| 主权项 |
1. A method for genotyping N loci in a target nucleic acid molecule present in a sample, wherein N is at least 1 and each locus is located in a respective genotype marker region of the nucleic acid molecule, and corresponds to two or more genotypes, the method comprising the steps of:
a) performing a pre-amplification of each genotype marker region utilizing primer-dependent enzymatic reactions, wherein the amplification is performed with a first group of amplification primer pairs consisting of a first set of one or more different forward primers and a first set of one or more different reverse primers, yielding one or more amplicons encompassing the genotype marker region(s), and wherein each amplicon contains a region with at least one nucleotide sequence that can act as a primer binding target distinct from the genotype marker region; b) performing on the amplicon(s) from step a) a two-level nucleic acid amplification reaction utilizing primer-dependent enzymatic reactions, wherein
b1) the first level is nucleic acid amplification utilizing a second group of primer pairs binding to each amplicon produced in step a), wherein said group of primer pairs consists of a second set of one or more different forward primers and a second set of one or more different reverse primers, wherein each sequence variant of each locus is targeted by at least one primer from said second sets of primers, said at least one primer forming a set of selection primers, the selection primers being bipartite having at the 3′-end a locus recognition sequence part binding to either of said N loci at the genotype marker region, and at the 5′-end an artificial one-piece tagging sequence part being genotype specific or an artificial two-piece tagging sequence part, one piece of which is genotype specific and the other one is non-genotype specific, and wherein each selection primer binding to the target locus of the genotype marker region forms a primer pair together with another primer belonging to said second sets of primers but targeting said nucleotide sequence that can act as a primer binding target of step a), and wherein each selection primer is present in a concentration of less than 100 nM, from 10 to 0.01 nM, or approximately 0.3 nM, and the other primer of said primer pair is present in a concentration of at least 100 times or between 100 and 100 000 times that of said selection primer, to yield amplicons each containing a respective one of said N loci;b2) the second level is nucleic acid amplification utilizing a third group of primer pairs binding to each amplicon produced in step bl), wherein said group of primer pairs, present in a concentration of at least 100 times or between 100 and 100 000 times that of said selection primers in step b1), and consists of a third set of one or more different forward primers and a third set of one or more different reverse primers, wherein each tagging sequence part of each selection primer is targeted by at least one primer from said third sets of primers, said at least one primer forming a set of detection primers, and wherein each detection primer binding to the tagging sequence part forms a primer pair together with another primer belonging to said third sets of primers but targeting said nucleotide sequence that can act as a primer binding target of step a), the detection primers having:
i) a genotype-specific sequence corresponding to the artificial one-piece tagging sequence part of the selection primers, wherein each detection primer is labelled with a genotype-specific detectable label; orii) a non-genotype specific sequence which is common to all detection primers and corresponds to the non-specific sequence of the artificial two-piece tagging sequence part of the selection primers, wherein each detection primer is optionally labelled at the 5′-end with a detectable label or a chemical moiety;which results in detectable amplicons each containing a respective one of said N loci labelled with genotype specific tagging sequences and, optionally, genotype specific labels; c) genotyping said N loci of the target nucleic acid molecule by
c1) detecting during, or after, the amplification thereof each label comprised in each amplicon produced in step b2i), and relating the predominant amount of detected label to a specific genotype for each locus; orc2) after amplification contacting each amplicon produced in step b2ii) with a detection array of genotype specific sequences, detecting the hybridization of each amplicon to the array, and relating the detected hybridization to a specific genotype for each locus. |
