JP2001516591A - Method for analyzing nucleic acids by mass spectrometry - Google Patents

Abstract

  (57) [Summary] The present invention provides a method of analyzing a population of nucleic acid fragments, each labeled with a mass label, the method comprising: i) ionizing the population of nucleic acid fragments; ii) ionizing the population using a mass spectrometer. Sorting the population of nucleic acid fragments into subpopulations, each containing at least one labeled nucleic acid fragment, according to mass; iii) cleaving the nucleic acid fragments of each subpopulation, Releasing the mass labels to be labeled; iv) determining the mass of each released mass label by mass spectrometry; and v) assigning each mass label to its labeled nucleic acid fragment.

Description

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method for analyzing a nucleic acid. The method is advantageous because different nucleic acid fragments can be analyzed simultaneously.

Methods for determining the mass of nucleic acids in a single step in a mass spectrometer have been developed primarily for sequencing (H. Koester et al., Nature Biotec).
hnology 14, 1123-1128, 1996). However, there are currently many problems with directly analyzing DNA with a mass spectrometer. One is DN
A fragmentation. The larger the molecule to be analyzed, the greater the degree of fragmentation. This results in a mass spectrum that is very difficult to interpret. However, it is envisaged to use modified nucleic acid analogs that are resistant to fragmentation in mass spectrometers, but this is not yet sufficient. .

[0003] A further and most significant problem is accurately measuring the mass of some large biomolecules. This resolution problem places a limit on the length of DNA sequence reads that can be achieved to a significant degree. At present, the absolute limit for direct mass spectrometry of the Sanger ladder is about 100 bases long, about 30-40 bases for practical purposes.

[0004] GB9719284.3 describes the use of nucleic acid hybridization probes cleavably linked to mass labels for the analysis of nucleic acids. GB9
7192844.3 describes a method of sequencing nucleic acids using mass-labeled primers or nucleotides to create a Sanger ladder.
In the present sequencing method, capillary electrophoresis mass spectrometry is used as a mass spectrometry method for analyzing the resulting mass-labeled Sanger ladder. These methods require a two-stage analysis. A sizing step to determine the length of each nucleic acid (ie, the number of nucleotides in a linear sequence) in the population, followed by the identification of the mass tag carried by each nucleic acid.

The present invention is a method for analyzing a population of nucleic acid fragments, each labeled with a mass label, comprising ionizing the population of nucleic acid fragments, using a mass spectrometer to analyze the population of ionized nucleic acid fragments. Subdividing, according to mass, into subpopulations each containing at least one labeled nucleic acid fragment, cleaving the nucleic acid fragments of each subpopulation, releasing mass labels that label the nucleic acid fragments, The present invention relates to a nucleic acid analysis method, comprising: determining the mass of each released mass label by mass spectrometry; and assigning each mass label to the labeled nucleic acid fragment.

[0006] The population of nucleic acid fragments can be ionized in any suitable manner. Electrospray ionization is particularly useful because it can be ionized directly from a solution of labeled nucleic acid fragments.

[0007] The ionized population is fractionated, and from the nucleic acid fragments contained in each sub-population,
Subsequent steps of cleaving the mass labels and determining the mass of each released mass label can be performed in designated areas of the mass spectrometer. Alternatively, in certain mass spectrometer configurations, such as those found in ion capture mass spectrometers or Fourier transform ion cyclotron resonance analyzers, the identification of each fractionated, cleaved, and released mass label is time-dependent. Are individual but occur in the same area.

[0008] The step of sorting the ionized population may be performed by applying a magnetic field, preferably an electromagnetic field such as a quadrupole, hexapole or twelve pole. Alternatively, the step of collecting the ionized population may be performed by an ion capture or ion cyclotron device. It is possible to combine electric and magnetic fields in order to carry out the preparative process. The step of cutting each sub-population may comprise collision or, for example,
This can be done in the cutting area by light cutting using a laser. Choosing how to perform the cleavage step will depend to some extent on how the mass label is linked to the nucleic acid fragment. Mass labels are generally photocleavable or digested easily, and are designed to be automatically cleavable upon collision with gas phase concentrations or solid surfaces in a mass spectrometer. It is linked to the nucleic acid fragment via a possible linker.

In the step of determining the mass of each emitted mass label, any suitable mass spectrometer arrangement can be used. In this step, the released mass labels are generally separated from one another, followed by detection. Separation can be achieved by any means used in mass spectrometers, such as magnetic fields, preferably electromagnetic fields including quadrupoles, hexapoles or dodecapoles. Alternatively, a time-of-flight arrangement can be used to use the time to separate the released mass labels from each other. Detection can be by any suitable means.

[0010] In a preferred configuration, the nucleic acid fragment and / or mass label is resistant to fragmentation.

[0011] In one embodiment, the population of nucleic acid fragments is generated by a method for sequencing DNA as disclosed in GB9719284.3. In this method, a template strand of DNA (generally a primed template) is contacted with nucleotides in the presence of a DNA polymerase to produce all possible lengths of DNA complementary to the template strand of DNA. A series of nucleic acid fragments containing the strands are generated. Thus, the population of nucleic acid fragments used in the analysis includes the set of nucleic acid fragments. Typically, each nucleic acid fragment is terminated in a mass spectrometer to identify nucleotides with a nucleotetide that cleavably binds to a corresponding specifically degradable mass label.
By sorting the ionized population containing a series of nucleic acid fragments according to mass, each length can be determined in each member of the series and / or the terminal nucleotides can be related. Thereby, the sequence of the DNA strand can be determined.

