Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1997-11-24
2001-07-31
Houtteman, Scott W. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C536S024300, C536S025300, C536S026600
Reexamination Certificate
active
06268129
ABSTRACT:
The present invention relates to a method of nucleic analysis, particularly to methods of DNA sequencing and detection of mutations.
The introduction of matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI MS)
1
has opened new possibilities for the application of mass spectrometric analysis in molecular biology and, increasingly, in DNA analysis
2
. DNA has proven to be significantly more difficult to analyze by MALDI MS than peptides and proteins
3
due to very different mass ranges and the quantitative number of negative charges that have to be controlled for mass spectrometric analysis. The displacement of sodium by ammonium counter ions or very stringent desalting protocols improve the resolution significantly
4,5
. Using this improvement restriction enzyme digested fragments can be analysed by matrix assisted laser desorption
6
. Chemically synthesized methylphosphonate oligonucleotides which have an uncharged backbone show good definition by MALDI MS
7,8
. They were detected in positive ion mode while for the analysis of negatively charged oligonucleotides negative ion mode detection was preferable.
The partial alkylation of phosphorothioate containing oligonucleotides for selective chemical backbone cleavage has been described
9,10
and fluorescent reporter groups have been linked to oligonucleotides via phosphorothioate groups
11,12
. Work on the enzymatic incorporation of &agr;-thio nucleotide triphosphates by DNA polymerases has been done for the development of alternative sequencing protocols
13,14
.
To date, a simple and effective method of making nucleic acid, particularly DNA, more suitable for analysis by mass spectroscopy, especially when the said nucleic acid has been enzymatically synthesised, has not been devised.
A first aspect of the invention provides a method of analysing a nucleic acid by mass spectrometry comprising the steps of (1) providing a nucleic acid molecule containing no or up to ten negative charges and no or up to ten positive charges; (2) introducing the said nucleic acid molecule into a mass spectrometer; and (3) determining the mass of the said nucleic acid molecule, wherein when the nucleic acid molecule has no negative charges has greater than 17 sugar-sugar linkages and when the nucleic acid has a charge there are fewer charges than there are sugar-sugar linkages.
Preferably, when the nucleic acid has no negative charges it has >20 sugar-sugar linkages; more preferably >30 sugar-sugar linkages; and still more preferably >50 sugar-sugar linkages.
A second aspect of the invention provides a method of analysing a nucleic acid by mass spectrometry comprising the steps of (1) preparing a nucleic acid molecule comprising a negatively charged non-phosphate sugar-sugar linkage; (2) eliminating the charge from all, or up to all but ten, of the sugar-sugar linkages of the said nucleic acid molecule; (3) introducing the said nucleic acid molecule in which the charge has been wholly or partly eliminated as said into a mass spectrometer; and (4) determining the mass of the said nucleic acid molecule.
It will be appreciated that, although a mass spectrometer primarily determines the mass or mass/charge ratio of a molecule, it is feasible that the output of the mass spectrometer will indicate some other feature of the molecule being analysed such as its relative, rather than its absolute, mass. Therefore, by “determining the mass of the said nucleic acid molecule” we include determination of any physical characteristic derivable from the mass or relative mass of the said nucleic acid molecule.
It is preferred if the nucleic acid provided in the first aspect of the invention or prepared in the second aspect of the invention has no or one charge.
Having more than one charge on the molecule may be useful as it provides a way of increasing the mass range of the mass spectrometer as a mass/charge ratio is analysed. Mixed charges (wherein some parts of the molecule have a positive charge and some parts have a negative charge) can be used for selective neutralisation.
It is further preferred that the nucleic acid is DNA.
By “DNA” we mean a molecule with a sugar-linkage-sugar backbone wherein the sugar residue comprises a 2′-deoxyribose (and therefore includes a DNA chain terminated with a nucleoside comprising a 2′,3′dideoxyribose moiety) and wherein, attached to the sugar residue at the 1 position is a base such as adenine (A), cytosine (C), guanine (G), thymidine (T), inosine (I), uridine (U) and the like. In normal DNA the linkage between sugar residues (the “sugar-sugar linkage”) is a phosphate moiety which forms a diester with the said sugar residues. However, as will be clear from the specification we include in the term “nucleic acid” (and more particularly in the term DNA) molecules with non-phosphate linkages.
Thus, by “a negatively charged non-phosphate sugar-sugar linkage” we include a phosphorothioate linkage and a phosphoroselenoate linkage. A phosphorothioate linkage is most preferred.
By the term “nucleic acid” we also include molecules with non-natural base analogues; molecules in which the 2′ and 3′ positions of the pentose sugar are independently any of —H, —OH or —NH
2
; and molecules in which an oxygen attached to the phosphorus atom but not in phosphodiester linkage is replaced by —SH, SeH, —BH
2
, —NH
2
, —PH
3
, —F, —Cl, —CH
3
, —OCH
3
, —CN and —H. Such molecules are exemplified in FIG.
12
. Replacement by —SH and —SeH is particularly preferred.
It is particularly preferred if the nucleic acid molecule has no phosphate sugar-sugar linkages.
It is also preferred if the nucleic acid molecule has any of one to ten phosphate sugar-sugar linkages; most preferably one.
It is most convenient if the mass spectrometry is matrix-assisted laser desorption ionization time-of-flight mass spectroscopy (MALDI MS) because the ionization conditions for the molecules are less severe than using other forms of mass spectroscopy, such as fast atom bombardment MS (FAB MS). Mass spectrometers, including MALDI mass spectrometers, are well known and are still improving in performance, particularly in mass resolution and sensitivity.
Conveniently, when the nucleic acid is uncharged or positively charged, the mass is determined in positive ion mode (PIM); similarly, when the nucleic acid is negatively charged, the mass is determined in negative ion mode. Indeed, when the nucleic acid molecule is uncharged no signal is detected in NIM.
The molecular masses of nucleic acid molecules as defined in the invention containing from two nucleoside moieties to around 1600 nucleoside moieties can be determined by mass spectroscopy and, as the spectrometers improve, it is envisaged that nucleic acid molecules of the invention of greater molecular mass could be used.
It is most preferred if the nucleic acid molecule whose mass is determined has between 2 and 500 nucleoside moieties; preferably between 5 and 300 nucleoside moieties; and more preferably between 10 and 200 nucleoside moieties.
Conveniently, the mass spectrometer is able to distinguish at least two nucleic acid molecules whose mass differs by the mass of a nucleoside moiety.
Although in some circumstances it may be useful to determine the molecular mass of a single nucleic acid species, it is preferred that a plurality of nucleic acid molecules with differing molecular mass are introduced into the mass spectrometer and the mass of at least one of the said molecules is determined.
As will be discussed in more detail below, it is particularly preferred if the nucleic acid is prepared using an enzymatic chain extension step. It is particularly preferred if a polymerase chain reaction or a chain terminating reagent is used in the preparation of the plurality of nucleic acids.
As is described in more detail below, a particularly preferred embodiment of the invention further comprises the step of determining a nucleotide sequence or detecting a mutation by comparing the masses or mass differences of the said plurality of nucleic acids molecules
Beck Stephan A.
Gut Ivo G.
Houtteman Scott W.
Imperial Cancer Research Technology Limited
Nixon & Vanderhye P.C.
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