencing.
  • Two complete sector mass analysers separated by a collision cell could be used for analysis of mass labelled nucleic acids.
  • a more typical geometry used is a triple quadrupole where the first quadrupole filters ions for collision.
  • the second quadrupole in a triple quadrupole acts as a collision chamber while the final quadrupole analyses the fragmentation products.
  • This geometry is quite favorable.
  • Another more favorable geometry is a Quadrupole/Orthogonal Time of Flight tandem instrument where the high scanning rate of a quadrupole is coupled to the greater sensitivity of a TOF mass analyser to identify the products of fragmentation.
  • Ion Trap mass spectrometers are a relative of the quadrupole spectrometer.
  • the ion trap generally has a 3 electrode construction - a cylindrical electrode with 'cap' electrodes at each end forming a cavity.
  • a sinusoidal radio frequency potential is applied to the cylindrical electrode while the cap electrodes are biased with DC or AC potentials.
  • Ions injected into the cavity are constrained to a stable circular trajectory by the oscillating electric field of the cylindrical electrode. However, for a given amplitude of the oscillating potential, certain ions will have an unstable trajectory and will be ejected from the trap.
  • a sample of ions injected into the trap can be sequentially ejected from the trap according to their mass/charge ratio by altering the oscillating radio frequency potential. The ejected ions can then be detected allowing a mass spectrum to be produced.
  • Ion traps are generally operated with a small quantity of a 'bath gas', such as helium, present in the ion trap cavity. This increases both the resolution and the sensitivity of the device by collision with trapped ions. Collisions both increase ionisation when a sample is introduced into the trap and damp the amplitude and velocity of ion trajectories keeping them nearer the centre of the trap. This means that when the oscillating potential is changed, ions whose trajectories become unstable gain energy more rapidly, relative to the damped circulating ions and exit the trap in a tighter bunch giving a narrower larger peaks.
  • a 'bath gas' such as helium
  • Ion traps can mimic tandem mass spectrometer geometries, in fact they can mimic multiple mass spectrometer geometries allowing complex analyses of trapped ions.
  • a single mass species from a sample can be retained in a trap, i.e. all other species can be ejected and then the retained species can be carefully excited by super-imposing a second oscillating frequency on the first.
  • the excited ions will then collide with the bath gas and will fragment if sufficiently excited.
  • the fragments can then be analysed further.
  • One can retain a fragment ion for further analysis by ejecting other ions and then exciting the fragment ion to fragment. This process can be repeated for as long as sufficient sample exists to permit further analysis.
  • an ion trap is quite a good instrument.
  • a sample of mass labelled population of nucleic acids can be injected into a spectrometer.
  • individual 'rungs' can be ejected specifically for cleavage in a collision chamber followed by further analysis in a second mass analyser of a tandem geometry instrument.
  • samples of a mass labelled nucleic acid population can be injected into a trap.
  • a single rung of a ladder, i.e. all species falling within about 100 daltons, or a mass labelled tandem satellite repeat linkage marker could be retained and the labels could be removed by collision induced fragmentation. Specific label species can then be scanned for and ejected from the trap for detection.
  • FTICR mass spectrometry has similar features to ion traps in that a sample of ions is retained within a cavity but in FTICR MS the ions are trapped in a high vacuum chamber by crossed electric and magnetic fields.
  • the electric field is generated by a pair of plate electrodes that form two sides of a box.
  • the box is contained in the field of a superconducting magnet which in conjunction with the two plates, the trapping plates, constrain injected ions to a circular trajectory between the trapping plates, perpendicular to the applied magnetic field.
  • the ions are excited to larger orbits by applying a radiofrequency pulse to two 'transmitter plates 'which form two further opposing sides of the box.
  • the cycloidal motion of the ions generate corresponding electric fields in the remaining two opposing sides of the box which comprise the 'receiver plates'.
  • the excitation pulses excite ions to larger orbits which decay as the coherent motions of the ions is lost through collisions.
  • the corresponding signals detected by the receiver plates are converted to a mass spectrum by fourier transform analysis.
  • these instruments can perform in a similar manner to an ion trap - all ions except a single species of interest can be ejected from the trap.
  • a collision gas can be introduced into the trap and fragmentation can be induced.
  • the fragment ions can be subsequently analysed.
  • fragmentation products and bath gas combine to give poor resolution if analysed by FT of signals detected by the 'receiver plates', however the fragment ions can be ejected from the cavity and analysed in a tandem configuration with a quadrupole, for example.
  • Mass labels that can be used in the present invention include those disclosed in GB 9700746.2, GB 9718255.4, GB 9726953.4, PCT/GB98/00127 and the UK application having Page White and Farrer file number 87820. The contents of these applications are incorporated herein by reference.