[0012] In a further embodiment of the present invention, a conventional Sanger sequencing strategy is used with modifications, which include digestion of DNA nucleic acid fragments containing thiophosphates.
This sequencing method utilizes α-thio dNTPs instead of the ddNTPs used in conventional Sanger sequencing reactions. These are included with normal dNTPs in a DNA polymerase-mediated primer extension reaction. Inclusion of one of the four α-thio dNTPs in four amplification reactions followed by restriction digestion with exonuclease III or snake venom phosphodiesterase yielded a ladder population with four bases at the ends. Can be (Labeit et al., D
NA 5, 173-177, 1986; Amersham, PCT-Ap
application GB86 / 00349; Ecksten et al., Nuclei.
c Acids Research 16, 9947, 1988). In this embodiment, each ladder is identified using a mass label instead of identifying a primer or α-thiodNTP by a radioisotope, as disclosed in the above-mentioned literature, and the obtained ladder is , Analyzed by tandem mass spectrometry.

The present sequencing method has the advantage that a DNA fragment having a high molecular weight is predominantly formed. In contrast, in the conventional Sanger sequencing, as the length of the DNA fragment increases, the DNA fragment containing each end decreases exponentially. Since mass spectrometers have reduced sensitivity to high molecular weight species, sequencing methods that increase their concentration will improve the sensitivity of such nucleic acid fragments for mass spectrometry.

In a preferred embodiment, the population of nucleic acid fragments is provided on a chip, typically a glass chip, each member of the population being at a remote location on the chip. The chip can be treated with a MALDI matrix material. By irradiating the population with laser light to ionize it, the nucleic acid fragments may be advantageous. In the present method, the nucleic acid fragments located in distant regions on the chip or the group consisting of the nucleic acid fragments can be released from the chip by appropriately assigning a laser beam spatially. Laser release of nucleic acid fragments can be performed in an exhaust chamber, which is typically part of a mass spectrometer.

[0015] The present invention describes the use of tandem mass spectrometry techniques and the use of cleavable mass labels as detection methods for nucleic acid sequencing and analyzing the molecular weight of nucleic acids. Capillary electrophoresis mass spectrometry uses capillary electrophoresis to determine the length of nucleic acids in a population, followed by ionization of the capillary electrophoretically separated eluate and cleavage of mass labels from the nucleic acids, and then The mass label is analyzed by mass spectrometry. Similarly, size separation by molecular weight, label cleavage and label analysis steps can be performed in a tandem mass spectrometer. Tandem mass spectrometry refers to various techniques in which ion vapor passes through multiple mass spectrometry steps. For the purposes of the present invention, a large number of labeled nucleic acids can be separated by length in a first mass spectrometer in tandem arrangement. Thereafter, the mass label is cleaved from the nucleic acids of interest between the first and second mass spectrometers. The cleaved mass labels are finally analyzed in the second mass spectrometry stage of the means.

The tandem mass spectrometry approach is highly desirable, and separation occurs in a fraction of a second, compared to capillary electrophoresis mass spectrometry separations which take on the order of tens of minutes to one hour. Therefore, a significant improvement can be expected over the system described in PCT / GB98 / 02048. Methods based on capillary electrophoresis
While facing the same problems as gel electrophoresis based separations for nucleic acid sizing, such problems can be significantly suppressed in capillary systems. These problems include broadening of the band due to temperature effects, compression due to the secondary structure of the template nucleic acid, and heterogeneity of the separation gel. When determining the mass of a nucleic acid molecule, these problems could be avoided even at a low resolution for determining the length of the nucleic acid molecule.

The present invention can solve the problems associated with direct analysis of DNA molecules by mass spectrometry. The problem of fragmentation producing complex spectra is that
Although some can be solved by improving nucleic acid analogs resistant to DNA fragmentation, further improvements are achieved with mass-labeled molecules. The mass label can be selected to have a different charge than the DNA in the mass spectrometer. This means that after cleaving the label from the corresponding DNA molecule, the label can be exclusively selected for analysis in the second mass spectrometer by using an appropriate mode of analysis. . DNA tends to form ions with a net positive charge, so the negative ion mode is generally more effective.
Furthermore, if a scanning mass spectrometer, such as a quadrupole, is used for the second mass spectrometric component, the analyzer is optional because it can filter out any nucleic acid fragment noise. Since the labels are molecules that have been characterized in detail, picking up signals from these labels in tandem analysis is generally simplified. Since ionization is a statistical process in nature, small amounts of background noise will result from fragmented DNA carrying the label. However, by modifying the energy imparted to the ions, it is potentially possible to advantageously fragment neutral labeled nucleic acid fragments that do not appear in any of the spectra.
Alternatively, one can simply select mass labels that accommodate the same charge as the corresponding DNA molecule, but their peaks in the mass spectrum do not correspond to DNA fragmentation products.

The present invention provides improvements in current technology on these issues. The problem of mass resolution is particularly acute in sequencing DNA length by single-stage mass spectrometry, whose terminal bases are determined by accurately measuring the mass of the molecule. The measurement requires that the mass of one dalton be accurately determined. The present invention is directed to a tandem scheme in which a first mass spectrometer determines the length of a DNA ladder. The analyzer only needs to have a mass resolution on the order of 300 daltons, and then cuts labels to identify terminal nucleotides in the collision chamber, or
Or it has another fragmentation induction step. The cleaved label is then identified on a second mass spectrometer. The label may be a small molecule and can be analyzed in a second mass spectrometer at high resolution.

