WO2019090971A1 - 利用二甲基亚砜改进基于实时荧光定量pcr的拷贝数分析 - Google Patents

利用二甲基亚砜改进基于实时荧光定量pcr的拷贝数分析 Download PDF

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WO2019090971A1
WO2019090971A1 PCT/CN2018/073047 CN2018073047W WO2019090971A1 WO 2019090971 A1 WO2019090971 A1 WO 2019090971A1 CN 2018073047 W CN2018073047 W CN 2018073047W WO 2019090971 A1 WO2019090971 A1 WO 2019090971A1
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copy number
gene
dmso
copy
number analysis
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魏娟
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先正达参股股份有限公司
先正达生物科技（中国）有限公司
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    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification

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  • the invention belongs to the field of genetic engineering, especially in the field of plant genetic engineering.
  • the present invention relates to the use of dimethyl sulfoxide (DMSO) in plant genetic transformation technology for real-time quantitative PCR (qPCR) studies and improved qPCR-based copy number analysis in plants application.
  • DMSO dimethyl sulfoxide
  • Plant genetic transformation is a technique widely used in crop transformation and gene function research. Usually, the expression level and biological trait of the introduced gene in crops are closely related to the number of copies.
  • the traditional Southern hybridization method [1] has the advantage of high accuracy as an absolute quantitative method, but it is not suitable for the rapid identification of the copy number of early transformed plants due to its large workload and long cycle.
  • digital PCR (ddPCR, digital droplet PCR) technology [2] [3] although accurate absolute quantitation can be achieved, it is based on accurate concentration dilution of DNA samples, making it difficult to achieve high-throughput sample preparation.
  • the TaqMan probe method in the real-time fluorescent quantitative PCR (qPCR) method is a commonly used real-time fluorescence quantification method using the 5' nuclease activity of Taq DNA polymerase.
  • the TaqMan probe set consists of a forward primer, a reverse primer, and a probe that labels the reporter group at the 5' end and the quencher group at the 3' end.
  • the qPCR reaction based on TaqMan probe method can be roughly divided into: double-stranded DNA is degenerated into single-stranded DNA under high temperature conditions; probes and primers specifically bind to template; activated Taq DNA polymerase starts DNA strand Synthesis; as the DNA strand extends, the 5' nuclease activity of Taq DNA polymerase decomposes the downstream-bound TaqMan probe; the reporter group labeled at the 5' end of the TaqMan probe is separated from the quencher group at the 3' end, thereby A fluorescent signal is emitted; the detector of the real-time PCR instrument quantifies it by receiving a fluorescent signal.
  • qPCR is widely recognized and applied because it can use the internal reference gene to achieve rapid homogenization of DNA samples and has high-throughput, rapid and sensitive detection of gene copy number.
  • low-cost, high-throughput genomic DNA extraction methods and high-throughput sample preparation methods have also challenged qPCR.
  • the PCR system needs to be optimized, and the common method is to add the corresponding concentration of chemical reagents.
  • chemical reagents such as DMSO, betaine, etc.
  • This application reports for the first time the use of dimethyl sulfoxide (DMSO) in real-time quantitative PCR (qPCR), improves the accuracy of qPCR-based copy number analysis, and improves qPCR-based copy number analysis in plants. Applications.
  • DMSO dimethyl sulfoxide
  • the inventors of the present invention have found for the first time that the addition of DMSO to a reaction system of real-time fluorescent quantitative PCR (qPCR) can improve the gene copy number analysis based on real-time fluorescent quantitative PCR, thereby completing the present invention.
  • qPCR real-time fluorescent quantitative PCR
  • a first aspect of the invention relates to the use of dimethyl sulfoxide for improving gene copy number analysis based on real-time fluorescent quantitative PCR (qPCR).
  • the addition of dimethyl sulfoxide improves the efficiency of PCR amplification of the internal reference gene and the target gene probe set in different samples (to maximize the desired amplification efficiency), thereby improving the gene.
  • a data set of samples in copy number analysis such as a degree of separation of data sets that improve different copy numbers.
