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Dec 2020

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Measurement of Transgene Copy Number in Plants Using Droplet Digital PCR
应用微滴数字PCR技术测定植物中的转基因拷贝数   

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Abstract

Transgenic plants are produced both to investigate gene function and to confer desirable traits into crops. Transgene copy number is known to influence expression levels, and consequently, phenotypes. Similarly, knowledge of transgene zygosity is desirable for making quantitative assessments of phenotype and tracking the inheritance of transgenes in progeny generations. Since the first transgenic plants were produced, several methods for determining copy number have been applied, including Southern blotting, quantitative real-time PCR, and more recently, sequencing methods; however, each method has specific disadvantages, compromising throughput, accuracy, or expense. Digital PCR (dPCR) divides reactions into partitions, converting the exponential, analogue nature of PCR into a linear, digital signal that allows the frequency of occurrence of specific sequences to be accurately estimated. Confidence increases with the number of partitions; therefore, the availability of emulsion technologies that enable reactions to be divided into tens of thousands of nanodroplets allows accurate determination of copy number in what has become known as digital droplet PCR (ddPCR). ddPCR offers similar benefits of low costs and scalability as other PCR techniques but with superior accuracy and reliability.


Graphic abstract:



Digital PCR (dPCR) divides reactions into partitions, converting the exponential, analogue nature of PCR into a linear, digital signal that allows the frequency of transgene copy number to be accurately assessed.


Keywords: Digital PCR (数字PCR), Digital droplet PCR (微滴式数字PCR), Transgene copy number (转基因拷贝数), Plants (植物), T-DNA (转移DNA)

Background

Plant transformation is used as a research tool to investigate gene function as well as for the production of genetically engineered crops. Several methods are used to deliver DNA into plant cells, including direct delivery into isolated protoplasts, biolistic delivery using high-density microparticles, Agrobacterium tumefaciens-mediated delivery, and most recently, nanoparticles (Altpeter et al., 2016; Wang et al., 2019). DNA delivery techniques can be optimized to obtain a higher frequency of transgenic lines with single or low-copy insertions; however, the exact number of transgenes transferred to the plant genome cannot be controlled or limited, and transgenic lines must be individually assessed to determine transgene copy number (Sivamani et al., 2015). Since expression levels, and consequently phenotypes, have long been acknowledged to be influenced by copy number, accurate determination is essential (Hobbs et al., 1993). Copy number determination can also be used to track inheritance and zygosity. Most methods of plant transformation deliver transgenes to somatic cells from which transgenic plants (referred to as T0) are regenerated, each with one more hemizygous transgenic locus. In progeny resulting from self-fertilization, transgenic loci usually follow classical Mendelian inheritance; although, non-Mendelian segregation has also been reported (Ahuja and Fladung, 2014). Accurate determination of copy number can be used to track transgenes through progeny generations, being particularly useful in lines with multiple transgene copies and in obligate outcrossing species.


In the decades since plant transformation became routine, a number of different technologies have been used to determine copy number. Initially, copy number was determined by Southern hybridization analysis (Southern, 1975). In this technique, genomic DNA is digested with a restriction endonuclease, separated by electrophoresis, and following transfer to a membrane, fragments containing the transgene are detected using a labeled DNA probe. As well as being laborious, it can be challenging to detect transgenes inserted close together (in tandem repeats), which are likely to be on the same fragment. Further, unless there is sufficient heterozygosity so that homologous chromosomes have different fragmentation patterns after restriction digestion, detection of zygosity is reliant on differences in band intensity, which is considered unreliable (Cantsilieris et al., 2013).


In the first decades of the twenty-first century, quantitative real-time PCR (qPCR) became the method of choice for estimating plant transgene copy number (Bubner and Baldwin, 2004; Li et al., 2004; Weng et al., 2004; Yang et al., 2005a and 2005b; Yi and Hong, 2019). The concentration of the transgene in each sample is compared with either a standard curve or a validated reference gene (an endogenous gene of known copy number) (Pfaffl, 2001). However, since each cycle of PCR is a doubling reaction, the two-fold increase required to differentiate one copy from two copies, or a hemizygote from a homozygote, is at the detection limit. Even with optimization, it can be challenging to achieve the levels of accuracy required to distinguish single copy and two-copy lines (Cantsilieris et al., 2013; Mieog et al., 2013).


Next-generation sequencing (NGS) technologies have reduced the cost of sequencing; however, even low-pass shallow sequencing, sometimes called ‘skim-sequencing,’ is expensive for large plant genomes – prohibitively so for routine copy number detection (Golicz et al., 2015; Kim et al., 2016). Targeted-capture sequencing, in which regions of the genome containing sequences of interest are isolated, has allowed the generation of accurate copy number data together with information on the sequence identity of insertion sites at reduced costs (Guttikonda et al., 2016). Initially, short-read next-generation sequencing technologies were applied (Polkoa et al., 2012; Lepage et al., 2013; Guo et al., 2016), but more recently, long-read nanopore-based sequencing platforms have also been utilized (Li et al., 2019; Boutigny et al., 2020). However, compared with PCR-based methods, even capture sequencing techniques have higher costs and require more time for sample preparation and data analysis.