An advantageous embodiment of the present technology is to use fluorinated mass labels when high-resolution mass spectrometry of the labels after cleavage from nucleic acids. Hydrogenated molecules with an integrated mass of 100 have a slightly higher net mass when measured at very high resolution. In contrast, hydrogenated molecules with an integrated mass of 100 have a slightly lower net mass. Such mass differences are identifiable in highly accurate mass spectrometry, where two molecules with the same integral mass but different compositions have mass spectra where they have different degrees of hydrogenation and fluorination. Shows different peaks. Because fluorinated molecules are not common in biological systems, fluorinated mass labels are used in mass spectrometry, even if there are fragmentation or contaminant peaks due to buffers, unless the nucleic acids and reagents used are fluorinated. Can be identified.

An important feature of the present invention is a mechanism for cleaving a label from a nucleic acid labeled with a mass label, which is performed after the first mass spectrometry step. Collision-induced dissociation of labels from the corresponding DNA is one method of cleavage currently used for peptide sequencing. An alternative is photon-induced cleavage of mass labels from DNA.

From an equipment standpoint, tandem mass spectrometers typically have a linear configuration in which individual components perform their respective processes and ion vapor goes from one component to the next. As described below, multiple arrangements of the linear device are possible. However, certain instruments, such as ion capture instruments and Fourier transform ion cyclotron mass spectrometers, allow all of these steps to occur in a single component.

Sizing Application of Tandem MS of Mass-Labeled Nucleic Acids Various sizing assays based on labeling of nucleic acids are also applicable with the present technology. A DNA sizing assay equivalent to capillary electrophoresis mass spectrometry described in PCT / US97 / 01046 is equally applicable to tandem mass spectrometry applications. These include, but are not limited to, differential display, restriction fragment length polymorphism, and linkage analysis.

Patent Documents (GB971475.1, GB9707980.0, GB9714
The DNA sizing method described in 716.9) is equally applicable to tandem MS.

The present invention is extremely advantageous for high-power analysis of mass-labeled DNA molecules. This is because the molecule can be analyzed very quickly. Further, in the present invention, multiple labeled nucleic acids can be multiplexed and delivered. The degree of multiplex delivery is limited only by the number of mass labels available and resolvable in the mass spectrometer.

Multiplexed Sanger Ladder Detection In the presence of many mass labels, the analysis of a series of Sanger sequencing reactions can be multiplexed. Sanger ladders derived from different templates can be analyzed simultaneously, as long as the terminal bases are labeled with a different set of labels or the terminal bases can be identified by specifically labeled primers. Multiplexed Sanger ladders can occur simultaneously in the same reaction or spatially different reactions, followed by pooling of templates depending on the format used.

Labeled nucleotides In a simultaneous reaction, the four terminal nucleotides can be labeled with a different set of four mass labels for each reaction to be delivered. In the simplest model, each template and its corresponding label must be spatially separate. Each sequencing reaction is performed separately, and then all templates are combined at the end of the sequencing reaction. Next, the generated Sanger
The ladders are singulated together in a tandem mass spectrometer using the soft ionization technique described below. Each set of four mass labels then correlates to a single source template.

This approach is necessary when RNA polymerase is used with ribonucleotides or their analogs. Because most RNA polymerases use a promoter sequence rather than a primer, the incorporation of a label is affected by the labeled nucleotide.

[0028] The use of labeled nucleotides is a preferred embodiment in that labeled nucleotides avoid certain potential problems with primer labeled sequencing. The polymerase reaction is often terminated incompletely without the intervention of blocked nucleotides. This is a problem with primer labeled sequencing. This is because incomplete termination results in a background of labeled nucleic acid fragments where the termination is incorrect. Labeling the blocking nucleotides will label correctly terminated nucleic acid fragments, thus ensuring that they are detected by the mass spectrometer. This, in turn, allows for cyclic sequencing in which multiple rounds of primer are added to the template. The sequencing reaction is
This is performed using a thermostable polymerase. After each reaction, the mixture is heat denatured,
The additional primer is annealed to the template. The polymerase reaction is repeated as the primer-template complex is reformed. Repeating this process multiple times linearly amplifies the signal, increasing the reliability and quality of the resulting sequence. This is an advantage over direct mass spectrometry techniques, which have to deal with incomplete terminated products. The incompletely terminated product appears in the mass spectrum and may read the base incorrectly.

Although labeled primers can be used explicitly, this requires that each template be sequenced individually in four reactions, and one type of terminator is described below. As described above, it is disadvantageous except in the case of multiplex delivery of many templates in the same reaction.

Preparation of templates with specific primers or promoters: To allow simultaneous sequencing reactions by mass labeling, distinguish the Sanger ladder generated for each template from the Sanger ladder generated from other templates. is necessary. This can be achieved using sequencing primers that are specifically labeled for each template. To ensure that each template has a unique sequencing primer site, the set of cloning vectors used for sequencing should have different primer binding sequences at each end of the DNA cloning site. Each sequencing reaction is performed on a group of templates. Here, there is only one template derived from each vector type, so that all templates in the reaction have unique primers.

Adapter to Induce Primers to Restriction Enzyme Fragments However, to sequence many templates simultaneously, the subcloning step can be omitted from the sequencing method as follows. This method is particularly useful for large DNA nucleic acid fragments such as the mitochondrial genome or cosmid.
Such a large DNA molecule has many cleavage sites, and a fragment group of about several hundred bases can be prepared by using a restriction enzyme. For example, Sau3A1
By using a restriction enzyme such as described above, it is possible to bind a known sticky end to a known primer sequence.