  • the addition of dimethyl sulfoxide allows the sample to be located in a more accurate data set, and more samples are located in a low copy (ie, 1 copy) data set, thereby increasing gene copy number analysis. The ratio of medium to low copy (ie 1 copy) data set.
  • the present invention improves qPCR-based gene copy number analysis by adding dimethyl sulfoxide to the qPCR reaction system.
  • the present invention can add dimethyl sulfoxide to a conventional qPCR reaction system and employ the usual reaction parameters.
  • a two-step amplification method that is, denaturation at 95 ° C for 20-30 s and annealing at 60 ° C for 30 s can be employed while receiving a fluorescent signal.
  • the number of cycles is generally 40-50.
  • the invention employs the reaction procedure set forth in Table 3 below.
  • the concentration of dimethyl sulfoxide in the qPCR reaction system is from 2.5 to 7.5% v/v, preferably from 5 to 7.5% v/v, most preferably 5% v/v.
  • the data shows that 5% v/v DMSO can significantly improve qPCR-based copy number analysis for different plant varieties and genes of interest tested as well as different probe sets.
  • the gene copy number analysis is gene copy number analysis in plants.
  • the plant can be rice or corn, such as indica or japonica.
  • the gene analyzed can be a PMI, GUS or Red gene.
  • the primer and probe sequences of the PMI, GUS or Red genes are shown in Table 2 below.
  • the copy number of the determined gene is usually determined by a relative quantification method, that is, the same DNA template, two completely independent amplification reactions (including the internal reference gene and the target gene), wherein the ratio of the starting amount of the target gene to the starting amount of the internal reference gene is The copy number of the gene of interest.
  • the selection of the internal reference gene usually selects low-copy genes in vivo, such as the alcohol dehydrogenase gene of corn, the lectin gene of soybean, the sucrose phosphate synthase gene of rice, and some endogenous housekeeping genes such as ⁇ -actin and 2-2-microglobulin and the like.
  • Primers and probes useful in the present invention can be designed according to principles known in the art, and the length of the primers and probes can be selected.
  • the internal reference gene of the present invention is selected from the group consisting of OsADH1 and ZmADH1 shown in Table 1 below, and the corresponding primers and probe sets can be as shown in Table 1.
  • the internal reference gene and the corresponding primer and probe sequences may be selected from the internal reference genes shown in Table 1 and Corresponding corresponding primer and probe sequences.
  • Another aspect of the invention relates to a method of improving real-time quantitative PCR (qPCR)-based gene copy number analysis comprising adding dimethyl sulfoxide to a qPCR reaction system.
  • qPCR real-time quantitative PCR
  • DMSO can significantly improve gene copy number analysis based on real-time fluorescent quantitative PCR (qPCR) compared to other reagents.
  • qPCR real-time fluorescent quantitative PCR
  • the inventors further found that 5% and 7.5% concentration (v/v) of DMSO can improve the data set of the plant sample in the rice copy number analysis (the separation of the data set of 1 copy and 2 copies is more clear),
  • the 7.5% concentration of DMSO fluorescence signal intensity was lower than 5%; as more sample spots converge on one copy of the data set, the low copy ratio in rice increased from ⁇ 10% to >20%.
  • the data in maize NP2222 showed that 5% concentration of DMSO did not significantly increase the low copy ratio (from 29% to 32%), but a clearer sample data set made copy number analysis easier and more accurate.
  • the data showed that 5% concentration (v/v) of DMSO was able to significantly improve qPCR-based copy number analysis for different plant varieties and gene of interest tested, as well as different probe sets.
  • Figure 1 is a schematic diagram showing the analysis of copy number of a foreign gene in plants.
  • Figure 2 shows the data set of copy number analysis of PMI gene probe set in indica rice at 0% v/v, 2.5% v/v, 5% v/v, 7.5% v/v and 10% v/v DMSO concentrations. .
  • Figure 3 shows copy number analysis of the internal reference gene (TET) and PMI gene (FAM) probe sets in indica rice at 2.5% v/v, 5% v/v, 7.5% v/v, and 10% v/v DMSO concentrations Fluorescence signal intensity.
  • TET internal reference gene
  • FAM PMI gene
  • Figure 4 shows the data set for copy number analysis of GUS gene and Red gene probe set in indica at 0% and 5% v/v DMSO concentrations.
