Mutation analysis using mass spectrometry

Details Mutation analysis using mass spectrometry Mutation analysis using mass spectrometry

1999-05-27

2003-01-07

Fredman, Jeffrey (Department: 1634)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S091100, C536S025300, C536S025400, C436S173000, C436S175000

Reexamination Certificate

active

06503710

ABSTRACT:

FIELD OF INVENTION
The invention presents a method for examining genetic material (desoxyribonucleic acid, DNA) to detect the presence of pre-known mutations, especially single nucleotide polymorphisms (SNPs), using mass spectrometry with ionization by matrix-assisted laser desorption (MALDI).
PRIOR ART
Subject of the invention is a method for the identification of mutations within a certain sequence of the genomic DNA of an organism, either for single mutations or for several mutations simultaneously. These mutative changes of the DNA sequence may be base exchanges (“point mutations”, often called SNP=“single nucleotide polymorphisms”), the introduction of bases (“insertions”), loss of bases (“deletions”) or even changes in the chemical nature of a base by, for example, methylation.
In order to characterize mutations clearly, a DNA sequence in which a mutation is supposed to have taken place must be sequenced from the beginning. In order to find an identified mutation in another individual, resequencing the corresponding DNA section is the best defined form of analysis available. In practice, this would mean that the identification of a known mutation in a subject would cost the same as the original characterization. Different forms of gel electrophoreses are used for the sequencing, but these are slow, expensive and not fully automated.
For this reason, alternative methods were developed for the identification of known mutations. For example, for the identification of many known mutations simultaneously, corresponding DNA sequences could be collected by fixing them onto the surface of a DNA chip. Their hybridization or nonhybridization with added genetic material can be used for the simultaneous identification of various different mutations. Thus, chips with 64,000 fixed sequences have become known. This DNA chip technology, however, has a few significant disadvantages, the most important being the high cost of making the DNA chips. Because determination of a huge number of mutations of an individual is not always necessary, this is not exactly an economic diagnostic method for a certain defined disease.
There is still a need for a method for the rapid recognition of mutations where a moderate degree of multiplex capability would be desirable yet would not be absolutely necessary, if the speed of the individual process is high.
Mass spectrometry using matrix-assisted laser desorption and ionization (MALDI) is a very powerful tool for analyzing biomolecules. The ions can be analyzed for their masses spectrometrically, for example, in a time-of-flight mass spectrometer. Because the speed of flight of the ions is about 10
7
times faster in the mass spectrometer than the speed of migration of the molecules in the electrophoretic gel, the mass-spectrometric method is extremely fast in comparison to the electrophoretic method, even when measurement of the spectrum is repeated 10 to 100 times to achieve a good signal-to-noise ratio.
The whole MALDI preparation and measurement procedure consists first of embedding the analyte molecules on a sample carrier in a solid, UV-absorbent matrix which is usually an organic acid. The sample carrier is placed in the ion source of a mass spectrometer. The matrix is vaporized by a short laser pulse of around 3 nanoseconds and the analyte molecule is thereby transported into the gas phase in a nonfragmented state. The analyte molecule is ionized by colliding and reacting with the matrix ions generated at the same time. A voltage is applied which accelerates the ions in a field-free flight tube. Due to their different masses, the ions in the ion source are accelerated to different speeds— the smaller ions reaching the detector earlier than the larger ions. The time of flight is converted into the mass of the ions.
Technical innovations in the hardware have significantly improved the time-of-flight mass-spectroscopic method using MALDI. The delayed acceleration (or extraction) method of MALDI ions improves the signal resolution in one place on the spectrum (e.g. U.S. Pat. No. 5,510,613). By subjecting the acceleration voltage to additional dynamic changes, good resolution can be achieved within a wide range of measurement (DE 196 38 577).
MALDI is particularly suitable for the analysis of peptides and proteins; the analysis of nucleic acids is somewhat more difficult. For nucleic acids, the ionization yield in the MALDI process is approximately 100 times less than for peptides and decreases strongly with increasing mass. For the ionization of peptides and proteins, only a single proton needs to be captured. For nucleic acids, which carry many negative charges on their backbone, all these negative charges have to be neutralized, before a further proton creates a positive ion; the ionization by the matrix is therefore significantly less efficient.
For MALDI, the choice of matrix is important. For the desorption of peptides, there are many very efficient matrices. A few effective matrices have been discovered for DNA in the meantime but the extremely low sensitivity compared to proteins was not improved.
The low sensitivity for DNA can be improved by chemically modifying the DNA so that it resembles a peptide. As explained in WO96/2781, phosphorothioate nucleic acids, for example, for which the usual phosphates on the backbone are substituted with thiophosphates, can be converted to a neutral DNA through simple alkylation chemistry, and the chemical covalent bonding of a single positively or negatively charged chemical group (“charge tag”) to this modified DNA increases the sensitivity so that it is within the range found for peptides.
These modifications have made it possible to utilize similar matrices to those used for the desorption of peptides. Another advantage of “charge tagging” is the increased independence of the analysis upon impurities which significantly interfere with the identification of unmodified DNA analysis samples.
A new method of mutation diagnostics using MALDI mass spectrometry has recently become known (Little, D. P., Braun, A., Darnhofer-Demar, B., Frilling, A., Li, Y., McIver, R. T. and Köster, H: Detection of RET proto-oncogene codon 634 mutations using mass spectrometry. J. Mol. Med. 75, 745-750, 1997). The primer (a DNA chain which functions as an identification sequence) is synthesized so that it will attach itself near to a known point mutation on the template strand by hybridization. Between the position of this point mutation and the 3′ end of the primer (this end is elongated by a polymerase), the sequence of the template strand may consist of a maximum of three of the four nucleo-bases only. At the position of the point mutation, the fourth base appears for the first time. Using polymerase and a particular set of deoxynucleotide triphosphates (which complement the three nucleo-bases which occur between primer and point mutation) and a didesoxynucleotide triphosphate (which is complementary to the potential point mutation) the primer is elongated (or “extended”) by duplication. The didesoxynucleoside triphosphate terminates the chain elongation by the polymerase reaction. Depending on whether the point mutation is present or absent, the polymerase reaction is either terminated at the point mutation position or at the next appropriate base adjacent to the potential mutation site. This process (WO 96/29 431, claim 47), which includes attachment of the primer to the surface, has been designated as “PROBE” assay by the authors.
Because unmodified DNA was used for the analysis during this work, a significant disadvantage with this method seemed to be that a relatively large amount of enzymatically generated DNA material had to be made in order to produce signals that could be detected in the analysis which followed. In addition to this, the PCR product had to be immobilized in a solid phase so that the primer, the template strand and the salts and the detergents from the polymerase reaction which would greatly affect the final analysis, can be washed away.
OBJECTIVE OF THE INVENTION
The objective of the inv

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