As a result, the restriction enzyme fragment has an adapter at each end. If necessary, the restriction nucleic acid fragment to which the adapter has been added at this stage can be amplified. After adapter addition and amplification, the adapter-added nucleic acid fragments are denatured and these nucleic acid fragments are hybridized to "capture" primers. The capture primer is biotinylated and hybridizes in solution to the free adapter-added nucleic acid fragment. As a result, the DNA fragment can be captured on the solid support to which avidin is bound. Alternatively, in advance, the primer is immobilized on a solid support,
Thereafter, it can be reacted with the nucleic acid fragment to which the adapter has been added. At this stage,
The template may be split into four separate pools and each pool sequenced at a different terminal nucleotide.

[0033] The capture fragment is obtained in duplex by reaction with a polymerase. This means that all copies of the sequence are immobilized. The hybridized captured strands can be melted and released at this stage and removed if desired. Also, further hybridization with the capture primer can amplify the sequence present at this stage.

[0034] The free DNA from the immobilized copy of the template can be denatured and a series of "sequencing" primers added for a nucleic acid sequencing reaction. These primers are added with an adapter, a restriction enzyme cleavage site and a predetermined number of bases. When using 64 available labels, the added base is 3 bases. Each of the added three base overlaps can be identified by a unique mass label. 5
With a population on the order of 0-60, most would be expected to have different 3-mers flanked by ligated primers. Thus, most of the template is expected to hybridize to different primers. Even if there are different template DNAs that bind to the same primer, if the amount of each template can determine which template is assigned to the base reading, the sequence can be assigned to each template. it can.

If the majority of the template is primed with a unique primer, a Sanger ladder is created by adding a polymerase, nucleotide triphosphates and one of the four blocking nucleotides to each reaction. can do. If a thermostable polymerase is used, the ladder can be denatured at the end of each cycle and new primer can be added. When using cyclic sequencing, some means of selecting properly terminated nucleic acid fragments will almost certainly be sought. This is because cyclic sequencing not only amplifies the number of properly terminated nucleic acid fragments, but also the number of improperly terminated nucleic acid fragments.

The Sanger ladders, preferably from each of the four sequencing reactions, are then pooled, and the ES ladders are used to avoid ambiguity in base assignment due to experimental error.
Analyze simultaneously by tandem mass spectrometry. Thus, each pool of template
You should have primers labeled with different sets of mass labels. Therefore, a total of 256 mass labels are required. Thus, each primer has four labels and each of the four labels involves a terminator reaction. The labels assigned to each primer should be similar in mass and size and the differences in migration between each termination reaction should be minimal.

This approach is suitable for methods that use DNA polymerases, primers, and DNA analogs.

Multiple Delivery by Nucleotide Labeling Reaction A further embodiment of the present invention is to make multiple template ladders simultaneously with the same reaction using labeled nucleotides.

It should be taken into account that unmodified ATP, CTP, GTP and TTP are present with four corresponding specific mass labeled terminal nucleotides. In the same reaction vessel, Sanger ladders for many templates can be made simultaneously. If these different templates have a common sequence (either the sequencing primer or a sequence of a certain length after all RNA polymerase binding sites), then the base sequence immediately next to the common sequence Prior to separation on the basis of these, they can be classified into individual groups. That is, the nucleic acid fragments can be separated on the hybridization array. In this method, here, a complementary sequence common to the sequence used for hybridization and a predetermined number (N) of bases, and at each point of the array, only one possible N It has a base sequence. This means that if N is 4, then 256 different positions on the array are needed. In a group of templates, the sequences immediately adjacent to the primers will most likely be different.

It is expensive to classify molds from only one reaction vessel. However, given the large number of mass labels, it is possible to have different sets of four mass labels that identify blocking nucleotides in many reactions. Thus, a plurality of molds, preferably different molds, can be added to different reaction vessels. After creating the Sanger ladder in each reaction vessel, the reactants can be pooled and the templates from each reaction can be sorted simultaneously. The majority of ladders for each template from each reaction separate into different locations on the array, and each location on the array contains template ladders from many different reactions.

Alternatively, different primers can be linked to a “sorting sequence” and a length of oligonucleotide can be used to sort the ladder with different primers on a hybridization chip. Such classifier sequences should ideally be non-complementary to one another to prevent cross-hybridization with each other, and should also have minimal cross-hybridization with the complement of all other classifier sequences. A minimal cross-hybridization set of oligonucleotides is fully discussed in PCT / US95 / 12678. Using a series of sequencing templates identified by different primers linked to different taxonomic sequences, the same reaction in the same reaction with the same labeled nucleotide terminator
An angel ladder can be made. The resulting Sanger ladder can then be sorted on a hybridization array containing a sequence complementary to the sorting sequence. This allows each Sanger ladder identified by a particular primer to be classified at a different location on the array.

When the ladders are classified at different positions on the array, it is necessary to separate the ladders from each position and identify mass labels that terminate each set of nucleic acid fragments of each length. How this is done depends on the array used.

In practice, a hybridization array is created in an array of wells on a microtiter plate. Each well contains a single immobilized oligonucleotide. In this case, a sample of the pooled reaction is added to each well and hybridized to the immobilized oligonucleotide present in the well. After a predetermined time, unhybridized DNA is washed away. The hybridized DNA is then thawed and separated from the capture oligonucleotide and injected into an electrospray interface following a tandem mass spectrometer.

