Comparison of three PCR-based assays for SNP genotyping in plants.
Comparison of three PCR-based assays for SNP genotyping in plants.
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DOI：
10.1186/s13007-018-0295-6
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发表时间：
2018
期刊：
影响因子：
5.1
通讯作者：
Stevanato P
中科院分区：
文献类型：
--
作者：
Broccanello C;Chiodi C;Funk A;McGrath JM;Panella L;Stevanato P
PCR等位基因识别技术在遗传学和基因组学中的单核苷酸多态性（SNP）检测中有着广泛的应用。使用荧光标记探针是靶向SNP检测的主要方法，但可以改善测定成本和错误率以提高基因分型效率。一种基于RNase H2依赖性PCR（rhPCR）与通用报告系统相结合的新检测方法rhRIPE试图降低引物/引物和引物/探针二聚体的错误率，同时与现有技术相比降低成本。在rhetoric被广泛采用之前，需要更多的实验来验证其与现有方法相比的有效性。本研究的目的是比较的准确性，灵敏度和成本的TaqMan，KASP，和rhB-SNP基因分型方法在甜菜（甜菜L.）。对于每种方法，设计测定以在从12个亲本系获得的一组96个甜菜个体中对33个SNP进行基因分型。使用从100至0.1 ng/PCR反应的一系列稀释液测试测定灵敏度。在QuantStudio 12 K Flex实时PCR系统（Thermo Fisher Scientific，USA）上进行PCR。识别率定义为基因型识别相对于可能的识别数的百分比，TaqMan、KASP和rhesus的识别率分别为97.0%、97.6%和98.1%。对于rhG 1 SNP，33个SNP中的24个与其他两种技术显示100%一致性。其余9个基因型与两种方法的基因型一致率均在99%以上（99.34-99.89%）。灵敏度测试表明，TaqMan和rhodium能够成功地确定SNP基因型，每个反应使用低至0.2 ng DNA，而KASP无法确定SNP状态低于0.9 ng DNA每个反应。还分析了每个反应的比较成本，其中rhBSNP提供了每个反应的最低成本。总之，rhodium比TaqMan或KASP产生更多的调用，对NTC数据的信号更高，同时提供最低的每次反应成本。本文的在线版本（10.1186/s13007-018-0295-6）包含补充材料，可供授权用户使用。
PCR allelic discrimination technologies have broad applications in the detection of single nucleotide polymorphisms (SNPs) in genetics and genomics. The use of fluorescence-tagged probes is the leading method for targeted SNP detection, but assay costs and error rates could be improved to increase genotyping efficiency. A new assay, rhAmp, based on RNase H2-dependent PCR (rhPCR) combined with a universal reporter system attempts to reduce error rates from primer/primer and primer/probe dimers while lowering costs compared to existing technologies. Before rhAmp can be widely adopted, more experimentation is required to validate its effectiveness versus established methods. The aim of this study was to compare the accuracy, sensitivity and costs of TaqMan, KASP, and rhAmp SNP genotyping methods in sugar beet (Beta vulgaris L.). For each approach, assays were designed to genotype 33 SNPs in a set of 96 sugar beet individuals obtained from 12 parental lines. The assay sensitivity was tested using a series of dilutions from 100 to 0.1 ng per PCR reaction. PCR was carried out on the QuantStudio 12K Flex Real-Time PCR System (Thermo Fisher Scientific, USA). The call-rate, defined as the percentage of genotype calls relative to the possible number of calls, was 97.0, 97.6, and 98.1% for TaqMan, KASP, and rhAmp, respectively. For rhAmp SNP, 24 of the 33 SNPs demonstrated 100% concordance with other two technologies. The genotype concordance with either technologies for the other 9 targets was above 99% (99.34–99.89%). The sensitivity test demonstrated that TaqMan and rhAmp were able to successfully determine SNP genotypes using as little as 0.2 ng DNA per reaction, while the KASP was unable to ascertain SNP states below 0.9 ng of DNA per reaction. Comparative cost per reaction was also analyzed with rhAmp SNP offering the lowest cost per reaction. In conclusion, rhAmp produced more calls than either TaqMan or KASP, higher signal to NTC data while offering the lowest cost per reaction. The online version of this article (10.1186/s13007-018-0295-6) contains supplementary material, which is available to authorized users.
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影响因子：
--
作者：
Thomson, M. J.
通讯作者：
Thomson, M. J.
影响因子：
--
作者：
Landoulsi Z;Benromdhan S;Ben Djebara M;Damak M;Dallali H;Kefi R;Abdelhak S;Gargouri-Berrechid A;Mhiri C;Gouider R
通讯作者：
Gouider R
影响因子：
3.5
作者：
Dobosy JR;Rose SD;Beltz KR;Rupp SM;Powers KM;Behlke MA;Walder JA
通讯作者：
Walder JA
DOI：
10.1007/978-1-61737-954-3_6
发表时间：
2011-01-01
期刊：
DISEASE GENE IDENTIFICATION: METHODS AND PROTOCOLS
影响因子：
--
作者：
Schleinitz, Dorit;DiStefano, Johanna K.;Kovacs, Peter
通讯作者：
Kovacs, Peter
影响因子：
2.1
作者：
Stevanato, Piergiorgio;Broccanello, Chiara;Concheri, Giuseppe
通讯作者：
Concheri, Giuseppe