Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2002-03-15
2004-08-03
Fredman, Jeffrey (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S320100, C435S252800, C435S174000, C435S183000, C382S129000, C382S133000, C382S153000, C382S173000, C382S286000, C382S291000, C702S019000, C702S022000, C536S022100
Reexamination Certificate
active
06770443
ABSTRACT:
FIELD OF THE INVENTION
The invention concerns methods and equipment for the mass spectrometric measurement of a large number of genotyping profiles, each formed by many SNPs (single nucleotide polymorphisms) in a DNA sample, and for the associated sample preparation.
BACKGROUND OF THE INVENTION
There is a growing demand for easy, fast and economical generation of specified genotyping profiles, each involving the determination of many SNPs in a DNA sample, partially up to several tens or even to several hundreds of SNPs. Such a profile can, for instance, identify a person or an animal, to provide evidence for the responsibility for a crime, or to exclude fraudulent substitutions (for example, in the case of race horses, cattle, pedigree dogs or pigeons). With about 50 SNPs a person can be uniquely identified from a tiny sample of DNA. This kind of profile can also provide evidence of ancestry, as proof of paternity, or as proof of the pedigree of cattle.
A particularly important use of genotype profiles, however, is the preventive detection of predispositions to disease, for instance a tendency towards thrombosis, or for the purposes of individualized medication (the “personal pill”). It is to be expected that the measurement of such genotype profiles with high analytic reliability will play an important role in the medicine of the future. The analytic reliability required for this can, to date, only be guaranteed by mass spectrometry.
Thus the field of this invention is a method for the detection of a large number of specific mutational changes in the genomic DNA in the course of a single, easy analysis process, where the mutation sites themselves are known and are specified for the genotyping task. As far as the mutational sequence changes are concerned, particular attention is paid to the simple exchange of bases (“point mutation”), which has become known recently under the abbreviation “SNP” (single nucleotide polymorphism). For human beings it is believed that at least three million SNPs occur frequently, which characterize many of the individual differences between people and control the individual genetic predisposition.
Usually a “wild type” is defined for a genome, and this “wild type” is considered to be free from mutations. Bearing in mind the frequency of mutations, for instance of SNPs, and the equal validity of the mutated type (mutants) and of the wild type, the definition of the wild type is arbitrary, or at least a matter of chance.
All the mutations of DNA considered here result in a difference in the mass of the segment of DNA containing this mutation as compared with the mass of the corresponding section from the wild type. This means that precise determination of the mass of a segment of DNA can be used to determine a mutation.
Mass spectrometry is an extremely powerful method for measuring the masses of biomolecules. The mass of the ions can be analyzed by mass spectrometry, for instance in time-of-flight mass spectrometers using ionization by matrix assisted laser desorption (MALDI). But ionization by electrospray ionization (ESI) can also be used, although usually in association with mass spectrometers of a different type.
Polymerase chain reactions (PCR) can be used to manufacture selected double-strand DNA products with a minimum length of about 40 base pairs by application of a pair of “selection primers”, single-strand oligonucleotides with a length of about 20 bases, in a known manner. The mutation site must be included by corresponding selection of the sequence of the two selection primers.
The obvious process of using mass spectrometry is simply to measure the mass of DNA products multiplied by PCR and so to determine the mutations. This process has been found almost impossible to implement, because accurate measurement of the masses of DNA products with lengths of more than 40 base pairs has proved impossible in practice. The reasons for this are given below.
Methods have therefore been sought that yield shorter DNA fragments. For this purpose, the process of limited, mutation-dependent primer extension was first developed, which generates extended primers having a length of about 15 or 25 nucleotides, from whose mass the type of mutation can be determined more effectively. Other improvements consist in the removal of a large piece of this extended primer, for instance by enzymatic digestion of a piece; the details of this will not be described any more closely here.
The invention of photo-cleavable linkers brought further progress. The linkers are integrated into the extension primers, and bridge one nucleotide without disturbing either the hybridization or the enzymatic extension, and can be cleaved by means of UV light following preparation of the sample. This allows small fragments with lengths of only 4, 5 or 6 nucleotides to be obtained, and these can be very effectively ionized using matrix assisted laser desorption and ionization (MALDI).
The MALDI preparation and measurement procedure consists in first embedding the analyte molecules on a sample carrier in a solid, UV-absorbing matrix, usually an organic acid. The sample carrier is inserted into the ion source of a mass spectrometer. A short laser pulse, about three nanoseconds in duration, is used to vaporize the matrix into the vacuum; during this process the analyte molecules are transported into the gaseous phase largely, though unfortunately not completely, unfragmented. The molecules of analyte are ionized by proton transfer as a result of impacts with matrix ions that are created at the same time. An applied voltage accelerates the ions into a field-free flight tube. Because of their different masses, the ions are accelerated in the ion source to different velocities. Smaller ions reach the detector earlier than larger ions. The measured flight time is used to calculate the masses of the ions.
MALDI is particularly suitable for the analysis of peptides and proteins. The analysis of nucleic acid chains is more difficult, and is only adequately effective for short-chain nucleic acids. The reason for this is that only a single proton needs to be captured to ionize peptides or proteins to form a positive ion, whereas nucleic acids form a poly-anion with multiple negative charges at the sugar phosphate backbone (one negative charge for each nucleotide), and the ionization process to form a positive ion is significantly less efficient because it needs the transfer of a multitude of protons from a multitude of matrix ions. It is only of adequate efficiency for very short chains, such as for the cleavage products of the extended primers, as can be created with the aid of photo-cleavable linkers.
It is a well-known and favorable embodiment for the analysis of genotyping profiles of DNA samples to use chips on which sufficient numbers of different types of extension primers are bonded in separate locations as probes for the selected SNPs of the genotyping profile. In association with the use of cleavable primers, as discussed above, this therefore provides a powerful tool for the mass spectrometric analysis of genotyping profiles.
SUMMARY OF THE INVENTION
The invention consists of providing a multitude of chips held simultaneously in a combining structure, as well for the sample preparation in a matching multitude of processing wells and as for the joint mass spectrometric analysis for the determination of numerous genotyping profiles. For this purpose the chips are held by the combining structure in such a way that they can be fed as a rigid unit to the multitude of wells with DNA samples as well as to the mass spectrometric analysis. The multitude of wells can, for instance, be a microtitre plate.
The combining structure can be a flat plate containing the chips as parts of its surface and which is pressed closely onto the processing wells, such as the wells of a microtitre plate, so that it comes into contact with the liquid inside the wells when the structure with the processing wells is inverted.
The linking structure is, however, preferably a plate on which the chips sit rigidly on sm
Bruker Daltonik GmbH
Chakrabarti Arun
Fredman Jeffrey
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