Note: Descriptions are shown in the official language in which they were submitted.
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CHARACTERISING NUCLEIC ACID BY MASS SPECTROMETRY
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.
Methods of single step determination of the mass of nucleic acids
in the mass spectrometer have been developed mainly for
sequencing (H. Koster et al., Nature Biotechnology 14, 1123 -
1128, 1996). There are, however, a number of problems with the
direct analysis of DNA in a mass spectrometer at present. One is
fragmentation of the DNA. The longer a molecule to be analysed
is, the greater the degree of fragmentation. This gives rise to
mass spectra that are very difficult to interpret. However
improvements are envisaged, using modified nucleotide analogues
that are resistant to fragmentation within a mass spectrometer.
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
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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:
(i) ionising the population;
(ii) sorting the ionised population in a mass spectrometer
according to mass into sub-populations each containing
at least one labelled fragment;
(iii)cleaving each sub-population to release the mass label
associated with each labelled fragment;
(iv) determining the mass of each released mass label by
mass spectroscopy; and
(v) assigning each mass label to its associated fragment.
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. Alternatively, in certain 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".
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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.
- Alternatively, 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.
In the step of determining the mass of each released mass label
by mass spectroscopy 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. Alternatively,
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.
In a preferred arrangement, the nucleic acid fragments and/or
mass labels are fragmentation resistant.
In one embodiment, the population of nucleic acid fragments is
produced from a method of DNA sequencing such as disclosed in GB
9719284.3. In such a method, a template strand of DNA., typically
a primed template, is contacted with nucleotides in the presence
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of DNA polymerase to produce a series of fragments containing all
possible lengths of a strand of DNA complementary to the template
strand of DNA. Thus, the population of nucleic acid fragments
- for analysis comprises the series of fragments. Typically, 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. By sorting the
ionised population comprising the series of fragments according
to mass, the respective length of each member of the series can
be determined and/or related to the nucleotide. This enables the
sequence of the strand of DNA to be determined.
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-thin dNTPs instead of the ddNTPs
used in a conventional Sanger sequencing reaction. These axe
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., DNA 5, 173-177, 1986; Amersham, PCT-Application GB86/00349;
Eckstein et al., Nucleic Acids Research 16, 9947, 1988). Rather
than labelling the primers or the alpha-thio dNTPs with a
radioisotope, as disclosed in these previous documents, a mass
label is used to identify each ladder and the resultant ladders
are analysed by tandem mass spectrometry in this embodiment.
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
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sequencing method that increases their concentration will improve
the sensitivity of the mass spectrometry analysis of these
fragments.
In a preferred embodiment 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. In this way, 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. For the purposes of this
invention 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.
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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. Thus one can
anticipate, further orders of magnitude improvements in
sequencing capacity in such a system over that described in
PCT/GB98/02048. 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.
The problems associated with methods that exploit direct analysis
of DNA molecules by mass spectrometry can be overcome by this
invention. The problem of complex spectra due to fragmentation
can be partially solved by improved fragmentation resistant
analogues of DNA but further improvement is achievable with mass
labelled molecules. 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. Since 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
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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.
This invention offers improvements over present techniques with
regard to these problems. 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. In contrast a fluorinated molecule whose integral
mass is 100 will tend to have a fractionally lower real mass.
These differences in mass are distinguishable in a high accuracy
mass analysis and two molecules with the same integral mass but
different compositions will produce distinct peaks in the mass
spectrum if they have different degrees o~f hydrogenation and
fluorination. Since fluorinated molecules are not common in
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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.
From the point of view of instrumentation, 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.
Sizing applications of Tandem MS of mass labelled nucleic acids
A variety of sizing assays based on labelling nucleic acids is
applicable with this technology. 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.
DNA sizing methods described in earlier patents that are also
compatible with tandem MS.
GB 9714715.1
GB 9707980.0
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GB 9714716.9.
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.
