Characterizing nucleic acid by mass spectrometry

Details Characterizing nucleic acid by mass spectrometry Characterizing nucleic acid by mass spectrometry

2000-05-04

2001-08-07

Fredman, Jeffrey (Department: 1655)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S091100, C436S173000, C536S023100

Reexamination Certificate

active

06270976

ABSTRACT:

FIELD OF THE INVENTION
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.
BACKGROUND OF THE INVENTION
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 each nucleic acid carries.
BRIEF SUMMARY OF THE INVENTION
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”.
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 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-thio dNTPs instead of the ddNTPs used in a conventional Sanger sequencing reaction. These are included with the normal dNTPs in a primer extension reaction mediated by a DNA polymerase. The four sets of base terminating ladders is obtained by including one of the 4 alpha-thio dNTPs in 4 amplification reactions followed by limited digestion with exonuclease III or snake venom phosphodiesterase. (Labeit et al., 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 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.


REFERENCES:
patent: 94/16101 (1994-07-01), None
patent: WO 94/21822 (1994-09-01), None
patent: WO 96/27681 (1996-09-01), None

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