Digital PCR (dPCR) converts the exponential, analogue nature of PCR into a linear, digital signal allowing accurate estimations of the frequency of occurrence of specific sequences (Vogelstein and Kinzler, 1999). To do this, each sample is divided into a large number of partitions, and PCR reactions are carried out on each partition individually. This was initially difficult and laborious as reactions needed to be divided across multiple compartments (tubes), but the development of emulsion technologies enabled the reaction to be compartmentalized in nanodroplets within a single tube (Diehl et al., 2006). In common with qPCR protocols, fluorescence is incorporated into the target amplicon; however, instead of monitoring fluorescence intensity at a specific cycle during the exponential phase of the reaction, the presence or absence of fluorescence (and therefore the target) at the reaction endpoint is determined for each partition. The copy number of the target gene is calculated by determining the fraction of partitions in which the target gene was amplified relative to a reference gene of known copy number. As each partition is scored for the presence of fluorescence, dPCR achieves precise quantitation of the transgene copy number. Confidence is improved by increasing population size (counting more partitions), resulting in the accuracy of copy number estimation by dPCR being reported as superior to qPCR and comparative with sequencing techniques (Abyzov et al., 2012). In recent years, a number of commercial platforms that divide reactions into tens of thousands of compartments using emulsion technology (digital droplet PCR; ddPCR) or microfluidics chips have been successfully applied for the accurate determination of transgene copy number in different plant species (Glowacka et al., 2016; Collier et al., 2017; Giraldo et al., 2019; Cai et al., 2020). Below, we describe a ddPCR protocol used to determine the transgene copy number in Arabidopsis plants (Cai et al., 2020).

Materials and Reagents

ddPCR set-up and droplet generation

  1. PCR Plate Heat Seal, foil, pierceable (Bio-Rad, catalog number: 1814040)

  2. DG8TM Cartridge for QX200TM/QX100TM Droplet Generator (Bio-Rad, catalog number: 1864008)

  3. Droplet Generator DG8TM Gasket (Bio-Rad, catalog number: 1863009)

  4. ddPCR Plates, 96-Well, Semi-Skirted (Bio-Rad, catalog number: 12001925)

  5. QubitTM dsDNA HS Assay kit (ThermoFisher Scientific, catalog number: Q32851)

  6. Restriction endonuclease with a recognition site within the transgene but not between the primer sites (see procedure for details) and compatible digestion buffer

  7. QX200TM ddPCR TM EvaGreen Supermix (Bio-Rad, catalog number: 1864033)

  8. QX200 Droplet Generation Oil for EvaGreen® (Bio-Rad, catalog number: 1864005)

Equipment

ddPCR set-up and droplet generation

  1. QubitTM 4 fluorometer (ThermoFisher Scientific, catalog number: Q33238)

  2. QX200TM droplet generator (Bio-Rad, catalog number: 1864002)

  3. Thermal cycler (e.g., Bio-Rad C1000 Touch thermal cycler, catalog number: 1851148)

  4. Multichannel pipettes (10-100 µl)

  5. Heat sealer (e.g., Eppendorf S100, catalog number: 5391000036)

  6. DG8 cartridge holder (Bio-Rad, catalog number: 1863051)

  7. QX200 droplet reader (Bio-Rad, catalog number: 1864003)

  8. Droplet reader plate holder (Bio-rad, catalog number: 12006834)

Software

  1. Primer design software, e.g., Primer 3 (https://primer3.ut.ee/)

  2. Sequence analysis software, e.g., Benchling (https://benchling.com)

  3. QuantaSoft software (Bio-Rad, catalog number: 1863007)

  4. Software capable of reading comma-separated values (.csv) files (e.g., Microsoft Excel)

  5. Droplet DigitalTM PCR Applications Guide

    http://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_6407.pdf

Procedure

In this procedure, transgene copy number is determined by performing two PCR reactions for each sample. The first amplifies a region of the transgene (target gene), and the second amplifies a region of an endogenous gene of known copy number (reference gene). An alternative method is multiplex amplification of the target and reference gene using hydrolysis probes for detection; primers and probes for several plant reference genes have been previously reported (Collier et al., 2017).


  1. Primer design and selection of restriction endonuclease

    1. Use primer design software (e.g., Primer 3) to select primers that amplify a 100-120 base pair region of both the transgene target and reference gene. Primers should optimally have annealing temperatures of ~60°C, similar estimated binding kinetics, and minimal dimerization.

    2. Check that each primer pair efficiently amplifies the desired target in a test PCR reaction. Include control reactions with (i) genomic DNA from a wild-type non-transgenic plant, and (ii) no DNA. Assemble the reaction as shown in Table 1.


      Table 1. PCR reaction for testing ddPCR primer pairs

      Components Stock concentration Final concentration Volume
      2× QX200TM ddPCRTM EvaGreen® Supermix 5 μl
      Forward Primer 2 μM 250 nM 1.25 μl
      Reverse Primer 2 μM 250 nM 1.25 μl
      Genomic DNA 5-50 ng (≤ 2.5 μl)
      Nuclease-free Water Bring reaction volume to 10 μl
      Total 10 µl


    3. Incubate the PCR reaction using the cycling conditions recommended by the reagent manufacturer, provided in Table 2.


      Table 2. PCR cycling conditions for testing ddPCR primer pairs. Use a 2°C/s ramping rate for all steps.