Multiplexing Sanger Ladder Detection
Given a large number of mass labels one can multiplex the
analysis of a series of Sanger sequencing reactions. One can
analyse Sanger ladders derived from different templates
simultaneously as long as their terminating bases are labelled
with a discrete set of labels or they are identifiable by
uniquely labelled primers. Multiplexed Sanger ladders may be
generated simultaneously in the same reaction or in spatially
discrete reactions followed by pooling of templates depending on
the format used.
Labelled Nucleotides
One can label the 4 terminating nucleotides with a different set
of 4 mass labels in each reaction that is to be multiplexed. In
the simplest scenario one must spatially separate each template
and its corresponding labels. 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.
This approach is necessary if RNA polymerases are used in
conjunction with ribonucleotides or their analogues since most
RNA polymerases use promoter sequences rather than primers and
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so incorporation of labels would have to be effected via labelled
nucleotides.
- The use of labelled nucleotides is 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.
One can clearly use labelled primers as well, but this requires
that each template be sequenced separately in four reactions, one
for each terminator which is less advantageous except for
multiplexing numerous templates in the same reactions which is
discussed below.
Preparation of templates With unique primers or promoters:
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
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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. Consider a large DNA fragment such as a
mitochondrial genome or a cosmid. One can cleave such a large
molecule with a frequently cutting restriction enzyme to generate
fragments of the order of a few hundred bases in length. If one
uses a restriction endonuclease like Sau3Al one is left with
fragments with a known sticky end to which one can ligate
adapters bearing a known primer sequence.
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. After adaptering and any amplification,
one denatures the adaptered fragments and hybridises these
fragments to a 'capture' primer. 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. At this stage
one would divide one's template into four separate pools in order
to sequence each pool with a different terminating nucleotide.
The captured fragments are made double stranded at this stage by
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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. One can also amplify the sequence present at this
stage by further hybridisation with capture primer.
After denaturing free DNA from the immobilised copies of the
template and disposing of free DNA, one can add a series of
'sequencing' primers to the reaction. These 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.
With the majority of templates primed with a unique primer one
can add polymerase, nucleotide triphosphates and one of the four
blocking nucleotides to each reaction and can generate Sanger
ladders. If a thermostable polymerase is used, then the ladders
can be denatured at the end of each cycle and fresh primers can
be added. If cycle sequencing is used then one would almost
certainly want some means to select for properly terminated
fragments since cycle sequencing not only amplifies the number
of properly terminated fragments but also the number of
improperly terminated fragments.
The Sanger ladders from each of the four sequencing reactions are
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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.
This approach is appropriate for use with DNA analogues which use
a DNA polymerase and a primer sequences.
Multiplexing with nucleotide labelled reactions
A further embodiment of this invention is generating multiple
template ladders simultaneously in the same reactions with
labelled nucleotides.
Consider a reaction in which unmodified ATP, CTP, GTP and TTP are
present with the four corresponding uniquely mass labelled
terminating nucleotides. One can generate Sanger ladders for a
number of templates simultaneously in the same reaction vessel.
If these different templates share a common sequence, either the
sequencing primer or a length of sequence after the RNA
polymerase that is common to all templates, they can be
subsequently sorted into separate groups prior to separation on
the basis of the sequence immediately adjacent to the common
sequence. One could separate the fragments onto a hybridisation
array where the array bears a sequence complementary to the
common sequence at all points and an additional predetermined
number of bases, N, such that each location on the array bears
just one of the possible N base sequences. This means if N is 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.
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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. Thus
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.
Alternatively 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. Such
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.
Having sorted ladders to discrete locations on an array one needs
to separate the ladders from each location and identify the mass
labels that terminate each set of fragments of each length. How
one does this would depend on the array used.
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Practically speaking 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. In this situation a sample of the pooled
reactions is added to each well and allowed to hybridise to the
immobilised oligonucleotide present in the well. After a
predetermined time 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.
Equally, and preferably, 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. (A.C.Pease et a1. Proc. Natl. Acad. Sci.