  • Figure 5 shows the data set for copy number analysis of PMI and Red gene probe sets in maize at 0% and 5% v/v DMSO concentrations.
  • Figure 6 shows the data set for copy number analysis of the PMI gene probe set in rice at 0M, 0.8M, 1M, 1.3M and 1.6M betaine concentrations.
  • Transgenic materials T0 generation transformed plants of indica, japonica and maize NP2222.
  • DMSO fetal sulfate
  • concentration of DMSO in the present application is a volume percent concentration (% v/v) unless otherwise specified.
  • Plant genomic DNA extraction using the improved Tris-SDS method [11] . Approximately 10 mg of leaves per plant were placed in a 96-well deep well plate and stored in an ultra-low temperature freezer at -80 °C for more than 30 minutes before extraction.
  • Primers and probes The TaqMan primers and probes of the internal reference gene and the target gene used in the present invention were designed by Primer Express 3.0 software of Applied Biosystems of the United States, and synthesized by Invitrogen, and the sequence information is shown in Table 1 and Table 2.
  • the total system of qPCR reaction was 25 ⁇ l, which contained 12.5 ⁇ l of JumpStart TM Taq ReadyMix TM , 5 ⁇ l of plant genomic DNA, 0.4 ⁇ l of internal reference gene and target gene primer probe set (final primer concentration was 300 nM, and the final probe concentration was 100 nM). ), 0% to 10% concentration (v/v) of DMSO and nuclease-free purified water.
  • TaqMan real-time PCR is performed using standard standard Applied 7900 Fast Real-Time PCR System. Table 3 shows the PCR reaction procedure.
  • a plant sample with a copy number of 1 in the previous sample was selected (the partial probe group did not have a copy positive control, such as the Red probe set in Figure 4), and the genomic DNA was extracted and subjected to the following gradient. Dilution: 1:1, 1:2, 1:4, 1:8 (see green dot (large circle) in Figure 1).
  • This sample will serve as a reference for copy number analysis (partially diluted samples may deviate, or may not be shown in the graph due to lower concentrations), but ultimately the separation of the actual data set is used as the basis for copy number analysis.
  • the fluorescence signal of the first 15 cycles of the PCR reaction is used as the fluorescence background signal, and the fluorescence threshold is set in the exponential region of all amplification curves, and the cycle of the fluorescence signal in each reaction tube reaches the set threshold.
  • the number is the Ct value (Cycle threshold)
  • the derived data contains the Ct values of the reference and target genes, labeled Ct TET and Ct FAM .
  • Each of these values can be used to position each sample at a point on the XY coordinate axis (see the blue dot in Figure 1 (square Point)), the points of all samples form a data set (see all blue dots (square dots) in Figure 1).
  • the data set may contain multiple data sets (1 copy set, 2 copy sets, 3 copy sets, etc.), respectively, with 1 copy, 2 copies, and 3 movable by mathematical simulation in the figure.
  • the copied lines coincide.
  • the data of one copy positive control and the data of one copy set of all the sample data sets were set as points of one copy control (yellow point (small circular point) in Fig. 1).
  • qPCR-based copy number analysis requires relatively high amplification of PCR, except that the efficiency of a single probe set is between 0.9 and 1.1, and the difference between the internal reference gene and the target gene probe set is controlled to a certain extent. (Slope difference ⁇ 0.2). As shown in Tables 5 and 6, in the 5% v/v and 7.5% v/v concentration DMSO systems, the PCR efficiency and the difference between the two probe sets can reach the copy number. Analysis requirements.
  • Example 4 Effect of different concentrations of betaine on qPCR-based copy number analysis and comparison with DMSO
  • qPCR real-time fluorescent quantitative PCR
  • 5% and 7.5% concentration (v/v) of DMSO can improve the data set of plant samples in rice copy number analysis (the separation of 1 copy and 2 copies of the data set is more clear); The sample points are concentrated in a single copy of the data set, increasing the low copy ratio in rice from ⁇ 10% to >20%.