      Step Temperature, °C Time Number of cycles
      Enzyme Activation and Thermosetting 95 5 min 1
      Denaturation 95 30 s 40
      Annealing 60 50 s
      Extension 72 50 s
      Signal Stabilization 4 5 min 1
      90 5 min 1
      Hold 4
      1


    4. Visualize the PCR products by agarose gel electrophoresis, ensuring that single clear bands of the expected sizes are obtained for both sets of primers.

    5. Digesting genomic DNA before amplification will ensure that, even if there are two copies of the transgene inserted in tandem, each copy will be on a separate DNA molecule and therefore in a separate droplet. Analyze the transgene sequence for the presence of restriction endonuclease (RE) recognition sequences using sequence analysis software (e.g., Benchling). Select an RE with at least one recognition site within the transgene but no sites between the primers of either the target or reference gene. Avoid the use of restriction endonucleases sensitive to methylation.


  2. ddPCR set-up

    1. Good quality genomic DNA is required for ddPCR. A cetyltrimethylammonium bromide (CTAB)-based method or a commercial plant DNA extraction kit will provide sufficiently pure genomic DNA. Each ddPCR reaction will generally require 5-50 ng genomic DNA.

      Notes:

      1. To accurately quantitate DNA concentration, we recommend the use of a Qubit fluorometer.

      2. The exact quantity of genomic DNA used in each reaction needs to be optimized for the genome size of the organism.

    2. Digest each sample of genomic DNA using the selected RE. An example restriction digestion reaction is shown in Table 3. Different quantities of enzyme and buffer will be required depending on the manufacturer and concentration of the enzyme. Aim to include ~10-30 U enzyme per g genomic DNA.

      Note: The EvaGreen® product notes indicate that digestion of genomic DNA can also be performed by adding 1 μl (2-5 U) desired restriction endonuclease to the PCR reaction.


      Table 3. Example reaction for restriction endonuclease digestion of genomic DNA

      Components Volume
      10× Restriction Enzyme Buffer 1 μl
      Restriction Enzyme (10-20 U/μl) 0.5-3 μl
      Genomic DNA 200 ng
      Nuclease-free Water Bring reaction volume to 10 μl
      Total 10 μl


    3. Inactivate the RE by heating. The exact temperature and length of incubation will depend on the specific enzyme and manufacturer. Following inactivation, the digested genomic DNA can be used directly in the next step without further clean-up.

    4. Assemble the PCR reactions in the order shown in Table 4. In addition to the sample reactions, set up control reactions with (i) genomic DNA from a wild-type non-transgenic plant and (ii) no DNA. It is critical that the assembled ddPCR reactions are well mixed prior to proceeding to droplet generation, as every droplet must contain the same concentration of each component.


      Table 4. Example ddPCR reaction

      Components Stock concentration Final concentration Volume
      2× QX200TM ddPCRTM EvaGreen® Supermix 11.25 μl
      Forward Primer 2 μM 250 nM 2.8 μl
      Reverse Primer 2 μM 250 nM 2.8 μl
      Genomic DNA (pre- digested) 5-50 ng (≤ 5.65 μl)
      Nuclease-free Water Bring reaction volume to 22.5 µl
      Total 22.5 µl

      Note: If multiple samples are being analyzed, as for other PCR methods, it is recommended to make a master mix and dilute concentrated DNA prior to assembling the ddPCR reaction.


  3. Droplet generation

    1. Open a DG8 TM Cartridge Holder by pressing the middle latches.

    2. Insert the DG8TM Cartridge into the holder and close the holder by pressing the two halves together (Figure 1).



      Figure 1. Workflow of droplet generation. The PCR reactions and oil are added to a cartridge, which is inserted into the holder and held in place by the gasket. Following droplet generation, the emulsified reactions are transferred to a microtiter plate for thermal cycling.


    3. Load each 20-µl reaction assembled in the previous step into individual sample wells of a DG8TM Cartridge using a multichannel pipette.

    4. Add 70 μl QX200 Droplet Generation Oil for EvaGreen® into the oil wells.

    5. Cover the cartridge with the DG8TM Gasket. Loop the holes of the gasket over the four hooks on the cartridge holder to hold the gasket in place.

    6. Open the DG8 droplet generator and insert the cartridge holder. Close the lid by pressing the button. The generator will start automatically.

    7. Transfer the entire volume of the droplet emulsion (typically 40 μl) to the desired wells of a semi-skirted 96-well ddPCR plate (Figure 1). Cover the plate with a pierceable foil plate heat seal and attach using a plate-sealer according to the manufacturer’s instructions. Rotate the plate and repeat this step to ensure the foil is fully attached.

      Note: To avoid disrupting the emulsified droplets, take care to pipette very slowly. Wide-bore pipette tips (or pipette tips with the extremity removed using a blade) can be used.