Similarly, and preferably, Southern methodology or Affymet
Rix (Santa Clara, California) methodology, or one of the hybridization arrays in discrete locations on a glass chip.
Using related inkjet technology to guide one member, the glass "
Arrays can be synthesized combinatorially on a "chip". (A.C.
Pease et al., Proc. Natl. Acad. Sci. USA. 91, 50
22-5206, 1994, according to the South method: U.S. Pat. Maskos and E
. M. Southern, Nucleic Acids Research 2
1, 2269-2270, 1993; M. Southern et al., Nuc
leic Acids Research 22, 1368-1373, 1
994). The pooled Sanger ladder is hybridized to the chip, and unhybridized material can be washed away. The chip can then be treated with a MALDI matrix material such as 3-hydroxypicolinic acid. Once a chip has been prepared by this method, it is loaded into a MALDI-based tandem mass spectrometer, and Sanger ladders from individual locations on the array are applied by applying laser light to the desired locations on the array. Can be released.
For direct release of DNA from the hybridization matrix, Ko
Demonstrated by Ster et al. (Nature Biotech. 14,
1123-1128). Analyzing the length of the nucleic acid fragment in a first mass spectrometer;
Subsequently, the label can be cleaved and analyzed in a second mass spectrometer.

Further, the advantage of multiple delivery and classification of templates lies in the ability to avoid many subcloning steps required in large-scale sequencing schemes. In the primer-labeled multiplex delivery, the template is prepared as described above, but at the stage of adding the sequencing primer, the primer used is not mass-labeled. If an RNA polymerase is to be used, the adapter has a promoter sequence for the polymerase rather than a primer sequence. An additional length consensus sequence after the promoter is also
Required for classification purposes.

The modified vector can also be used so that each template has a promoter with a specific sequence adjacent to or adjacent to a specific sequencing primer site. A group of cloning vectors used for sequencing is DNA
It is necessary to have different primer binding sequences at both ends of the cloning site. Each sequencing reaction is performed on a group of templates. Here, there is only one template derived from each vector type, such that all templates in the reaction have good primers.

DNA Fragmentation The mechanism of nucleic acid fragmentation in mass spectrometry is currently thought to involve nucleobase protonation, which results in cleavage of the N-glycosidic bond and thereby loss of the base. ing. This exposes the exposed sugar phosphate backbone for further cleavage, resulting in overall nucleic acid analysis fragmentation. (L. Zhu et al., J. Am
. Chem. Soc. 117, 6048-6056, 1995).

Various chemical modifications to sugars and nucleobases allow D
It has been shown that NA stabilization increases. (Tang, Zhu and Smith, Anal. Chem. 69, 302-312, 1997).
. Modifications that have been shown to be effective include modification of the deoxyribose sugar ring at the 2′-hydrogen, where it is apparent that the electron leaving group stabilizes the N-glycoside bond. Have been. 2'-hydroxyl and 2'-
Fluoro groups have each been found to partially or almost completely block fragmentation. The 2'-hydroxyl group results in RNA or nucleic acids having arabinose as the sugar component. These modifications were tested with chemically synthesized oligonucleotides in the references above. These modified nucleotides are not accepted by currently available enzymes, and their manipulation probably requires the manipulation of a polymerase, but mass spectrometry of nucleic acids with these modifications is extremely high. Resolution separation is possible. Similarly, this
The mass-labeled Sanger ladders of the classes described herein can be separated by a direct mass spectrometer with reduced fragmentation and significantly increased power. Other chemical modifications that suppress base loss are N-deaza modifications of adenine and guanine groups, which are accepted by the polymerase. (F, Kirpekar et al., Rapi.
d Commun. Mass Spectrom. 8, 727-730,
1994 and H.C. Koster et al., Nature Biotechnolo
gy 14, 1123-1128, 1996).

The above considerations regarding DNA fragmentation apply particularly to the use of MALDI technology, in that the mechanism of protonation leading to cleavage is believed to be exacerbated by the matrix used to ionize nucleic acids. . Because many of these are mildly acidic compounds such as cinnamic acid derivatives, 2,5'-dihydroxybenzoic acid. The matrix, 3-hydroxypicolinic acid, is
It has been shown that there is less fragmentation than most that improve the performance of the ALDI-based approach. However, mass labeling techniques are also highly comparable to ESI-based approaches, where the control of buffers and ionization conditions can suppress protonation problems.

The complex spectral problem of fragmentation can be solved to some extent by improving fragmentation resistant analogs of DNA, but further improvements are achieved with mass-labeled molecules. The mass label can be selected to have a different charge than DNA in the mass spectrometer. This means that after cleaving the label from the corresponding DNA molecule, the label can be exclusively selected for analysis in the second mass spectrometer. Since only the label is analyzed in the second mass spectrometer, most DNA nucleic acid fragments do not appear on the spectrum or can be distinguished from DNA fragmentation products if the label has the same charge as DNA. They are selected to have mass so that they can be easily identified. However, there is still a small background from DNA nucleic acid fragments that carry a label that can be handled to some extent by the present invention. Fragmentation of a single charged species occurs by "mild" ionization techniques, such as electrospray, MALDI and FAB, generally forming charged and uncharged nucleic acid fragments.

[0051] In the positive mass spectrometry, which _{is, (1) [R 1 -R} 2 - ^{_{label] + -> R 1 + +}} R 2 - label or _{(2) [R 1 -R 2} - label ] +-> R ₁ + R ₂ -labeled substance + Or, in negative mass spectrometry, (1) [R ₁ -R ₂ -labeled substance]--> R ₁ + R ₂ -labeled substance or [R ₁ -R ₂ label] ----> R ₁ + R ₂ -label-

A DNA nucleic acid fragment without a label, whether charged or uncharged, is not recognized in the second mass analysis layer, or has a mass dependent on the label used. It should be possible to resolve from the peak of the label. Uncharged species with labels are also not found in the final spectrum. If the fragmentation pathways in (1) and (2) are of equal probability and unambiguous, the fragmentation noise is expected to be halved compared to the noise observed in Sanger ladder direct mass spectrometry, Ion formation is not determined with equal probability but by the heat of formation of the species involved. In general, the stability of the bond is analyzed by comparing the heat of formation of the ionic species on the left side of the above equation with the heat of formation of the neutral species on the right side. For the purpose of sequencing, it is possible to label either the 3 'end when using a labeled nucleotide terminator, or to label the 5' end by using a labeled primer. be able to. Thus, by favoring the fragmentation path of equation (1), one can choose a form that minimizes noise. Further, fragmentation of molecular ions can be controlled by determining the energy imparted to the ions in the ionization process. While control in technology based on MALDI, which is essentially a relatively high energy process, is not easy, electrospray, APCI (Atmosphere)
ic Pressure Chemical Ionization) and F
In AB-based technology, by controlling the accelerating potential used,
The energy applied to the ions can be controlled relatively easily.