  • the data in maize NP222222 showed that 5% concentration (v/v) of DMSO did not significantly improve the low copy ratio (from 29% to 32%), but a clearer sample data set made copy number analysis easier. And accurate.
  • 5% concentration (v/v) of DMSO was able to significantly improve qPCR-based copy number analysis for different plant varieties and gene of interest tested, as well as different probe sets.

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Abstract

利用二甲基亚砜改进基于实时荧光定量PCR的拷贝数分析。具体而言，2.5–7.5％v/v、优选5％v/v的DMSO对于测试的不同植物品种和目的基因均能够显著改善基于qPCR的拷贝数分析。

Description

利用二甲基亚砜改进基于实时荧光定量PCR的拷贝数分析 技术领域
本发明属于基因工程领域，尤其是植物基因工程领域。具体而言，本发明涉及二甲基亚砜(DMSO)在植物遗传转化技术中的应用，其用于实时荧光定量PCR(qPCR)的研究，并改进了基于qPCR的拷贝数分析在植物中的应用。
背景技术
植物遗传转化是一种广泛应用于作物改造和基因功能研究的的技术，通常导入基因在作物中的表达水平和生物学性状与其拷贝数的多少密切相关。传统的Southern杂交法 [1]作为一种绝对定量的方法，具有准确性高的优点，但由于其工作量大、周期长，并不适用于早期转化植株拷贝数的快速鉴定。近几年兴起的数字PCR(ddPCR，digital droplet PCR)技术 [2][3]，虽然可以实现精准的绝对定量，但它基于对DNA样品进行准确的浓度稀释，不易实现高通量样品制备。
实时荧光定量PCR(qPCR)方法中的TaqMan探针法，是一种常用的利用Taq DNA聚合酶的5′核酸酶活性的实时荧光定量方法。TaqMan探针组由一条正向引物、一条反向引物和一条在5′端标记报告基团及3′端标记猝灭基团的探针组成。基于TaqMan探针法的qPCR反应大致可分为：双链DNA在高温条件下变性解离成单链DNA；探针和引物与模板特异性结合；被激活的Taq DNA聚合酶起始DNA链的合成；随着DNA链的延伸，Taq DNA聚合酶的5′核酸酶活性将下游结合的TaqMan探针分解；TaqMan探针5′端标记的报告基团与3′端的猝灭基团分离，从而发出荧光信号；实时荧光定量PCR仪的检测器通过接收荧光信号对其进行定量。
随着植物遗传转化技术的普及和广泛应用，对转基因植株拷贝数的快速检测提出了更高的要求。qPCR [4]作为一种相对定量的方法，由于可以使用内参基因实现DNA样品的快速均一化，具有高通量、快速、灵敏的检测基因拷贝数的特点，得到广泛的认可和应用。与此同时，低成本、高通量基因组DNA的提取方法，高通量样品制备方法，也对qPCR提出了挑战。
对于不同质量的DNA或不同GC含量的基因序列，PCR体系需要进行优化，其中常用的方法就是添加相应浓度的化学试剂。虽然有文章报道，一些化学试剂，如 DMSO、甜菜碱等，可以提高PCR的特异性、产量和成功率，但仅限于针对终端PCR(End-point PCR，即PCR反应结束后检测PCR产物的量)的研究。
早在1990年，Bachmann等就报道了利用二甲基亚砜和非离子洗脱剂改进PCR扩增 [5]。随后，甜菜碱被报道可以提高高GC含量基因的PCR扩增 [6]。2006年，Musso等利用DMSO、甜菜碱和7-脱氮-2-脱氧鸟苷三磷酸扩增GC含量高达67％到79％的基因序列 [7]。2010年，Jensen等报道DMSO和甜菜碱可以减少DNA二级结构的形成和扩增的引导错误，从而大大提高了高GC含量基因PCR扩增过程中特异性和产量，而且具有很好的兼容性 [8]。除此以外，牛血清白蛋白(BSA) [9]、甲酰胺 [10]等，也可以提高PCR的特异性和产量，并最终提高PCR的成功率。