    8. Place the sealed plate into a thermal cycler and use exactly the same cycling conditions as those used in the test PCR (see Table 2). During the initial heat cycle, the oil surrounding each droplet will thermoset.


  4. Droplet counting

    1. When the PCR cycle is complete, switch on the QX200TM droplet reader and open the QuantaSoftTM software.

    2. In the QuantaSoftTM software, start a new experiment (Template > New).

    3. Select the wells to be detected and enter the sample names or record the well positions for target and reference genes for each sample.

    4. Select the key parameters in the well editor as follows:

      1. Experiment: ABS (absolute quantitation).

        Note: The QuantaSoftTM software provides three default experiments: Absolute Quantitation (ABS), Rare Event Detection (RED), and Copy Number Variation (CNV). Both ABS and CNV can be used for copy number determination. For CNV mode, a known reference gene copy number needs to be provided. The ABS mode is more flexible. For example, the copy number of the reference gene is not required, and the relative ratio of target and reference can be calculated.

      2. Supermix: QX200TM ddPCR EvaGreen Supermix.

      3. Target 1 type: Ch1 Unknown.

      4. Target 2 type: Ch2 Unknown.

      5. Name: Enter sample names, e.g., transgenic line x, reference gene. This can help to distinguish which wells are for transgene and which are for reference genes.

    5. Apply the chosen parameters to the selected wells by clicking ‘OK’.

    6. Remove the PCR plate from the thermocycler and insert into the base of a plate-reader holder. Cover the plate with the lid and press the latches to lock the holder.

    7. Open the QX200TM droplet reader, insert the plate-reader holder, and close the lid.

    8. Check that the indicator lights for ‘power’, ‘bottle levels’, and ‘plate in place’ are green.

    9. Click ‘Run’ in the QuantaSoftTM software. Select EvaGreen in the ‘Run Option’ dialog. Click OK to start the reading.

Data analysis

  1. When reading is complete, click on ‘Analysis’.

  2. Click the ‘1D amplitude’ button to view the distribution of positive and negative droplets. An example distribution is shown in Figure 2.



    Figure 2. Example plot showing positive (blue) and negative (grey) droplets for eight samples (A01-H01). The X-axis (event number) indicates the number of droplets measured across the total experiment, and the Y-axis shows the amplitude of fluorescence.


    Note: A single reaction should produce 12,000-20,000 droplets. The calculation of copy number relies on the ratio of positive to negative droplets. If the positive droplets saturate (most droplets are positive), this means that every droplet had the target molecule and it is likely that either the primers are non-specific or too much DNA was used in the reaction. To obtain an accurate result, primer specificity should be checked and/or the reactions repeated with less DNA.

  3. The threshold, which distinguishes positive droplets from negative droplets, as well as the concentration value, is automatically determined; however, if desired, the threshold can be manually adjusted.

  4. Click the “Concentration” button. Export the concentration data as a comma-separated values (.csv) file.

  5. Open the .csv file in the appropriate software (e.g., Microsoft Excel). Any sample names provided in the QuantaSoftTM software will be in the ‘Sample’ column. The ‘Concentration’ column contains the number of PCR amplicons per μl required to calculate copy number.

  6. The copy number of the target gene is calculated by (Ctarget/Cref) * Nref, where Ctarget is the concentration of the transgene, Cref is the concentration of the endogenous gene, and Nref is the copy number of the reference gene. In the example provided in Table 5, the reference gene is a single copy homozygous gene in the Arabidopsis genome; thus, there are two copies in each of the diploid somatic cells from which DNA was extracted. In line 1, the concentration of the target (Ctarget) is 115 copies/μl, and the concentration of the reference is (Cref) is 227 copies/μl. Therefore, the copy number of the transgene is (115/227)*2 = 1.0, meaning that line 1 has a single copy of the transgene (hemizygous). In line 2, Ctarget = 784 copies/μl and Cref = 173 copies/μl. Therefore, the copy number of the transgene is (784/173)*2 = 9.1, meaning that line 2 has nine copies of the transgene. In line 3, Ctarget = 11.7 copies/μl and Cref = 333 copies/μl. Therefore, the copy number of the target transgene is (11.7/333)*2 = 0.07 copies, which means that line 3 is unlikely to contain a transgene.

    Note: When determining transgene copy numbers in polyploid plants, many endogenous genes typically presenting as a single-copy gene may be present on multiple genomes. For example, there may be four copies of a typical single-copy reference gene in the diploid cells of a tetraploid plant; therefore, the concentration value for a single-copy hemizygous transgene will be one quarter of the reference gene. The method described above can also be used for determining transgene zygosity in progeny generations; however, since the ddPCR protocol will only provide raw values, knowledge of the copy number of the parent line(s) will be required. For example, if the diploid cells of a progeny plant are estimated to contain six copies, knowledge of the copy number of any parental line(s) is required to determine whether the plant has six independent hemizygous loci or three homozygous loci.


    Table 5. Example ddPCR data from three transgenic plants (Lines 1-3) showing the number of positive and negative droplets obtained in which the transgene (target) and a known-copy endogenous gene (ref) were amplified. The column showing the concentration (number of PCR amplicons per μl) is used to calculate the copy number.