USA. 91, 5022-5206, 1994. according to South method: U. Maskos
and E.M. Southern, Nucleic Acids Research 21, 2269-2270, 1993.
E.M. Southern et al, Nucleic Acids Research 22, 1368-1373, 1994).
One could hybridise the pooled Sanger ladders to the chip and
wash away unhybridised material. 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.
Again, the advantage of multiplexing and sorting templates is the
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ability to -avoid a number of sub-cloning steps in a large scale
sequencing project. One would prepare template as described above
for primer labelled multiplexing but at the stage when sequencing
- primer is added, the primers used would not be mass labelled. If
RNA polymerases are to be used then the adaptors would bear a
promoter sequence for the polymerase rather than a primer
sequence. An additional length of common sequence after the
promoter would also be needed for sorting purposes.
One can also use engineered vectors to ensure that each template
bears a unique sequencing primer site or a promoter with a unique
sequence adjacent to it. 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.
Fragmentation of DNA
The mechanism of fragmentation of nucleic acids in the mass
spectrometer is currently thought to involve protonation of the
nucleobase, which leads to cleavage of the N-glycosidic bond and
consequent loss of the base. This leaves the exposed sugar
phosphate backbone exposed and prone to further cleavage
resulting in fragmentation of the nucleic acid molecule as a
whole. (L. Zhu et al, J. Am. Chem. Soc. 117,6048 - 6056, 1995).
Various chemical modifications to the sugar and nucleobases have
been shown to increase stability of DNA in the mass spectrometer.
(Tang, Zhu and Smith, Anal. Chem. 69, 302 -312, 1997).
Modifications shown to be effective include modifications at the
2'-hydrogen of the deoxyribose sugar ring, where electron
withdrawing groups are seen to stabilise the N-glycosidic bond.
2'-hydroxyl and 2'-fluoro groups~are seen to partially and almost
completely block fragmentation, respectively. 2'-hydroxyl groups
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give RNA or a nucleic acid with arabinose as the sugar component.
These modifications were tested in chemically synthesised
oligonucleotides in the reference above. These modified
- nucleotides are not accepted by currently available enzymes and
will probably require engineering of polymerases to accept them
but will permit much higher resolution separation in the mass
spectrometer of nucleic acid bearing these modifications. This
in turn will permit mass labelled Sanger ladders of the sort
described here to be separated by direct mass spectrometry with
less fragmentation, massively increasing throughput. Other
modifications that reduce base loss are N7-deaza modifications
of adenine and guanosine groups which are accepted by
polymerases. (F, Kirpekar et al, Rapid Commun. Mass Spectrom. 8,
727 -730, 1994 and H. Ktister et al, Nature Biotechnology 14, 1123
- 1128, 1996) .
It should be noted that the discussion above regarding
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.
The problem of complex spectra due to fragmentation can be
partially solved by improved fragmentation resistant analogues
of DNA but further improvement is achievable with mass labelled
molecules. Mass labels can be chosen to take a different charge
to DNA in the mass spectrometer. This means that after cleavage
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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.
In positive mass spectrometry this gives:
( 1 ) [R1-RZ-label ] + -> Rl+ + RZ-label or
(2 ) [Rl-RZ-label ] + -> Rl + RZ-label+
Or alternatively in negative ion spectrometry:
( 1 ) [R1-RZ-label ] - -> Rl- + RZ-label or
(2) [Rl-R21abe1]- > Rl + RZ-label-
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
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the neutral species on the right, as discussed below. For the
purposes of sequencing one can label either the 3' terminus, if
labelled nucleotide terminators are used, or one can label the
5' terminus, by using labelled primers. One can thus choose the
format which minimises noise through favouring fragmentation
pathway in equation (1). Furthermore, 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.
This analysis is over-simplified but is sufficient to illustrate
the principle that mass labels can offer an advantage in avoiding
some of the problems with fragmentation. Oligonucleotide
fragmentation is a reasonably complicated process and is not
fully understood although L. Zhu et al (J. Am. Chem. Soc.
