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1. 发明申请

WO2013113885A1 METHOD FOR VARIANT DIFFERENTIATION UTILIZING A PYROSEQUENCING DISPENSATION ORDER 审中-公开
标题翻译：使用色差分配订单的变异差分法
公开(公告)号：WO2013113885A1
公开(公告)日：2013-08-08
申请号：PCT/EP2013/052051
申请日：2013-02-01
申请人： RUPRECHT-KARLS-UNIVERISTÄT HEIDELBERG
发明人： SKOROKHOD, Alexander
IPC分类号： C12Q1/68
CPC分类号： C12Q1/6869 , C12Q2565/301
摘要： The present invention relates to a method for differentiating between at least two sequence variants of a polynucleotide by pyrosequencing, wherein at least one sequence variant comprises at least one mutation, said method comprising a) generating a dispensation order based on a nucleotide sequence encompassing the nucleotides affected by said variants, said dispensation order comprising dispensations corresponding to each nucleotide occurring in at least one of the variants, said nucleotide dispensations ordered in a sequence i) generating on average a pyrogram peak for at least every second nucleotide dispensation for each variant and ii) generating the maximal possible number of sequence pyrogram peaks for each variant; b) generating according to the dispensation order of a) a first pyrogram comprising expected intensity values for a first variant and at least a second pyrogram comprising expected intensity values for at least one further variant; c) comparing a sample pyrogram comprising measured intensity values, said sample pyrogram being derived from pyrosequencing analysis of a sample polynucleotide according to the nucleotide dispensation order of a), with the said first and at least the said second pyrogram generated in step b); and d) differentiating between said at least two sequence variants, wherein the sample polynucleotide corresponds to the first variant if the sample pyrogram is essentially identical with the first pyrogram and wherein the sample polynucleotide corresponds to a further variant if the sample pyrogram is essentially identical with a further pyrogram.
摘要翻译：本发明涉及通过焦磷酸测序来区分多核苷酸的至少两个序列变体的方法，其中至少一个序列变体包含至少一个突变，所述方法包括a）基于包含核苷酸的核苷酸序列产生分配顺序所述分配顺序包括对应于至少一个变体中出现的每个核苷酸的分配，所述核苷酸分配在序列中排序i）平均每个变体产生至少每第二个核苷酸分配的热克峰，ii ）为每个变体产生最大可能数量的序列辉度峰值; b）根据分配顺序生成a）包括第一变体的预期强度值的第一热解图和至少一个包含至少一个其它变体的预期强度值的第二热解图; c）比较包含测量强度值的样品热解图，所述样品热解图是根据a）的核苷酸分配顺序衍生自样品多核苷酸的焦磷酸测序分析与步骤b）中产生的所述第一和至少所述第二热解图; 和d）在所述至少两个序列变体之间进行区分，其中如果样品热解图与第一热解图基本相同，则样品多核苷酸对应于第一变体，并且其中样品多核苷酸对应于另一变体，如果样品焦点图与另一个焦点。

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