    Well ExptType Sample Status Concentration Supermix Positives Negatives
    A01 Absolute Quantitation Line 1 target OK 115 QX200 ddPCR EvaGreen Supermix 1648 16017
    A02 Absolute Quantitation Line 1 ref OK 227 QX200 ddPCR EvaGreen Supermix 3283 15412
    B01 Absolute Quantitation Line 2 target OK 784 QX200 ddPCR EvaGreen Supermix 8398 8869
    B02 Absolute Quantitation Line 2 ref OK 173 QX200 ddPCR EvaGreen Supermix 2321 14615
    C01 Absolute Quantitation Line 3 target OK 11.7 QX200 ddPCR EvaGreen Supermix 113 11313
    C02 Absolute Quantitation Line 3 ref OK 333 QX200 ddPCR EvaGreen Supermix 3061 9346

Acknowledgments

We thank Oleg Raitskin for technical help and Simon Foster for equipment maintenance. We acknowledge financial support from the UK Biotechnology and Biological Sciences and the Engineering and Physics Research Councils (BBSRC and EPSRC) synthetic biology for growth program (OpenPlant Synthetic Biology Research Centre BB/L014130/1), as well as grants BBS/E/T/000PR9816 and BB/R021554/1. This protocol was developed to generate the transgene copy number data presented in Cai (2020).

Competing interests

No competing interests to declare.

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简介

[摘要]生产转基因植物既可以研究基因功能,又可以赋予作物所需的性状。众所周知,转基因拷贝数会影响表达水平,从而影响表型。同样,转基因接合性的知识对于对表型进行定量评估和跟踪后代中转基因的遗传是可取的。自从第一批转基因植物问世以来,已经应用了多种确定拷贝数的方法,包括Southern印迹、定量实时PCR ,以及最近的测序方法;ħ H但是,每个方法具有特定的缺点,影响吞吐量,准确性,或费用。数字 PCR ( dPCR ) 将反应分成多个部分,将 PCR 的指数、模拟性质转换为线性数字信号,从而可以准确估计特定序列的出现频率。置信度随着分区数量的增加而增加;因此,能够将反应分成数万个纳米液滴的乳液技术的可用性允许在众所周知的数字液滴 PCR ( ddPCR ) 中准确确定拷贝数。ddPCR具有与其他 PCR 技术类似的低成本和可扩展性优势,但具有卓越的准确性和可靠性。


图文摘要:

数字 PCR ( dPCR ) 将反应分成多个部分,将 PCR 的指数、模拟性质转换为线性数字信号,从而可以准确评估转基因拷贝数的频率。

[背景]植物转化被用作研究基因功能以及生产基因工程作物的研究工具。有几种方法用于递送DNA导入植物细胞,其中包括直接递送到分离的原生质体,生物射弹递送采用高密度微粒,农杆菌tumefac iens介导的递送,以及最近的纳米颗粒(Altpeter等人,2016;王等人。, 2019) 。DNA递送技术可以优化解Ž编,得到的转基因品系具有单个或低更高的频率-拷贝插入; 然而,转移到植物基因组的转基因的确切数量无法控制或限制,必须单独评估转基因品系以确定转基因拷贝数(Sivamani等,2015)。因为表达水平,并因此表型,长期被公认由拷贝数的影响,准确的测定是必需的(霍布斯等人,1993)。拷贝数测定还可用于跟踪遗传和接合性。大多数植物转化方法将转基因传递到体细胞,从中再生转基因植物(称为 T 0 ),每一个都具有一个多半合的转基因基因座。在从自fertili得到的子代Ž通货膨胀,转基因座位通常遵循经典孟德尔遗传; 人虽然,非孟德尔分离也有报道(的Ahuja和Fladung ,2014)。拷贝数的准确测定可用于通过后代跟踪转基因,特别适用于具有多个转基因拷贝的品系和专性异交物种。

自从植物转化成为惯例以来的几十年里,许多不同的技术已被用于确定拷贝数。最初,拷贝数由Southern杂交分析确定(Southern,1975)。在这项技术中,基因组 DNA 用限制性内切核酸酶消化,通过电泳分离,然后转移到膜上,使用标记的 DNA 探针检测含有转基因的片段。除了费力之外,检测可能位于同一片段上的靠近插入(串联重复)的转基因也具有挑战性。此外,除非有足够的杂合度使得同源染色体在限制性消化后具有不同的片段化模式,否则合子性的检测依赖于条带强度的差异,这被认为是不可靠的(Cantsilieris等,2013)。

在 21 世纪的最初几十年,定量实时 PCR (qPCR) 成为估计植物转基因拷贝数的首选方法(Bubner和 Baldwin,2004;Li等,2004;Weng等,2004) ;Yang等人,2005a 和 2005b ;Yi 和 Hong,2019)。将每个样品中转基因的浓度与标准曲线或经过验证的参考基因(已知拷贝数的内源基因)进行比较(Pfaffl ,2001)。然而,因为每个PCR循环是加倍反应,所需的两个倍的增加来区分一个从两个拷贝,或半合子复制从纯合体,是在检测极限。即使优化解Ž通货膨胀,它可以是具有挑战性的,以实现高精度的水平所需区分单拷贝和两拷贝线(Cantsilieris等人,2013; Mieog 。等人,2013年)。