Although the analysis is oversimplified, it is sufficient to illustrate the principle that mass labels can be advantageous in avoiding some of the problems associated with fragmentation.
Oligonucleotide fragmentation is a rather complex process and is not fully understood, but is described in L. et al. Zhu et al. (J. Am. Chem. Soc. 117, 604).
8-6056, 1995) elucidate the possible mechanism of nucleotide fragmentation in MALDI-based systems. Although the distribution of charge on the fragmented ions could not be clearly determined from their results, cleavage was favored at the 3'CO bond between the deoxyribose and phosphodiester bonds and the 3 ' It appears to release phosphate groups on nucleic acid fragments. This may be advantageous for sequencing positive ion forms in the first mass spectrometer. This is because the appearance of negative ions is not detected. This would be advantageous for nucleotide labeled sequencing over primer labeled sequencing.

RNA-Based Sequencing As fragmentation resistant DNA “analogs” already having the appropriate polymerases,
RNA can be mentioned. RNA is more chemically unstable than DNA,
It is more resistant to fragmentation in a mass spectrometer than DNA. Generally, RNA is not preferred as a material to be handled because digestive enzymes are easily contaminated in manual experiments. However, this is not a significant issue in automated high-power sequencing, where contamination with RNAse and the like can be controlled fairly tightly. For use in sequencing, terminal ribonucleotides or analogs that are accepted by RNA polymerase are required. Such terminators can be made by synthesizing 3 'hydroxyl blocked ribonucleotides. The blocking group may be a linker to a cleavable material label that identifies the nucleotide.

To avoid the problem of RNA sensitivity to enzymatic digestion, 2′-fluorosugar analogs or 2′-O-, which are resistant to enzymatic digestion and fragmentation in a mass spectrometer.
RNA analogs such as methyl sugar analogs can be used. Terminators can be made as described for ribonucleotides above.

Mass Resolution The problems with mass resolution encountered with direct technology can be significantly reduced by using mass labels.

Charges Carrying Non-Cleavable Tags If it is desired to use a mass label that yields a different charge from DNA
One should ensure that the DNA carries the proper charge. It ensures that it is possible with very high probability to form the appropriate ions or to tag the DNA with a charged carrier that is already charged prior to ionization. Charge carriers include, for example, quaternary ammonium ions, which can be bound by fragmentation resistant ligation to sequencing primers.

[0058] Multiply charged tags attached to a sequencing sequence may be used to increase the charge on the DNA molecule such that the mass to charge ratio is reduced. This increases mass resolution by ensuring that higher mass molecules can be analyzed in the most sensitive detection range of a given mass spectrometer. Thus, while a DNA molecule having a mass on the order of 6000 daltons is outside most of the sensitivity range of most instruments, if it possesses three positive charges with a mass / charge ratio of about 2000, most mass spectrometers Well within the sensitivity range of

Base Mass Equalization Tandem separation of the mass-labeled Sanger ladder according to the present invention requires that the molecules be separated by length in a first analyzer. As noted above, the present invention requires less mass accuracy than conventional approaches. However, if one intends to analyze many labeled templates simultaneously, the mass of the bases, i.e., adenine, cytosine, guanine and guanine, will be such that adding any four nucleotides to the oligonucleotide will increase the mass by the same amount. It may be advantageous to normalize the synthetic nucleotide analog for thymine. This normalization avoids any overlap in mass between labeled molecules of different lengths and ensures that the labeled molecules arrive in sequence order prior to removal and analysis of mass labels identifying terminal nucleotides. Can be assured.

In addition, if it is desired to use a label having a mass greater than the mass of a single nucleotide, normalization is beneficial. The "correction ladder" pair with the lightest and heaviest mass labels can then be used to define the "reach" for a given length of labeled molecule, if desired. Such ranges overlap for molecules of different lengths, but as long as any given template is labeled with a set of labels of similar mass, it will always arrive in the correct order.

Mass Spectrometry Technology The current approach to direct analysis of the Sanger ladder is MALDI T
There is a tendency to use OF instruments preferably. Although the MALDI approach generally does not induce fragmentation at the ion, the acidic matrix used in many DNA studies is believed to be responsible for many fragmentations. Thus, the technology always faces this problem unless fragmentation resistant DNA analogs are available or a better matrix is found. In addition, TOF devices are limited in that high molecular weight species are required to achieve mass accuracy. MAL as ionization technology
The use of DI has an adverse effect on this. This is because this results in ions having a rather broad kinetic energy distribution. However, the reflector (ref
Electron instrument can suppress this problem to some extent.

[0062] Electrospray ionization produces ions with a very narrow energy distribution. Further, the electrospray ionization generally does not induce fragmentation at the molecular ions. Since DNA is subjected to mass spectrometry in solution, acid-induced fragmentation can also be avoided by using appropriate buffers. Similarly, very limited ion populations can be created using fast atom bombardment ionization techniques based on liquid phases. These techniques may be advantageous for improving the mass resolution of higher molecular mass species and inhibiting fragmentation.

