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Young Mee Yoon| Field Applications Scientist | Molecular Devices

Introduction

Genotyping is a process for analyzing genetic differences among individuals by examining their DNA sequences. The many methods available for genotyping enable researchers to investigate genomic diversity in humans, microorganisms, and plants. Single nucleotide polymorphisms (SNPs) are one of the most common types of genetic variation, consisting of a single nucleotide mutation at a specific locus. SNP genotyping has proven very useful in identifying disease-related mutations in various species, and as a result many techniques for SNP detection have been developed¹.

Kompetitive Allele Specific PCR (KASP) is one of the most widely used techniques for SNP genotyping as it is highly accurate, cost effective, and provides great flexibility in terms of assay design. KASP uses two allele-specific forward primers and one common reverse primer to amplify target DNA sequences via polymerase chain reaction (PCR) such that the reaction products are labeled with the fluorescent dye corresponding to their sequence. If a sample is homozygous, PCR products will be labeled with the fluorescent dye HEX or FAM only, but if it is heterozygous both HEX- and FAM-labeled products will be present. Unincorporated fluorescent dyes are quenched^{2, 3}. KASP assays are performed in a microplate format, and the results can be detected using a fluorescence microplate reader.

In this application note, we demonstrate how SpectraMax microplate readers can be used to read final KASP products using a KASP assay validation kit from LGC Genomics.

The first experiment was performed to validate that the readers generated distinctive signals for each fluorophore including FAM, HEX, and FAM/HEX. In the second experiment, the KASP assay was performed with known DNA samples provided by the vendor, and the PCR products were detected on the readers to verify correct genotyping of the samples.

Materials

• Standard ROX validation kit (LGC Genomics cat. KBS-1014-101)
• Thermal cycler (MJ Research cat. PTC-200)
• TempPlate semi-skirted 96-well PCR plate (USA Scientific cat. #1402-9700)
• 384-well low volume solid black microplate (Corning cat. #3676)
• Microseal B PCR plate sealing film (Bio-Rad cat. #MSB1001)
• SpectraMax i3xMulti-Mode Microplate Reader (Molecular Devices cat. #i3x)
• SpectraMax M5eMulti-Mode Microplate Reader (Molecular Devices cat. #M5e)

Methods

Microplate reader compatibility test

The validation kit included individual tubes of diluted fluorophores: FAM, HEX, and FAM+HEX. These were dispensed directly into a microplate and did not require any thermal cycling. Each tube of fluorophore also contained ROX, a passive reference dye that enables normalization of HEX and FAM signal, eliminating the effects of variation due to pipetting. 5 µL of each fluorophore (HEX, FAM, and HEX+FAM) was dispensed to triplicate wells of a 384-well plate. The plate was sealed with a clear film and then centrifuged at 560 x g for one minute. After centrifugation, the plate was immediately read on the M5e and i3x readers using the instrument settings listed in Table 1.

Detection of KASP genotyping reactions

For the second test, the validation kit contained 36 known samples, including 33 DNA samples and 3 no-template controls (NTC). 5 µL of each sample was pipetted into a 96-well plate for PCR. The same volume of genotyping mixture (2x KASP Master mix plus KASP Assay mix) was added to the each well. The passive reference dye ROX was included in the KASP Master mix. The plate was sealed with a clear film and centrifuged at 560 x g for one minute. A thermal cycling reaction was immediately initiated. Detailed thermal cycling conditions are listed in Table 2. After the reaction was complete, 5 µL of each sample was transferred into wells of a 384-well plate and detected on the microplate readers using the instrument settings listed in Table 1.

Results

For the first test, relative fluorescence units of each fluorophore, including FAM, HEX, and a mixture of both, were normalized to ROX and the normalized values were plotted in SoftMax® Pro Software. As shown in Figure 1, three distinct clusters were generated.