下一步-克eneration小号equencing(NGS)技术已经降低了测序成本; 然而,即使是低通浅层测序,有时也称为“脱脂测序” ,对于大型植物基因组来说也是昂贵的 –对于常规拷贝数检测而言,这是令人望而却步的(Golicz等人,2015 年;Kim等人,2016 年)。目标-捕获测序,其中将含有感兴趣的序列的基因组区域是分离的,已允许的产生与在降低成本的插入位点的序列同一性信息准确拷贝数数据一起(Guttikonda等人,2016) 。最初,短读被应用新一代测序技术(Polkoa等,2012;勒帕等人; 2013年郭等,2016) ,但最近,长期阅读的纳米孔-基于测序平台也已utili ž ED (李等人,2019;布蒂尼。等人,2020) 。然而,与基于PCR 的方法相比,即使是捕获测序技术也具有更高的成本,并且需要更多的时间进行样品制备和数据分析。

数字 PCR ( dPCR ) 将PCR的指数、模拟性质转换为线性数字信号,允许准确估计特定序列的出现频率(Vogelstein和Kinzler ,1999)。为此,每个样品被分成大量分区,并在每个分区上单独进行PCR 反应。这是最初困难和费力的根据需要在多个区室(管)进行划分反应,但乳液技术的发展使反应是compartmentali ž在单管内纳米液滴ED(Diehl的等人,2006)。与 qPCR 协​​议一样,荧光被纳入目标扩增子中;然而,不是在反应的指数阶段监测特定循环的荧光强度,而是确定每个分区在反应终点处是否存在荧光(以及目标)。目标基因的拷贝数是通过确定相对于已知拷贝数的参考基因扩增目标基因的部分的分数来计算的。由于每个分区被打进荧光的存在,DPCR实现精确孔定量吨的转基因拷贝数的通货膨胀。通过增加种群大小(计算更多分区)提高了信心,导致通过dPCR估计拷贝数的准确性被报告为优于 qPCR 并与测序技术相比较(Abyzov等,2012)。近年来,一些商业平台,分反应到使用乳化技术的隔间数万(数滴PCR; ddPCR )或微小号芯片已成功地应用于准确测定不同种类植物的转基因拷贝数(Glowacka等,2016;Collier等,2017;Giraldo等,2019;Cai等,2020)。下面,我们描述一个ddPCR用于确定转基因拷贝数协议在拟南芥植物(蔡等人,2020) 。

关键字:数字PCR, 微滴式数字PCR, 转基因拷贝数, 植物, 转移DNA


材料和试剂

ddPCR设置和液滴生成
PCR板热封,箔,可刺穿(Bio- R ad,目录号:1814040)
用于 QX200 TM /QX100 TM液滴发生器的DG8 TM墨盒(Bio- R ad,目录号:1864008)
液滴发生器 DG8 TM垫片(Bio- R ad,目录号:1863009)
ddPCR板,96 孔,半裙(Bio- R ad,目录号:12001925)
QubitTM的dsDNA HS测定试剂盒(赛默飞小号系统求解,目录号:Q32851)
限制性核酸内切酶在转基因内具有识别位点,但不在引物位点之间(详见程序)和兼容的消化缓冲液
QX200 TM ddPCR TM EvaGreen Supermix (Bio- R ad,目录号:1864033)
用于EvaGreen ® 的QX200 液滴生成油(Bio- R ad,目录号:1864005)

设备

ddPCR设置和液滴生成
Qubit TM 4荧光计(ThermoFisher Scientific,目录号:Q33238)
QX200 TM液滴发生器(Bio- R ad,目录号:1864002)
热循环仪(例如,Bio- R ad C1000 Touch 热循环仪,目录号:1851148)
多通道移液管小号(10-100微升)
热封机(例如,Eppendorf S100,目录号:5391000036)
DG8 筒架(Bio- R ad,目录号:1863051)
QX200 液滴阅读器(Bio- R ad,目录号:1864003)
液滴读取器板架(Bio-rad ,目录号:12006834)

软件

引物设计软件,例如引物 3 ( https://primer3.ut.ee/ )
序列分析软件例如,Benchling (https://benchling.com)
QuantaSoft软件(Bio- R ad,目录号:1863007)
能够读取逗号分隔值 (.csv) 文件的软件(例如Microsoft Excel)
Droplet Digital TM PCR 应用指南
http://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_6407.pdf

程序

在此过程中,通过对每个样品进行两次 PCR 反应来确定转基因拷贝数。第一个扩增转基因区域(靶基因),第二个扩增已知拷贝数的内源基因(参考基因)区域。另一种方法是使用水解探针检测目标基因和参考基因的多重扩增;之前已经报道了几种植物参考基因的引物和探针(Collier等,2017)。