Mass Spectrometer Geometry Mass spectrometry has a very diverse field, many mass spectrometer configurations exist, often can be combined in a variety of geometries, and include complexities such as peptide tags generated in the present invention. Enables the analysis of various organic molecules.

Analysis of Mass-Labeled Nucleic Acids by Tandem Mass Spectrometry Many techniques have been described for tandem mass spectrometry, in which a first mass spectrometer based on mass-to-charge ratio selects ions from a sample, Further analysis is performed by inductive fragmentation of the ion selected by. The fragmentation products are analyzed by a second mass spectrometer. The first mass spectrometer of the tandem instrument acts as a filter to select ions to enter the second mass spectrometer based on the mass-to-charge ratio, and thus has essentially a single mass / charge ratio The species enters the second mass spectrometer at a particular time. On leaving the first mass spectrometer, the selected ions pass through the collision chamber, causing molecular fragmentation. Ion supply source->MS1-> Collision cell->MS2-> Ion detector

When using a suitable fragmentation-resistant analog and attaching a mass label to a nucleic acid molecule using a suitable fragmentation-labile linker, the mass-labeled nucleic acid molecule, or group of molecules, comprises a first A relatively low resolution mass filtration step in a mass spectrometer can separate from other molecules of different lengths. The mass label for the selected species can then be cleaved from the DNA in a collision-induced fragmentation step. The label can then be analyzed in the second mass spectrometer of the tandem instrument.

Various tandem geometries are possible. Conventional "sector" equipment can be used. Here, the electrical sector provides a first mass spectrometer stage, the magnetic sector provides a second mass spectrometer, and a collision cell is located between the two sectors. This geometry is not ideal for peptide sequencing. Two complete sector mass spectrometers separated by a collision cell can be used for the analysis of mass labeled nucleic acids. The more typical geometry used is the first
Are quadrupoles that filter ions for collision. The second of the triple quads acts as a collision chamber, and the last quadrupole analyzes the fragmentation products. This geometry is quite favorable. Another more preferred geometry is a quadrupole / orthogonal time-of-flight tandem instrument, where the high operating speed of the quadrupole is combined with a more sensitive TOF mass spectrometer to generate fragmentation. Identify things.

Ion Capture Ion capture mass spectrometers are related to quadrupole mass spectrometers. Ion capture types generally have a three electrode construction (a cylindrical electrode with a "cap" electrode forming a gap at each end). A sinusoidal radio frequency potential is applied to the cylindrical electrode, while the cap electrode is biased with a DC or AC potential.
By vibrating the electric field of the cylindrical electrode, ions implanted in the gap are restrained in a stable circulation path. However, for a given amplitude of the oscillating potential, certain ions have an unstable path and emit from capture. By altering the oscillating radio frequency potential, a sample of the ions injected into the capture can be emitted from the capture in sequence according to the mass / charge ratio. The released ions can then be detected and a mass spectrum can be generated.

[0068] Ion capture is generally operated by a small amount of "bath gas" such as helium present in the ion capture cavity. This increases the resolution and sensitivity of the device due to the impact of the captured ions. Both collisions increase ionization as the sample is introduced into the capture, attenuating the amplitude and velocity of the ion path and holding it near the center of the capture. This means that as the oscillating potential changes, the ions whose path has become unstable compared to the attenuated circulating ions gain energy more quickly and escape to the capture in tighter bundles, making them narrower and larger. It means that a peak occurs.

Ion capture can approximate the geometry of tandem mass spectrometers, and indeed they can approximate the geometry of multiple mass spectrometers, allowing for complex analysis of the captured ions. A single mass species from the sample can be retained in the capture, i.e., radiating all other species and then carefully superimposing the retained species by superimposing the second vibration frequency on the first vibration frequency. Can be excited. The excited ions then collide with the bath gas and fragment if sufficiently excited. The nucleic acid fragments can then be further analyzed. The release of other ions and subsequent fragmentation into nucleic acid fragments can retain nucleic acid fragment ions for further analysis. This process can be repeated as long as there are enough samples to allow for further analysis. It should be noted that these devices generally retain a high percentage of nucleic acid fragment ions after induction of fragmentation. These instruments and the FTICR mass spectrometer (discussed below) use a temporally resolved tandem mass spectrometer instead of the spatially resolved tandem mass spectrometer found in linear mass spectrometers. Present.

For nucleic acid sequencing and other nucleic acid sizing applications, ion capture is a fairly good instrument. A sample of the mass-labeled population of nucleic acids can be injected into the analyzer. For the Sanger ladder, for cutting in the collision chamber followed by further analysis in a second mass spectrometer of the tandem geometry instrument,
Individual "bars" can be specifically released. Alternatively, a sample of the mass-labeled nucleic acid population can be injected into the capture. The ladder can carry a single bar (ie, all species fall within about 100 daltons) or a mass-labeled tandem satellite repeat ligation marker, and the label can be removed by collision-induced fragmentation. The specific labeled species can then be scanned and emitted from the capture for detection.