而这些报道大多数是针对终端PCR展开的。迄今为止，尚无关于二甲基亚砜(DMSO)在实时荧光定量PCR(qPCR)中的应用。
本申请则首次报道了二甲基亚砜(DMSO)在实时荧光定量PCR(qPCR)中的应用，提高了基于qPCR的拷贝数分析的精确度，并改进了基于qPCR的拷贝数分析在植物中的应用。
发明内容
本发明的发明人首次发现将DMSO添加至实时荧光定量PCR(qPCR)的反应体系中可改进基于实时荧光定量PCR的基因拷贝数分析，由此完成了本发明。
本发明的第一方面涉及二甲基亚砜在改进基于实时荧光定量PCR(qPCR)的基因拷贝数分析中的应用。在一个实施方案中，二甲基亚砜的加入，可以同时改进内参基因和目的基因探针组在不同样品中的PCR扩增效率(以最大限度接近理想的扩增效率)，从而改进了基因拷贝数分析中样品的数据组，例如改进不同拷贝数的数据集合的彼此分离程度。在一个实施方案中，二甲基亚砜的加入，使样品定位在更加准确的数据集合，且更多的样品定位在了低拷贝(即1个拷贝)数据集合，从而提高了基因拷贝数分析中低拷贝(即1个拷贝)数据集合的比例。
本发明通过向qPCR反应体系添加二甲基亚砜来改进基于qPCR的基因拷贝数分析。例如，本发明可以向通常的qPCR反应体系添加二甲基亚砜并采用通常的反应参数。例如，可采用两步扩增法，即95℃变性20-30s，60℃退火延伸30s，同时接收荧光信号。循环数一般为40-50。在一个实施方式中，本发明采用下文表3所示的反应程序。
在一个具体实施方案中，二甲基亚砜在qPCR反应体系中的浓度为2.5-7.5％v/v，优选5-7.5％v/v，最优选5％v/v。数据显示，5％v/v的DMSO对于测试的不同植物品种和目的基因以及不同的探针组均能够显著改善基于qPCR的拷贝数分析。
在一个具体实施方案中，所述基因拷贝数分析为在植物中的基因拷贝数分析。植物可以是水稻或玉米，例如粳稻或籼稻。
在一个具体实施方案中，所分析的基因可以为PMI、GUS或Red基因。在一个更加具体的实施方式中，PMI、GUS或Red基因的引物和探针序列如下文表2所示。
测定基因的拷贝数通常通过相对定量的方法，即同一DNA模板、两个完全独立的扩增反应(包括内参基因和目的基因)，其中目的基因起始量与内参基因起始量的比值即为目的基因的拷贝数。
内参基因的选择通常选取生物体内源的低拷贝基因，如玉米的乙醇脱氢酶基因、大豆的凝集素基因、水稻的蔗糖磷酸合酶基因及一些内源性管家基因如β-肌动蛋白和β2-微球蛋白等。
可遵循本领域已知的原则设计可用于本发明的引物和探针，并选择引物和探针的长度。
在一个更加具体的实施方式中，本发明的内参基因选自下文表1所示的OsADH1和ZmADH1，其相应的引物和探针组可如表1所示。在一个更加具体的实施方式中，当目的基因及其相应的引物和探针序列如表2所示时，其内参基因和相应的引物和探针序列可选自表1所示的内参基因和相应的相应的引物和探针序列。
本发明的另一方面涉及一种改进基于实时荧光定量PCR(qPCR)的基因拷贝数分析的方法，其包括向qPCR反应体系添加二甲基亚砜。
发明的有益效果
本研究中，本发明人令人惊讶地发现DMSO相比于其它试剂可显著改进基于实时荧光定量PCR(qPCR)的基因拷贝数分析。本发明人进一步发现，5％和7.5％浓度(v/v)的DMSO在水稻拷贝数分析中，可以改进植物样品的数据组(1个拷贝和2个拷贝的数据集合分离的更加清晰)，但是7.5％浓度的DMSO荧光信号强度较5％有所降低；由于更多的样品点汇聚在1个拷贝的数据集合，使水稻中的低拷贝比例从<10％提高到>20％。在玉米NP2222中的数据表明，5％浓度的DMSO虽然没有明显提高低拷贝比例的作用(从29％提高到32％)，但更加清晰的样品数据 组使拷贝数分析更加简单和准确。数据显示，5％浓度(v/v)的DMSO对于测试的不同植物品种和目的基因以及不同的探针组均能够显著改善基于qPCR的拷贝数分析。
本研究首次利用DMSO改进基于qPCR的拷贝数分析在植物中的应用，具有开创性的意义。