For the second test, the ROX-normalized fluorescence of amplified DNA samples were plotted in SoftMax Pro software(Figure 2). Three well-defined clusters were detected. FAM homozygotes were located near the X axis, whereas HEX homozygotes were located close to the Y axis, and the heterozygote samples containing both FAM and HEX formed a cluster between the two homozygotes clusters. No-template controls formed a cluster near the origin as expected. The data closely matched the genotyping results provided by the vendor for the validation kit.

Table 1. Plate reader settings used for data acquisitions.

Table 2. Thermal cycle settings for KASP genotyping reactions

Figure 1. Reader validation using three fluorophores. HEX/ROX and FAM/ROX values obtained from both M5e and i3x formed three distinctive clusters.

Figure 2. KASP genotyping results. PCR products read on the plate readers clustered into three different groups including FAM homozygotes (blue), HEX homozygotes (red), and FAM/HEX heterozygotes (green). No-template controls (NTC) clustered near the origin.

Conclusion

SpectraMax M5eand i3x plate readers have been fully validated for use with KASP genotyping assays. Monochromatorbased optics easily enable setup of the three excitation/emission wavelength pairs required to detect fluorescent PCR products representing all three genotypes present in the reaction. Consistent results are obtained on both instruments. Data analysis and graphing of genotypic clusters by SoftMax Pro Software provides rapid visualization of the normalized results.

References

1. Chen, X., and Sullivan, P.F. “Single nucleotide polymorphism genotyping: biochemistry, protocol, cost and throughput.” The Pharmacogenomics Journal 3, (2003): 77-96.

2. Patterson, E.L., Fleming, M.B., Kessler, K.C., Nissen, S.J., and Gaines, T.A. “A KASP genotyping method to identify nothern watermilfoil, eurasian watermilfoil, and their interspecific hybrids.” Frontiers in Plant Science 8, (2017): 752.

3. Smith, S.M., Maughan, P.J. “SNP genotyping using KASPar assays.” Plant Genotyping 1245, (2015): 243-256.

Young Mee Yoon| Field Applications Scientist | Molecular Devices

前言

基因分型是一种在不同个体间检测他们的 DNA 序列以分析遗传差异的过程。用于基因 分型的方法很多，使得研究人员有能力研究 人类、微生物和植物中的基因多样性。单核苷 酸多态性 (SNPs) 是其中一种最为普遍的遗传 变异类型，包括某个特定位点的单核苷酸突 变。SNP 基因分型已经被证实在识别许多物 种的疾病相关突变中非常有用，因此也已开 发出很多针对 SNP 检测的技术1 。

Kompetitive 等位基因特异性 PCR 因其高准 确性、高性价比并在分析设计方面的高度灵 活性而成为一种最为广泛使用的 SNP 基因分 型技术。KASP使用两条等位基因特异的正向 引物和一条通用反向引物，通过聚合酶链式 反应 (PCR) 扩增目标 DNA 序列，与其序列相 对应的反应产物则被标记上荧光染料。如果 一个样品是纯合子，PCR 产物将只会被 HEX 或 FAM 荧光染料标记，但如果是杂合体，则 会出现被 HEX 和 FAM 同时标记的产物。未参 与标记的荧光染料会被淬灭2,3。KASP 可在微 孔板中进行操作，并且可使用荧光微孔读板 机进行检测。

在本篇应用文献中，我们展示了使用来自 LGC 基因组学的 KASP 检测验证试剂如何在 SpectraMax 微孔读板机上读取最终的 KASP 产物。

第一个实验是为了验证每种荧光染料都可 在读板机中得到特定的信号，染料包括 FAM、HEX 和 FAM/HEX。第二个实验中， 使用由供应商提供的已知 DNA 样品进行 KASP 实验，PCR 产物在读板机中检测以 验证样品的正确基因型。

材料

• 标准 ROX 验证试剂盒 (LGC Genomics cat. KBS-1014-101)
• 热循环仪 (MJ Research cat. PTC-200)
• TempPlate 半裙 96- 孔 PCR 板 (USA Scientific cat. #1402-9700)
• 384- 孔低体积全黑微孔板 (Corning cat. #3676)
• Microseal B PCR 板密封薄膜 (Bio-Rad cat. #MSB1001)
• SpectraMax i3x 多功能微孔读板机 (Molecular Devices cat. #i3x)
• SpectraMax M5e 多功能微孔读板机 (Molecular Devices cat. #M5e)