Fourier Transform Ion Cyclotron Resonance Mass Spectroscopy (FTICR MS) FTICR mass spectroscopy has features similar to ion capture in that a sample of ions is retained in the void, whereas FTICR MS uses an electric and magnetic field By crossing the ions are captured in the high vacuum chamber. The electric field is generated by a pair of plate electrodes forming both sides of the box. The box is contained within a field of a superconducting magnet, and the capture plate is combined with the two plates in the field and injected into the circulation between the capture plates perpendicular to the applied magnetic field. Restrain ions. By applying radio frequency pulses to two "transmitting plates" that form two further opposing faces of the box, the ions are excited into larger orbits. The circulating movement of the ions produces corresponding electric fields in the remaining two opposing faces of the box containing the "receiver plate". The excitation pulse excites the ions into larger orbitals and decay as a consistent movement of the ions lost through collisions. The corresponding signal detected by the receiver plate is converted to a mass spectrum by Fourier transform analysis.

For derivation of fragmentation experiments, these instruments operate in a manner similar to the ion capture type, and can emit from the capture all but a single species of interest.
Impacting gases can be introduced into the capture to induce fragmentation. Thereafter, the nucleic acid fragment ions can be analyzed. When the signal detected by the "receiver plate" is analyzed by FT, the resolution is generally reduced when the fragmentation product and the bath gas are combined, but the nucleic acid fragment ions are emitted from the gap, for example, to form a quadrupole. Can be analyzed in a tandem configuration.

For nucleic acid sequencing and other nucleic acid sizing applications, the FTI
Advantageously, a CR MS can be used. Because these instruments have very high mass resolution for molecules of significant size.

The mass label that can be used in the present invention includes GB970074
6.2, GB9718255.4, GB9726953.4, PCT / GB98
/ 00127 and Page White and Farrer file number 8
No. 7820. The mass labels disclosed in the UK application having No. 7820. The contents of these applications are incorporated herein by reference.

[Procedural Amendment] Submission of translation of Article 34 Amendment of the Patent Cooperation Treaty

[Submission date] March 15, 2000 (2000.3.15)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Claims

[Correction method] Change

[Correction contents]

[Claims]

──────────────────────────────────────────────────続 き Continuation of front page (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE ), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GE, GH, GM, HR, HU, ID, IL, IS, JP, KE, KG, KP , KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZWF term (reference) 4B024 AA11 AA20 CA01 CA20 HA08 HA11 HA19 4B063 QA01 QA12 QA13 QQ42 QR08 QR13 QR14 QR32 QR42 QR62 QS25

Claims (16)

[Claims] 1. A method for analyzing a population of nucleic acid fragments each labeled with a mass label, comprising ionizing the population of nucleic acid fragments, using a mass spectrometer to analyze the population of ionized nucleic acid fragments. Sorting according to mass into sub-populations, each containing at least one labeled nucleic acid fragment, cleaving the nucleic acid fragments of each sub-population to release a mass label that labels the nucleic acid fragment, releasing A nucleic acid analysis method, comprising: determining the mass of each of the identified mass labels by mass spectrometry; and assigning each of the mass labels to the labeled nucleic acid fragment. 2. The method according to claim 1, wherein the step of cleaving the nucleic acid fragments of each subpopulation is performed in a mass spectrometer. 3. The nucleic acid analysis method according to claim 1, wherein the nucleic acid population is generated by performing a Sanger nucleic acid sequencing reaction using the nucleic acid as a template. 4. The Sanger so that each nucleic acid fragment generated in the Sanger sequencing reaction contains a specific mass label at its terminal nucleotide.
The nucleic acid analysis method according to claim 3, wherein each terminal nucleotide used in the er sequencing reaction is labeled with a mass label specific to the nucleotide. 5. The method according to claim 5, wherein the nucleic acid population is prepared using a plurality of nucleic acids as a template.
The nucleic acid analysis method according to claim 4, comprising a nucleic acid fragment generated from an angel sequencing reaction. 6. The nucleic acid analysis method according to claim 5, wherein the nucleic acid population is generated by pooling the nucleic acid fragments generated from a plurality of individual Sanger sequencing reactions. 7. The set of labels used in each Sanger sequencing reaction is specific to that Sanger sequencing reaction, and
The nucleic acid analysis method according to claim 6, wherein a template for the Sanger sequencing reaction is identified. 8. The method according to claim 5, wherein each Sanger sequencing reaction is performed simultaneously in the same reaction, and the template is identified by sorting the nucleic acid fragment according to the nucleotide sequence of the nucleic acid fragment. Nucleic acid analysis method. 9. The nucleic acid analysis method according to claim 1, wherein each of the Sanger sequencing reactions is performed using all four terminal nucleotides in the same reaction. 10. The nucleic acid analysis method according to claim 3, wherein the terminal nucleotide used in the Sanger sequencing reaction includes ddNTP. 11. The nucleic acid population is subjected to a PCR reaction of a nucleic acid template in the presence of dNTP and α-thio-dNTP to generate a nucleic acid containing dNTP and α-thio-dNTP, and the resulting nucleic acid is converted to an endonuclease or an endonuclease. The nucleic acid analysis method according to claim 1 or 2, which is a nucleic acid fragment obtained by treating with snake venom phosphodiesterase and digesting the nucleic acid. 12. Each α-thio-dNTP used in a PCR reaction is specific for said nucleic acid such that each nucleic acid fragment produced after digestion contains a specific mass label at the terminal nucleotide of the nucleic acid fragment. The nucleic acid analysis method according to claim 11, comprising a mass label. 13. The nucleic acid analysis method according to claim 1, wherein the nucleic acid fragment and / or the labeled product is resistant to fragmentation. 14. The mass label for labeling a nucleic acid fragment such that the label has a different charge from the nucleic acid fragment when subjected to mass spectrometry. 3. The nucleic acid analysis method according to 1. 15. The nucleic acid analysis method according to claim 1, wherein the mass label has a negative charge when subjected to mass spectrometry. 16. The nucleic acid analysis method according to claim 1, wherein the mass label is a fluorinated mass label.