限制性内切酶的引物设计与选择
使用引物设计软件(例如,Primer 3)选择可扩增转基因目标基因和参考基因的 100-120 碱基对区域的引物。引物应最佳地已退火〜60的温度下℃下,类似的估计结合动力学,以及最小dimeri ž通货膨胀。
检查每个引物对是否在测试 PCR 反应中有效地扩增了所需的目标。包括与 ( i ) 来自野生型非转基因植物的基因组 DNA 和 (ii) 无 DNA 的对照反应。组装反应如表 1 所示。


表 1. 用于测试ddPCR引物对的PCR 反应

使用表 2 中提供的试剂制造商推荐的循环条件孵育 PCR 反应。

表 2. 用于测试ddPCR引物对的PCR 循环条件。对所有步骤使用2°C/s 的升温速率。

Visuali ž ËPCR产物通过琼脂糖凝胶电泳,从而确保这两组引物获得了预期大小的单条带清晰。
在扩增前消化基因组 DNA 将确保,即使有两个转基因副本串联插入,每个副本也将位于单独的 DNA 分子上,因此位于单独的液滴中。ANALY ž e是转基因序列为限制性内切酶(RE)使用识别序列的存在的序列分析软件例如,Benchling 。选择在转基因中至少有一个识别位点的n RE,但在目标或参考基因的引物之间没有位点。避免使用对甲基化敏感的限制性内切核酸酶。

ddPCR设置
ddPCR需要高质量的基因组 DNA 。甲十六烷基三甲基溴化铵(CTAB)为基础的方法或市售的植物DNA提取试剂盒将提供足够纯的基因组DNA。每个ddPCR反应通常需要 5-50 ng 基因组 DNA。
注意小号:
为了准确孔定量泰特DNA浓度,我们建议使用一个量子位荧光计。
在每个反应中所用的基因组DNA的确切数量必须是优化解ž的生物体的基因组大小编辑。
使用选定的 RE 消化每个基因组 DNA 样本。一个例子限制性消化反应示于表3.不同量的酶和缓冲液将根据制造商和浓度需要的酶。目标是每 g 基因组 DNA包含 ~10-30 U 酶。
注意:EvaGree n ®产品说明表明,也可以通过向PCR 反应中加入 1 μl (2-5 U) 所需的限制性内切酶来消化基因组 DNA 。

表 3. 基因组 DNA 限制性内切酶消化的示例反应

通过加热灭活 RE。温育的精确温度和长度将取决于在特定的酶和制造商。灭活后,消化的基因组 DNA 可直接用于下一步,无需进一步清理。
按照表 4 中所示的顺序组装 PCR 反应。除了样品反应之外,还设置了与 ( i ) 来自野生型非转基因植物的基因组 DNA 和 (ii) 无 DNA 的对照反应。在进行液滴生成之前,组装好的ddPCR反应必须充分混合,因为每个液滴必须包含相同浓度的每种成分。


表 4. ddPCR反应示例

注意:如果多个样品正在ANALY Ž版,作为其他的PCR方法,建议向组装使主混合物和稀浓缩DNA之前ddPCR反应。

液滴生成
按下中间的闩锁,打开 DG8 TM墨盒固定器。
将 DG8 TM Cartridge 插入支架并通过将两半压在一起来关闭支架(图 1)。
图 1.液滴生成的工作流程。PCR 反应和油被添加到墨盒中,墨盒插入支架并由垫圈固定到位。液滴生成后,乳化反应被转移到微量滴定板进行热循环。

加载每个20 -在先前步骤中成DG8的单个样品池组装微升反应TM使用多道移液器墨盒。
将 70 μl QX200 用于EvaGreen ® 的液滴生成油加入油井中。
用 DG8 TM垫圈盖住滤芯。将垫圈的孔绕在墨盒支架上的四个挂钩上,以将垫圈固定到位。
打开 DG8 液滴发生器并插入墨盒支架。按下按钮关闭盖子。发电机将自动启动。
将整个体积的液滴乳液(通常为 40 μl )转移到半裙边 96 孔ddPCR板的所需孔中(图 1)。盖上刺穿箔片热封的板和附连使用根据一个板密封器的制造商的说明。旋转板并重复此步骤以确保箔完全贴合。
注意:为避免破坏乳化液滴,请注意移液速度非常缓慢。可以使用大口径移液器吸头(或使用刀片去除末端的移液器吸头)。
密封板放置到热循环仪和我们Ë完全相同的循环条件为那些在测试PCR中使用(见表2)。在最初的热循环过程中,每个液滴周围的油都会热固。