117,6048 - 6056, 1995) have elucidated a possible mechanism of
nucleotide fragmentation in MALDI based systems. The distribution
of charge on fragmentation ions was not clearly determinable from
their results, however cleavage appears to be favoured at the 3'
C-0 bond between deoxyribose and the phosphodiester linkage,
leaving a phosphate group on the 3' end fragment. For positive
ion mode sequencing in the first mass analyser, this may be
advantageous as the appearance of a negative ion will not be
detected. This would favour nucleotide labelled sequencing over
primer labelled sequencing.
RNA based sequencing
One possible fragmentation resistant DNA 'analogue' that already
has appropriate polymerases is of course RNA. RNA is chemically
less stable than DNA but is more resistant to fragmentation in
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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.
To avoid the problems of RNA sensitivity to enzymatic
degradation, one can use RNA analogues that are resistant to
enzymatic degradation and are fragmentation resistant in a mass
spectrometer such as 2'-fluoro sugar analogues or 2'-O-methyl
sugar analogues. Terminators could be generated as.described for
ribonucleotides above.
Mass Resolution
The problem of mass resolution faced by direct techniques can be
greatly reduced by the use of mass labels.
Charge carrying non-cleavable tags
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.
One might also use multiply charged tags attached to sequencing
primers to increase the charge on a DNA molecule so that its mass
charge ratio is reduced. This would increase mass resolution by
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ensuring that higher mass molecules can be analysed in the most
sensitive detection range of a given mass spectrometer. Thus 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.
Equalisation of Base Masses
Tandem separation of mass labelled Sanger Ladders according to
this invention 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.
Furthermore, if one wishes to use labels with masses greater than
the mass of a single nucleotide, normalisation would be
beneficial. One could then use a pair of 'calibration ladders'
bearing the lightest and heaviest mass labels to demarcate the
'arrival envelope' of labelled molecules of a given length if
desired. Such envelopes could overlap for molecules of differing
lengths, but as long as any given template is labelled with a set
of labels that are close in mass, they will always arrive in the
correct order.
Mass Spectrometry Techniques
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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. Furthermore 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 Analyser Geometries
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.
Analysis of Mass Labelled Nucleic Acids by Tandem Mass
Spectroircetry
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
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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. On
leaving the first mass analyser, the selected ion passes through
a collision chamber, which results in fragmentation of the
molecule.
ION SOURCE -> MS1 -> COLLISION CELL -> MS2 -> ION DETECTOR
If appropriate fragmentation resistant analogues are used and a
suitably fragmentation labile linker is used to couple a mass
label to a nucleic acid 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.
Various 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
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high scanning rate of a quadrupole is coupled to the greater
sensitivity of a TOF mass analyser to identify the products of
fragmentation.
Ion Traps
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.
Ion traps can mimic tandem mass spectrometer geometries, in fact
they can mimic multiple mass spectrometer geometries allowing
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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. It should
be noted that these instruments generally retain a high
proportion of fragment ions after induced fragmentation. These
instruments and FTICR mass spectrometers (discussed below)
represent a form of temporally resolved tandem mass spectrometry
rather than spatially resolved tandem mass spectrometry which is
found in linear mass spectrometers.
For nucleic acid sequencing and other nucleic acid sizing
applications, an ion trap is quite a good instrument. A sample
of mass labelled population of nucleic acids can be injected into
a spectrometer. For a Sanger ladder, 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. Alternatively samples of a mass labelled
nucleic acid population can be inj ected 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.
Fourier Transform Ion Cyc.Iotron Resonance Mass Spectrometry
(FTICR MS)
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
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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.
For induced fragmentation experiments 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.
Generally 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.
For nucleic acid sequencing and other nucleic acid sizing
applications FTICR MS could be used and may be advantageous as
these instruments have a very high mass resolution for molecules
of significant size.
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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.