附图说明
图1为植物中外源基因拷贝数分析示意图。
图2显示在0％v/v、2.5％v/v、5％v/v、7.5％v/v和10％v/v DMSO浓度下PMI基因探针组在粳稻中拷贝数分析的数据组。
图3显示在2.5％v/v、5％v/v、7.5％v/v和10％v/v DMSO浓度下内参基因(TET)和PMI基因(FAM)探针组在粳稻中拷贝数分析的荧光信号强度。
图4显示在0％和5％v/v DMSO浓度下GUS基因和Red基因探针组在粳稻中拷贝数分析的数据组。
图5显示在0％和5％v/v DMSO浓度下PMI基因和Red基因探针组在玉米中拷贝数分析的数据组。
图6显示在0M、0.8M、1M、1.3M和1.6M甜菜碱浓度下PMI基因探针组在水稻中拷贝数分析的数据组。
具体实施方式
下面将结合实施例对本发明的实施方案进行详细描述，但是本领域技术人员将会理解，下列实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
实施例1.材料与方法
材料
转基因材料：籼稻、粳稻和玉米NP2222的T0代转化植株。
试剂：JumpStart TM Taq ReadyMix TM(Sigma-Aldrich)、植物基因组DNA、内参基因和目的基因引物探针组(引物终浓度为300nM，探针终浓度为100nM)、DMSO和无核酸酶的纯净水。除非特殊说明，本申请中DMSO的浓度为体积百分比浓度(％v/v)。
方法
植物基因组DNA提取：采用改进的Tris-SDS法 [11]。每株植物取大约10mg的叶片放入96孔深孔板，提取前需在-80℃超低温冰箱中保存30分钟以上。
引物和探针：本文中使用的内参基因和目的基因的TaqMan引物和探针均利用美国Applied Biosystem公司的Primer Express 3.0软件设计，并由Invitrogen公司合成，序列信息见表1和表2。
表1 内参基因实时荧光定量PCR的引物和探针序列
Figure PCTCN2018073047-appb-000001
表2 目的基因实时荧光定量PCR的引物和探针序列
Figure PCTCN2018073047-appb-000002
实时荧光定量PCR(qPCR)反应体系和反应条件
反应体系：qPCR反应总体系为25μl，其中含有12.5μl JumpStart TM Taq ReadyMix TM、5μl植物基因组DNA、各0.4μl内参基因和目的基因引物探针组(引物终浓度为300nM，探针终浓度为100nM)、0％至10％浓度(v/v)的DMSO和无核酸酶的纯净水。
反应程序：TaqMan实时荧光定量PCR使用标准制式的Applied 
Figure PCTCN2018073047-appb-000003
 7900快速实时PCR系统。表3为PCR反应程序。
表3 PCR反应程序
Figure PCTCN2018073047-appb-000004
一个拷贝阳性对照的制备：选取之前样品中拷贝数为1的植物样品(部分探针组无一个拷贝阳性对照，如图4中的Red探针组)，提取基因组DNA，再对其进行以下梯度稀释：1:1、1:2、1:4、1:8(见图1中的绿点(大圆形点))。该样品将作为拷贝数分析的参照(部分稀释样品可能有所偏离，或由于浓度较低无法显示在图中)，但最终以实际数据集合的分离作为拷贝数分析的依据。
数据分析：PCR结束后，使用SDS 2.4软件分析结果。PCR反应的前15个循环的荧光信号作为荧光本底信号，将荧光阈值(threshold)设置在所有扩增曲线的指数型区域内，每个反应管内的荧光信号到达设定阈值时所经历的循环数即为Ct值(Cycle threshold，循环阈值)，将基线结束点(baseline end cycle)设置在阈值线穿过第一条扩增曲线时的循环数的前3-5个循环(如：最早的Ct＝24，那么基线为24-3＝21)，将数据导出，用于下一步相对定量分析。导出的数据中包含内参基因和目的基因的Ct值，标记为Ct TET和Ct FAM，通过这两个数值可以将每个样品定位在XY坐标轴的一个点(见图1中的蓝点(方形点))，所有样品的点形成了数据组(见图1中所有蓝点(方形点))。该数据组可包含多个数据集合(1个拷贝集合，2个拷贝集合，3个拷贝集合，等)，分别与图中的三条通过数学模拟可移动的1个拷贝、2个拷贝和3个拷贝的线相重合。参照一个拷贝阳性对照的数据和所有样品数据组中的1个拷贝集合的数据，设定为1个拷贝对照的点(图1中的黄点(小圆形点))。
