Simple SNP analysis using mass spectrometry

Details Simple SNP analysis using mass spectrometry Simple SNP analysis using mass spectrometry

1999-11-12

2003-07-08

Riley, Jezia (Department: 1687)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S091100, C435S091200, C536S022100, C536S025300

Reexamination Certificate

active

06589735

ABSTRACT:

The invention relates to a method of investigating by mass spectrometry the genetic material deoxyribonucleic acid (DNA) replicated by polymerase chain reaction (PCR), for the identification of known mutations and polymorphisms; it particularly relates to the analysis of single nucleotide polymorphisms (SNPs) by matrix assisted laser desorption and ionization (MALDI).
The invention consists of using a set of nucleoside triphosphates for the selective PCR replication of the DNA in which one or more of the nucleoside triphosphates have been made much heavier by attaching a chemical group, but in such a way that the replication is not disturbed by the polymerase. In this way a single nucleotide polymorphism in DNA pieces with a length of about 40 to 50 bases can very easily be made visible by mass spectrometry without any further manipulation.
BACKGROUND OF THE INVENTION
Subject of this invention is a method for easily and quickly detecting mutative changes at certain known points of the genomic DNA of an organism. Special consideration is here given to polymorphisms where with a statistical frequency a single base exchange is to be found at a certain point in the genome. This type of polymorphism has in recent years been given the designation “single nucleotide polymorphism” (SNP).
SNPs have in the meantime acquired considerable importance for genotyping. It is assumed that the human genome contains about 3 million such SNPs. Therefore there are about 3 million points at which with a statistical frequency a base is exchanged for a different base. Such a base exchange can take place within a gene or in non expressed areas between the genes. Therefore, and due to the large redundancy of the genetic code, an SNP can be without any phenotypical effect. Certain forms (so-called alleles) of SNPs can, however, also be linked to a phenotypical variation, e.g. by the exchange of an amino acid in a protein, by a change in the gene expression or its regulation etc. The phenotypical variation can, for example, be expressed in a changed tolerance to environmental influences, a changed pharmaceutical effect, or, under extreme circumstances, in a genetically conditioned disease. SNPs inherit half from the father and half from the mother so SNPs can also be applied in individual analysis (genetic passport).
SNPs acquire increasing importance for genotyping and particularly for the coupling analysis of multicausal diseases. The higher frequency in the genome and the thus possible denser marker network, as well as the lower mutation rate compared with the STR markers (short tandem repeats) used to date represent a considerable advantage.
The basis for detecting such and other mutations is the selective PCR (polymerase chain reaction), a replication method for DNA pieces in the test tube, which was only developed by K. B. Mullis in 1983 (who was awarded the Nobel Prize for it in 1993) and after introduction of temperature-stable polymerases began an unprecedented march to victory through the genetic laboratories.
PCR is the targeted replication of a piece of the double-stranded DNA (dsDNA) accurately selected by the replication method itself. Selection of the DNA segment is performed by a pair of so-called primers, two single-stranded DNA pieces (ssDNA) each having a length of about 20 nucleotides, which (described somewhat briefly and simplified) hybridize at both ends (the future ends) of the selected DNA piece. Enzymatic replication is performed by a DNA polymerase, which represents a chemical factory inside a molecule, by passing through a simple temperature cycle. The PCR reaction takes place in aqueous solution in which a few molecules of the original DNA and sufficient quantities of DNA polymerase, primers, nucleoside triphosphates, activators, and stabilizers are present. In each thermal cycle (for example the melting of the double helix at 94° C., hybridization of the primers at 55° C., reconstitution to a double helix by attachment of new DNA building blocks by the polymerase at 72° C.) the number of selected DNA segments is basically doubled. Therefore, in 30 cycles, around 1 billion DNA segments are generated from one single double strand of the DNA as original material. (In a more exact description, both primers hybridize on the two different single strands of the DNA and the shortening to the selected DNA segment including the two attached primers only occurs statistically during further replication).
Mass spectrometry with ionization of heavy molecules either by matrix-assisted laser desorption (MALDI) or by electrospray (ESI) is a very efficient method of analyzing biomolecules. For instance, the ions can be analyzed with regard to their mass in time-of-flight mass spectrometers. Since the flight velocity of the ions in the mass spectrometer is about 10
7
faster than the migration velocity of the molecules in the gel of electrophoresis, the mass spectrometry method is exceptionally faster than the previously used gel electrophoresis method, even if the spectrum measurement is repeated 10 to 100 times in order to achieve a good signal-to-noise ratio.
Due to the capability of a higher sample throughput the MALDI method has become more widespread than ESI for analyzing DNA. The MALDI method consists of first embedding the analyte molecules on a sample support in a UV-absorbing matrix, usually an organic acid. The sample support is introduced to the ion source of a mass spectrometer. Due to a short UV laser pulse of about 3 nanoseconds in length the matrix is evaporated into the vacuum; largely unfragmented, the analyte molecule is transported into the gaseous phase. Ionization of the analyte molecule is achieved by collisions with matrix ions forming simultaneously. An applied voltage accelerates the ions into a field-free flight tube. Based on their various masses the ions in the ion source are accelerated to various velocities. Smaller ions reach the detector earlier than large ones. The time of flight is converted to the mass of the ions.
Technical innovations in hardware have significantly improved the method of time-of-flight mass spectrometry with MALDI ionization. Worth mentioning is the delayed acceleration (Delayed Extraction) with which an improved resolution of the signals is achieved at a point in the spectrum, but also an even more reduced fragmentation. By means of an additional dynamic change in acceleration voltage it is possible to achieve a good resolution in a large mass range (for example see DE 196 38 577).
Naturally the MALDI method of ionization can be also coupled to other types of mass spectrometry such as RF quadrupole ion traps or ion cyclotron resonance spectrometers.
MALDI is ideally suitable for analyzing peptides and proteins. The analysis of nucleic acids is much more difficult. For nucleic acids ionization in the MALDI process is about 100 times lower than for peptides and decreases disproportionately with increasing mass. On the one hand, DNA pieces are very fragile and easily decompose in the MALDI process, while on the other hand they tend to form adducts with numerous alkali ions. Both processes of fragmentation and adduct formation cause the determination of mass to become increasingly inaccurate as mass increases.
Although one can determine a DNA piece with a length of 20 to 25 bases (around 6,000 to 8,000 atomic mass units) accurately to within three to five atomic mass units, it is no longer the case for DNA having a length of about 40 to 50 bases (around 12,000 to 16,000 atomic mass units). In the latter case the mass difference has to be about 40 to 60 mass units to ensure reliable differentiation. The four natural nucleobases of DNA, however, only have mass differences of 9 to a maximum of 40 atomic mass units so a base exchange can no longer be reliably detected at this length of DNA pieces. Only with extremely careful work and extremely efficient cleaning to keep adduct formation to a minimum is it possible to detect mass differences of 20 atomic mass units in this mass range.
The minimum length of a PCR amplified DNA product around an S

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