Method of amplifying multiple nucleic acid sequences for differentiation

Abstract

It is intended to provide a multiplex detection method using a plural number of tags which shows little variation in amplification amount from tag to tag. A method of amplifying a plural number of nucleic acid fragments, in which the nucleic acid fragments involve one nucleic acid sequence for differentiation that is selected from the first group comprising the first to n-th nucleic acid sequences for differentiation and one nucleic acid sequence for amplification that is selected from the second group comprising the first to m-th nucleic acid sequences for amplification, and which comprises amplifying a nucleic acid fragment mixture containing multiple kinds of such nucleic acid fragments under such conditions as allowing the amplification of the nucleic acids with the use of, as primers, sequences being complementary to at least a part of all of the nucleic acid sequences for differentiation and at least a part of the nucleic acid sequences for amplification possibly occurring in the nucleic acid fragment mixture as described above or sequences located in the same chain side thereof; wherein n is an integer of 2 or more, the first to n-th nucleic acid sequences for differentiation involved in the first group differ from each other in base sequence, m is an integer of 1 or more but not more than n, and the first to m-th nucleic acid sequences for amplification involved in the second group differ from each other in base sequence.

Description

technical field [0001] The invention relates to nucleic acid amplification reaction technology. Background technique [0002] Human SNP (Single Nucleotide Polymorphism, ie, 1 nucleotide polymorphism) is a genotype that occurs at a frequency of about 1 in several hundreds of nucleotides. Their variants, whether coding or non-coding, are widely scattered across the genome. In addition, the variation is not only the substitution of bases, but also the insertion (intercalation) or deletion (deficiency) of bases. The size of the human genome is 3 billion base pairs, so even if such a polytype exists in a ratio of 1 in 1000 base pairs, there are 3 million SNPs as a whole. Therefore, it is difficult to discover medically useful SNPs from such a large number of SNPs. For example, in order to clarify the correlation between a SNP and a disease or between a SNP and drug sensitivity, by testing as many samples as possible, the relevant SNP can be specified with high precision. For ...

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Problems solved by technology

[0013] In the multiplexing method using such a conventional tag sequence, when the tag sequence is amplified by PCR, the amplified tag sequence is biased toward a specific tag sequence and has a high amplification rate, or conversely, the specific tag sequence Sequences have a lower amplification rate such that there is a bias in the amplification rate and thus in the amount of the resulting amplified product

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