AbstractPCR is tolerant to single nucleotide mismatches. Therefore, genotyping of point mutations by PCR requires special conditions for the amplification of allele-specific PCR fragments. MS-PCR (mutagenically separated PCR) is an improved version of ARMS (amplification refractory mutation system) in which additional nucleotide mismatches near the mutation site are used to separate the wt fragments from the mutant fragments in a single-tube PCR.

摘要PCR对单核苷酸错配具有耐受性。因此，通过PCR对点突变进行基因分型需要特殊条件才能扩增等位基因特异性PCR片段。MS-PCR（诱变分离PCR）是ARMS（扩增难治性突变系统）的改进版本，其中突变位点附近的其他核苷酸错配用于在单管PCR中将wt片段与突变片段分离。

In the originally described procedure, the resulting fragments are resolved on agarose gels according to differences in size introduced by different lengths of the allele-specific primers. In order to evaluate the PCR fragments by melting curve analysis, we enlarged the difference in the melting temperatures of the fragments of the two alleles by increasing the GC content of the longer allele-specific primer resulting in a higher melting temperature of the corresponding fragment.

在最初描述的程序中，根据不同长度的等位基因特异性引物引入的大小差异，将所得片段在琼脂糖凝胶上分离。为了通过熔解曲线分析评估PCR片段，我们通过增加较长等位基因特异性引物的GC含量来扩大两个等位基因片段的熔解温度差异，从而提高相应片段的熔解温度。

Using the murine retinal degeneration mutations rd1 and rd8 as an example, we show that such primers result in an easy to handle genotyping procedure: qPCR followed by melting curve analysis. In summary, MS-PCR is a simple and easy-to-use method for detecting single nucleotide variants..

以鼠视网膜变性突变rd1和rd8为例，我们表明此类引物可产生易于处理的基因分型程序：qPCR，然后进行熔解曲线分析。总之，MS-PCR是一种简单易用的检测单核苷酸变异的方法。。

IntroductionMany diseases such as haemophilia, Duchenne muscular dystrophy, or retinal degeneration are caused by mutations of single nucleotides. Such diseases are rare but the effects are often very destructive to the patients. They are investigated in animal models that have the same point mutation in the corresponding gene, and a prerequisite is that the animals have to be genotyped correctly before developing a phenotype.

引言许多疾病如血友病，杜兴氏肌营养不良症或视网膜变性是由单核苷酸突变引起的。这种疾病很少见，但其影响通常对患者具有很大的破坏性。在相应基因中具有相同点突变的动物模型中对它们进行了研究，前提是在产生表型之前必须对动物进行正确的基因分型。

Therefore, simple and reliable detection methods are required.Genotyping of single nucleotide substitutions (SNPs, single nucleotide polymorphisms) can be achieved by different methods1. Most of them are based on polymerase chain reactions (PCR). In standard PCR procedures using one forward and reverse primer pair, the amplified fragment is evaluated by various methods such as digestion with a restriction enzyme that is sensitive to the mutated site (RFLP, restriction fragment length polymorphism), sequencing the PCR fragment, or high resolution melting analysis2,3 (Fig. 1A).

因此，需要简单可靠的检测方法。单核苷酸替代（SNP，单核苷酸多态性）的基因分型可以通过不同的方法实现1。其中大多数是基于聚合酶链反应（PCR）。在使用一个正向和反向引物对的标准PCR程序中，通过各种方法评估扩增的片段，例如用对突变位点敏感的限制性内切酶（RFLP，限制性片段长度多态性）消化，对PCR片段进行测序，或高分辨率熔解分析2,3（图1A）。

In other PCR based methods, two allele-specific forward and one reverse primers are used to produce specific fragments for different alleles (allele-specific PCR (AS-PCR)) followed by the evaluation of the PCR products in different ways such as agarose gel electrophoresis or various kinds of fluorescent labels (Fig. 1B).Fig.

在其他基于PCR的方法中，使用两个等位基因特异性正向引物和一个反向引物产生不同等位基因的特异性片段（等位基因特异性PCR（AS-PCR）），然后以不同方式评估PCR产物，例如琼脂糖凝胶电泳或各种荧光标记（图1B）。图。

1Principle of MS-PCR. (A) Standard PCR for evaluation via RFLP, high resolution melting curve analysis, or sequencing showing primers (arrows) and mutation site (SNP). (B) Allele-specific PCR for evaluation via agarose gel electrophoresis, melting curve analysis, or fluorescent labels showing primers (arrows) and mutation site (SNP).

1 MS-PCR原理。（A） 通过RFLP，高分辨率熔解曲线分析或显示引物（箭头）和突变位点（SNP）的测序进行评估的标准PCR。（B） 等位基因特异性PCR，用于通过琼脂糖凝胶电泳，熔解曲线分析或显示引物（箭头）和突变位点（SNP）的荧光标记进行评估。

(C) MS-PCR is explained for a het rd8 mouse with the primers Crb1-mF1 (wt) and Crb1-mF2 (mut). The mutated base is indicate.

（C） 。指示突变的碱基。

Data availability

数据可用性

Data is provided within the manuscript or supplementary information files. Data used and analysed during the current study to optimize the procedures are available from the corresponding author on reasonable request.

数据在手稿或补充信息文件中提供。在当前研究中使用和分析的数据可以在合理的要求下从通讯作者那里获得，以优化程序。

The authors declare no competing interests.

作者声明没有利益冲突。

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Download referencesAcknowledgementsWe would like to thank Sophie Krüger for valuable technical assistance and Dr. Ralph Birkenhäger for sequencing.FundingOpen Access funding enabled and organized by Projekt DEAL.Author informationAuthors and AffiliationsEye Center, Medical Center, Faculty of Medicine, University of Freiburg, Killianstr.

下载参考文献致谢我们要感谢SophieKrüger提供的宝贵技术援助和RalphBirkenhäger博士的测序。。作者信息作者和附属机构基里安斯特弗莱堡大学医学院医学中心Seye中心。

5, 79106, Freiburg, GermanyMelanie E. Schwämmle, Felicitas Bucher, Günther Schlunck & Gottfried MartinFaculty of Biology, University of Freiburg, Freiburg, GermanyMelanie E. SchwämmleAuthorsMelanie E. SchwämmleView author publicationsYou can also search for this author in.

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Reprints and permissionsAbout this articleCite this articleSchwämmle, M.E., Bucher, F., Schlunck, G. et al. Genotyping single point mutations in rd1 and rd8 mice using melting curve analysis of qPCR fragments.

转载和许可本文引用本文Schwämmle，M.E.，Bucher，F.，Schlunck，G。等人。使用qPCR片段的熔解曲线分析对rd1和rd8小鼠中的单点突变进行基因分型。

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KeywordsARMS PCRGenotypingSingle nucleotide variant

关键词SARMS PCRGenotypingSingle nucleotide variant

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