液滴计数
PCR 循环完成后,打开 QX200 TM液滴阅读器并打开QuantaSoft TM软件。
在QuantaSoft TM软件中,开始一个新实验(模板 > 新建)。
选择要检测的孔并输入样品名称或记录每个样品的目标和参考基因的孔位置。
在井编辑器中选择关键参数如下:
实验:ABS(绝对孔定量牛逼通货膨胀)。
注:QuantaSoft TM软件提供了三种默认实验:绝对孔定量牛逼通货膨胀(ABS),稀有事件检测(RED)和拷贝数变异(CNV)。ABS 和 CNV 均可用于确定拷贝数。对于 CNV 模式,需要提供已知的参考基因拷贝数。ABS模式更加灵活。例如,拷贝数的不需要参考基因,并且可以计算目标和参照的相对比率。
Supermix :QX200 TM ddPCR EvaGreen Supermix 。
目标 1 类型: Ch1 Unknown 。
目标 2 类型: Ch2 Unknown 。
名称:é NTER样品名称例如,转基因行x,参照基因。这有助于区分哪些孔用于转基因,哪些孔用于参考基因。
单击“确定”将所选参数应用于所选孔。
从热循环仪中取出 PCR 板并插入读板器支架的底座。盖上盖子上的板,然后按所述闩锁保持器。
打开 QX200 TM液滴读取器,插入读板器支架,然后合上盖子。
检查“电源”、“瓶位”和“盘子就位”的指示灯是否为绿色。
单击QuantaSoft TM软件中的“运行” 。在“运行选项”对话框中选择EvaGreen 。单击“确定”开始阅读。

数据分析

阅读完成后,单击“分析”。
单击“一维振幅”按钮以查看正负液滴的分布。示例分布如图 2 所示。

图 2. 示例图显示了八个样品 (A01-H01) 的阳性(蓝色)和阴性(灰色)液滴。X 轴(事件编号)表示整个实验中测量的液滴数量,Y 轴表示荧光幅度。

注意:单个反应应产生 12,000-20,000 个液滴。的拷贝数的计算依赖于正的比率到负液滴。如果阳性液滴饱和(大多数液滴是阳性的),这意味着每个液滴都有目标分子,很可能引物是非特异性的,或者反应中使用了过多的 DNA。为了获得准确的结果,应检查引物特异性和/或用较少的 DNA 重复反应。
的阈值,这区别于负液滴正液滴,以及浓度值,则自动地确定; ħ H但是,如果需要的话,该阈值可以手动调节。
单击“浓度”按钮。将浓度数据导出为逗号分隔值(.csv) 文件。
在打开.csv文件的适当的软件(例如,Microsoft Excel)中。QuantaSoft TM软件中提供的任何样品名称都将出现在“样品”列中。在“ ç oncentration”列包含每PCR扩增子的数量微升需要计算拷贝数。
靶基因的拷贝数是通过计算(ç目标/ Ç REF )* Ñ REF ,其中Ç目标是转基因的浓度,Ç REF是内源基因的浓度,和Ñ REF是的拷贝数参考基因。在表5提供的例子中,参考基因是拟南芥基因组中的单拷贝纯合基因;因此,在每个提取 DNA 的二倍体体细胞中都有两个拷贝。在第 1 行中,目标 ( C target )的浓度为 115 拷贝/ μl ,参考浓度 ( C ref ) 为 227 拷贝/ μl 。因此,转基因的拷贝数是(227分之115)* 2 = 1.0,这意味着该线1具有转基因(半合子)的单个副本。在第 2 行中,C target = 784 拷贝/ μl和C ref = 173 拷贝/ μl 。因此,转基因的拷贝数是(173分之784)* 2 = 9.1 ,这意味着该线2具有转基因的9份。在第 3 行中,C target = 11.7 拷贝/ μl和C ref = 333 拷贝/ μl 。因此,对拷贝数的目标转基因是(11.7 / 333)* 2 = 0.07份,这意味着线3是不太可能包含转基因。
注意:在确定多倍体植物中的转基因拷贝数时,许多通常表现为单拷贝基因的内源基因可能存在于多个基因组中。例如,在四倍体植物的二倍体细胞中,典型的单拷贝参考基因可能有四个拷贝;因此,单拷贝半合子转基因的浓度值将是参考基因的四分之一。上述方法也可用于确定后代中的转基因接合度;ħ H但是,因为该ddPCR协议将只提供原始值,父行(多个)的拷贝数的知识是必需的。例如,如果估计后代植物的二倍体细胞含有六个拷贝,则需要了解任何亲本系的拷贝数以确定植物是否具有六个独立的半合子位点或三个纯合子位点。


表 5.来自三个转基因植物(第 1-3 行)的示例ddPCR数据,显示了扩增转基因(目标)和已知拷贝的内源基因(参考)时获得的阳性和阴性液滴的数量。显示浓度(每微升PCR 扩增子数)的列用于计算拷贝数。

致谢

我们感谢 Oleg Raitskin的技术帮助和 Simon Foster 的设备维护。我们感谢英国生物技术和生物科学以及工程和物理研究委员会(BBSRC 和EPSRC )合成生物学增长计划(OpenPlant合成生物学研究中心 BB / L 014130/1)的财政支持,以及 BBS/E/ T/000PR9816 和 BB/R021554/1。该协议旨在生成 Cai (2020) 中提供的转基因拷贝数数据。

利益争夺

没有要申报的竞争利益。

参考

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引用:Cai, Y., Dudley, Q. M. and Patron, N. J. (2021). Measurement of Transgene Copy Number in Plants Using Droplet Digital PCR. Bio-protocol 11(13): e4075. DOI: 10.21769/BioProtoc.4075.
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