拷贝数计算：采用相对定量ΔΔCt法计算目的基因的拷贝数。由于在PCR的指数扩增时期，荧光信号的量与样品的初始浓度成正比，因此样品中目的基因相对于内参基因的数量为2^Ct FAM样品/2^Ct TET样品＝2^(Ct FAM样品-Ct TET样品)，即2^ΔCt样品。通过分析一个拷贝阳性对照的数据，可以设定一个拷贝阳性对照中目的基因相对于内参基因的数量为2^ΔCt对照，即1拷贝＝2^ΔCt对照，由此样品中目的基因的拷贝数计算公式为：拷贝数＝(2^ΔCt样品)/(2^ΔCt对照)＝2^ΔΔCt。
实施例2.不同浓度DMSO对样品数据组、扩增曲线和荧光信号强度ΔRn的影响
为了确定DMSO的最佳工作浓度，首先选用了PMI基因探针组在粳稻中测试了一系列的浓度梯度，分别为0％、2.5％、5％、7.5％和10％(见图2)。图2中样品数据组显示，在5％、7.5％和10％浓度的DMSO体系下更多的样品汇集在了1拷贝数据集合，且与1拷贝阳性对照重合的更好。而0％和2.5％v/v浓度DMSO体系下的样品则汇集的较为松散，且有很少的样品与1拷贝阳性对照重合。不同浓度DMSO对qPCR的扩增曲线也有不同程度的影响。如图3所示，随着DMSO浓度从2.5％v/v、5％v/v、7.5％v/v到10％v/v的增加，内参基因和PMI基因探针组的扩增曲线均变得松散且更快的到达平台期，终点的荧光信号强度降低，尤其是目的基因PMI探针组表现的尤为明显。因此，参照样品的数据组、扩增曲线和荧光信号强度，选择5％浓度的DMSO体系是比较好的工作浓度。
为了验证不同的探针组在5％v/v浓度DMSO体系中的表现，使用了GUS基因和Red基因探针组进行了测试。如图4显示，在5％浓度(v/v)DMSO体系中，这两个探针组均有更好的数据组，更多的样品点汇聚在1个拷贝数据集合，且其扩增曲线和荧光信号强度也较为正常(数据未显示)。
在玉米NP2222中的数据显示(图5)，5％v/v DMSO对样品数据组的改进虽然不如在水稻中明显，但仍然可以看到分离更加清楚的数据集合，使得拷贝数分析更加简单准确。
实施例3. 5％v/v浓度DMSO对转基因植株中低拷贝(1个拷贝)比例的影响
通过在水稻和玉米中qPCR中使用5％v/v DMSO后，进行了低拷贝(1个拷贝)比例的统计。如表4所示，在粳稻中低拷贝比例由7.5％提高到了25％，籼稻则由10％提高到了22％。在玉米中，低拷贝比例没有明显变化(29％到32％)。
表4. 0％和5％DMSO对水稻和玉米拷贝数分析中低拷贝(1个拷贝)的比例
Figure PCTCN2018073047-appb-000005
DMSO对PCR扩增效率的影响
基于qPCR的拷贝数分析对PCR的扩增要求相对较高，除了要求单个探针组的效率在0.9至1.1之间外，还要求内参基因和目的基因探针组之间的差异控制在一定范围(斜率差<0.2)。如表5和表6所示，在5％v/v和7.5％v/v浓度DMSO体系中，两个探针组的PCR效率和之间的斜率差(Difference of Slop)均可以达到拷贝数分析的要求。
表5. 0％、5％v/v和7.5％v/v浓度DMSO体系下内参基因和PMI探针组的qPCR效率及其之间的差异
Figure PCTCN2018073047-appb-000006
表6. 0％、5％v/v和7.5％v/v浓度DMSO体系下内参基因和Red探针组的qPCR效率及其之间的差异
Figure PCTCN2018073047-appb-000007
实施例4.不同浓度甜菜碱对基于qPCR的拷贝数分析的影响及与DMSO的比较
为了测试DMSO之外其他试剂，我们选用了甜菜碱并测试了在0M，0.8M，1M，1.3M，1.6M浓度下对PMI探针组qPCR的影响(见图6)。图6中样品数据组显示，虽然在1M和1.3M浓度的甜菜碱体系下数据组稍有改进但是效果不如5％浓度DMSO明显，且不同探针组下表现不稳定(数据未显示)。因此，相比较DMSO，甜菜碱改进基于qPCR的记忆拷贝数分析所表现的效果不明显且不稳定。