方法

微孔读板机兼容性测试

验证试剂盒包括单独的已稀释荧光染料 管：FAM、HEX 和 FAM + HEX。将它们直 接加入微孔板中无需任何热循环。每管荧 光染料同样包含 ROX，一种无源参考染 料用于标准化HEX和 FAM 信号，消除加 液 产 生 的 差 异 影 响 。 每 种 荧 光 染 料 (HEX、FAM 和 HEX + FAM) 加 5 µL 到 384 孔板中，且包含三个重复。孔板以干净薄 膜密封，然后以 560 x g 离心一分钟。离 心好后，立即将孔板放入 M5e 和 i3x 读板 机中使用表1中的仪器设置进行读数。

KASP 基因分型反应物检测

第二个测试中，验证试剂盒包含 36 个已 知样品，包括 33 个 DNA 样品和 3 个无模 板对照 (NTC)。每个样品以 5 µL 量加入 96 孔板中进行 PCR。相同体积的基因分 型混合物 ( 2x KASP Master 混合物加 KASP 测定混合物 ) 添加到每孔中。无源 参考染料 ROX 也包含在 KASP Master 混 合物中。用干净薄膜密封孔板并以 560 x g 离心一分钟。立即启动热循环反应。详细 的热循环条件列举在表2中。反应完成 后，每个样品取 5 µL 转移到 384 孔板中 并在微孔读板机中使用表1中的仪器设置 进行检测。

结果

第一个测试，FAM、HEX 和两者的混合物 这几种荧光染料的相对荧光单位与 ROX 进 行标准化且标准化值在 SoftMax® Pro 软件 中绘制曲线。如图1所示，得到了三个不 同的集群。

第二个测试中，ROX 标准化的扩增 DNA 样品荧光在 SoftMax Pro 软件中绘制曲线 (图2)。三个显著的集群被检测出来。 FAM 纯合子位置靠近X轴，而 HEX 纯合子 则位置靠近Y轴，包含FAM和HEX的杂合样 品所形成的的集群在两个纯合子集群之 间。 无模板对照形成的集群如预期靠近最初位 置。所得数据与供应商的验证试剂盒基因 分型结果高度一致。

表1 读板机设置用于数据采集

表 2 热循环设置用于 KASP 基因分型反应

***图 1 使用三种荧光染料进行读板机验证。*HEX/ROX 和 FAM/ROX 读值来 自于 M5e 和 i3x 且都形成三种不同的集群

***图 2 KASP 基因分型结果。*PCR 产物在读板机上得到三个不同集群，包括 FAM 纯合子 ( 蓝色 )，HEX 纯合子 ( 红色 ) 和 FAM/HEX 杂合子 ( 绿色 )。无模板对照 (NTC) 集群接近最初位置

结论

SpectraMax M5e 和 i3x 读板机通过使用 FASP 基因分型实验得到了充分的验证。 基于光栅单色器的光学系统可以轻松的建 立三种激发/发射波长对儿，满足 PCR 反 应产物中代表三种基因分型的荧光探测要 求。两款仪器上的结果也是一致的。通过 SoftMax Pro 软件对基因集群进行数据分 析和绘图实现了标准化结果的快速可视 化。

参考文献

1. Chen, X., and Sullivan, P.F. “Single nucleotide polymorphism genotyping: biochemistry, protocol, cost and throughput.” The Pharmacogenomics Journal 3, (2003): 77-96.

2. Patterson, E.L., Fleming, M.B., Kessler, K.C., Nissen, S.J., and Gaines, T.A. “A KASP genotyping method to identify nothern watermilfoil, eurasian watermilfoil, and their interspecific hybrids.” Frontiers in Plant Science 8, (2017): 752.

3. Smith, S.M., Maughan, P.J. “SNP genotyping using KASPar assays.” Plant Genotyping 1245, (2015): 243-256.

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