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  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention concerne un procédé permettant d'analyser une population de fragments d'acide nucléiques tous marqués par un marqueur de masse. Ce procédé consiste: i.) à ioniser la population; ii.) à trier en sous-populations, en fonction de la masse, la population ionisée dans un spectromètre de masse, chaque sous-population contenant au moins un fragment marqué; iii.) à cliver chaque population de façon à détacher le marqueur de masse associé à chaque fragment marqué; iv.) à évaluer par spectroscopie de masse la masse de chaque marqueur de masse détaché; et v.) à affecter chaque marqueur de masse au fragment qui lui est associé.
EP98942924A 1997-09-15 1998-09-15 Caracterisation d'acide nucleique par spectrometrie de masse Withdrawn EP1015632A1 (fr)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
GB9719638 1997-09-15
GBGB9719638.0A GB9719638D0 (en) 1997-09-15 1997-09-15 High throughput analysis of sanger dna ladders by tandem mass spectrometry
GB9725630 1997-12-03
GBGB9725630.9A GB9725630D0 (en) 1997-12-03 1997-12-03 Charecterising nucleic acid
PCT/GB1998/002789 WO1999014362A1 (fr) 1997-09-15 1998-09-15 Caracterisation d'acide nucleique par spectrometrie de masse

Publications (1)

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EP1015632A1 true EP1015632A1 (fr) 2000-07-05

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EP (1) EP1015632A1 (fr)
JP (1) JP2001516591A (fr)
AU (1) AU746443B2 (fr)
CA (1) CA2303790A1 (fr)
NZ (1) NZ503289A (fr)
WO (1) WO1999014362A1 (fr)

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Publication number Priority date Publication date Assignee Title
DE19824280B4 (de) * 1998-05-29 2004-08-19 Bruker Daltonik Gmbh Mutationsanalyse mittels Massenspektrometrie
GB0006141D0 (en) 2000-03-14 2000-05-03 Brax Group Ltd Mass labels
EP1373561B1 (fr) * 2000-06-13 2009-02-18 The Trustees of Boston University Utilisation de mass-matched nucleotidiques dans l'analyse de melanges d'oligonucleotides et le sequen age hautement multiplexe d'acides nucleiques
ES2296996T3 (es) 2001-09-14 2008-05-01 Electrophoretics Limited Marcaje de moleculas.
US7195751B2 (en) 2003-01-30 2007-03-27 Applera Corporation Compositions and kits pertaining to analyte determination
US20050148087A1 (en) 2004-01-05 2005-07-07 Applera Corporation Isobarically labeled analytes and fragment ions derived therefrom

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US5547835A (en) * 1993-01-07 1996-08-20 Sequenom, Inc. DNA sequencing by mass spectrometry
AU687801B2 (en) * 1993-03-19 1998-03-05 Sequenom, Inc. DNA sequencing by mass spectrometry via exonuclease degradation
GB9504598D0 (en) * 1995-03-03 1995-04-26 Imp Cancer Res Tech Method of nucleic acid analysis
DE69701671T3 (de) * 1996-01-23 2006-08-17 Qiagen Genomics, Inc., Bothell Verfahren und zusammenstellungen zur sequenzbestimmung von nukleinsäuremolekülen
EP0886681A1 (fr) * 1996-03-04 1998-12-30 Genetrace Systems, Inc. Methodes de criblage des acides nucleiques par spectrometrie de masse
WO1997037041A2 (fr) * 1996-03-18 1997-10-09 Sequenom, Inc. Sequençage d'adn par spectrometrie de masse
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AU725966B2 (en) * 1997-01-15 2000-10-26 Xzillion Gmbh & Co. Kg Mass label linked hybridisation probes
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See references of WO9914362A1 *

Also Published As

Publication number Publication date
NZ503289A (en) 2002-11-26
AU9088798A (en) 1999-04-05
JP2001516591A (ja) 2001-10-02
CA2303790A1 (fr) 1999-03-25
AU746443B2 (en) 2002-05-02
WO1999014362A1 (fr) 1999-03-25

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