Mutation analysis by PCR and mass spectrometry
AssigneeBruker Daltonik GmbH
InventorJochen Franzen
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Abstract
The invention concerns mass spectrometric analysis of known mutation sites in the genome, such as single nucleotide polymorphisms (SNPs). The invention uses minor amounts of primers with photocleavable linkers, intermixed with a major amounts of primers without linkers, to produce short mutation-containing DNA sequences by enzymatic amplification procedures such as polymerase chain reactions (PCR). After this single amplification procedure, the linker-containing PCR by-products are extracted, washed and photolytically cleaved. Short oligonucleotides are produced which facilitate mass spectrometric analysis. Additionally to the use of linkers, some types of primers may contain blockers which stop the polymerase copying process to achieve even shorter oligonucleotides for analysis.
Claims15
Method for mass-spectrometric analysis of known mutation sites in DNA, comprising the following steps:
- (a) amplifying a DNA sequence containing the mutation site by polymerase chain reaction, using a mixture of different types of primers wherein the primers of at least one type contain photocleavable linkers,
- (b) extracting linker-containing amplification products from the product solution,
- (c) cleaving the extracted linker-containing amplification products, and
- (d) analyzing the cleavage products by mass spectrometry.
Method as in any of the preceding claims, wherein the primers of at least one of the linker-containing types of primers contains also an affinity group, and wherein affinity bonding to a substrate is used to extract, in step (b), the linker-containing primers carrying the affinity groups.
Method as in one of the preceding claims, wherein a multiplexing PCR is used with more than one analytical primer pair to products with information about more than one mutation site.
Method as in one of the preceding claims, wherein the PCR amplification starts with higher annealing temperatures in the first thermocycles, and continues later with lower annealing temperatures.
Dependencies are inferred from the claim text.