以上结果表明，DMSO显著地改进了基于实时荧光定量PCR(qPCR)的基因拷贝数分析。其中，5％和7.5％浓度(v/v)的DMSO在水稻拷贝数分析中，可以改进植物样品的数据组(1个拷贝和2个拷贝的数据集合分离的更加清晰)；由于更多的样品点汇聚在1个拷贝的数据集合，使水稻中的低拷贝比例从<10％提高到>20％。在玉米NP2222中的数据表明，5％浓度(v/v)的DMSO虽然没有明显提高低拷贝比例的作用(从29％提高到32％)，但更加清晰的样品数据组使拷贝数分析更加简单和准确。综上所述，5％浓度(v/v)的DMSO对于测试的不同植物品种和目的基因以及不同的探针组均能够显著改善基于qPCR的拷贝数分析。
尽管本发明的具体实施方式已经得到详细的描述，本领域技术人员将会理解。根据已经公开的所有教导，可以对那些细节进行各种修改和替换，这些改变均在本发明的保护范围之内。本发明的全部范围由所附权利要求及其任何等同物给出。
参考文献
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[3]Hindson C.M.,Chevillet J.R.,BriggsH.A.,Gallichotte E.N.,Ruf I.K.,Hindson B.J.&TewariM.(2013)Absolute quantification by droplet digital PCR versus analog real-time PCR.Nature Methods 10,1003–1005.
[4]Ingham,DJ.,Beer,S.,Money,S.,Hansen G.(2001)Quantitative Real-Time PCR Assay for Determining Transgene Copy Number in Transformed Plants.BioTechniques 31,132-140.
[5]B.Bachmann,W.Luke,G.Hunsmann,Improvement of PCR amplified DNA sequencing with the aid of detergents,Nucleic Acids Res.18(1990)1309.
[6]W.Henke,K.Herdel,K.Jung,D.Schnorr,S.A.Loening,Betaine improves the PCR amplification of GC-rich DNA sequences,Nucl.Acids Res.25(1997)3957–3958.
[7]M.Musso,R.Bocciardi,S.Parodi,R.Ravazzolo,I.Ceccherini,Betaine,dimethylsulfoxide,and 7-deaza-dGTP,a powerful mixture for amplification of GC-rich DNA sequences,J.Mol.Diagn.8(2006)544–550.
[8]Jensen,M.,Fukushima,M.and Davis,R.(2010)DMSO and Betaine greatly improve amplification of GC-rich constructs in de novo synthesis.PLOS One 5(2010)11024.
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Claims (4)

  1. 一种基于实时荧光定量PCR(qPCR)的水稻基因拷贝数分析的方法，其包括向qPCR反应体系添加5.0-7.5％v/v的二甲基亚砜。
  2. 根据权利要求1所述的方法，其中二甲基亚砜在qPCR反应体系中的浓度为5％v/v。
  3. 根据权利要求1或2所述的方法，其中所述水稻为粳稻或籼稻。
  4. 根据权利要求1或2所述的方法，其中所述基因为PMI、GUS或Red基因。
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