REPLI-g Single Cell Kit

对单个细胞或有限的样本进行高度均一的全基因组扩增（WGA）

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REPLI-g Single Cell Kit (24)

Cat. No. / ID: 150343

REPLI-g sc Polymerase, Buffers, and Reagents for 24 whole genome amplification reactions (yields up to 40 µg/reaction)

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Reactions

REPLI-g Single Cell Kit 旨在用于分子生物学应用。该产品不能用于疾病诊断、预防和治疗。

✓ 全天候自动处理在线订单

✓ 博学专业的产品和技术支持

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特点

采用多重置换扩增技术对基因组位点进行无偏差的扩增
适用于二代测序等新技术应用
产量稳定，可达40 µg（产物平均长度大于>10 kb）
可用于癌症、干细胞或宏观基因组学（metagenomics）研究

产品详情

很多研究人员使用二代测序仪器对生物样本的DNA序列进行分析和基因分型，但经常受限于有限的样本量。REPLI-g Single Cell Kit可均一的扩增单个细胞（1至1000个细菌或肿瘤细胞）或纯化的基因组DNA，能够覆盖基因组所有位点。另有专用于干血或新鲜或冷冻的组织样本的实验方案。所有缓冲液和试剂生产都经过严格控制的流程，避免污染DNA，确保每次实验获得可靠的结果。该产品采用多重置换扩增（MDA）技术，对所有基因组位点进行无偏差的扩增。与基于PCR的全基因组扩增技术相比，该技术具有更好的扩增高保真度，避免出现假阳性或假阴性信号。

绩效

覆盖基因组所有位点，十分适用于二代测序和其他下游应用

REPLI-g Single Cell Kit扩增的DNA平均长度大于10 kb，并且覆盖所有基因组位点。该产品已经过验证，适用于二代测序、基于芯片技术的比较基因组杂交（aCGH）和real-time PCR应用等多种下游分析。使用该产品无需单独进行PCR扩增，减少了手动操作，获得的产物长度PCR方法更长（参见" Next-generation sequencing using REPLI-g amplified DNA requires less hands-on time and generates more sequence information than PCR-based methods"）。用扩增产物进行二代测序，获得高品质结果。这表明该试剂盒的位点覆盖率大，错配率低，其扩增的DNA（甚至从单一细菌细胞中扩增的DNA）能够与纯化获得的基因组DNA相媲美（参见" Comparable NGS results"）。另有研究对人类常染色体和X染色体上的多个标记物进行了分析，三个独立的实验均显示：基因组的所有位点被成功扩增，没有遗漏任何一个位点（参见" Complete genome coverage"）。

表1. 样本类型和研究领域
样本类型（细胞/DNA）	研究领域
人类/动物	生物标记物研究（SNP、突变、CNV）
	干细胞研究
	循环胚胎细胞分析
	嵌合体研究
	遗传易感性研究
	转基因动物基因分型
癌症	体细胞遗传变异分析
	肿瘤恶化
	肿瘤干细胞/进化
	循环肿瘤细胞分析
细菌	宏观基因组学研究
	病原体分析
	微生物基因分型
Plants*	植物气孔研究
Plants*	花粉分析

REPLI-g Single Cell Kit的表现优于其他供应商的试剂盒

其他供应商的试剂盒基于PCR技术进行全基因组扩增，获得的产物片段较短，带有PCR引物序列，可能影响下游应用。基于PCR的全基因组扩增容易出现错误，会导致碱基对突变、STR的减少或增多，使用低保真Taq DNA聚合酶会导致位点缺失。相反，REPLI-g Single Cell Kit能够进行高度均一的全基因组扩增，位点偏差小。对四个试剂盒进行了检测，其中两个利用多重置换扩增技术（包括REPLI-g Single Cell Kit），另外两个利用PCR技术。采用单一细胞扩增实验方案对所有试剂盒进行序列位点检测。除REPLI-g Single Cell Kit外的其他供应商的试剂盒都出现了位点的遗漏（参见" Unbiased DNA amplification from a single cell"）。

查看图表

Next-generation sequencing using REPLI-g amplified DNA requires less hands-on time and generates more sequence information than PCR-based methods.

Comparable NGS results.

Complete genome coverage.

Unbiased DNA amplification from a single cell.

原理

REPLI-g Single Cell Kit含有REPLI-g sc Polymerase，这是一种优化的新型高保真Phi 29聚合酶。该试剂盒采用多重置换扩增技术扩增复杂的基因组DNA，温和的碱孵育能够避免DNA片段化，促进对所有位点的无偏差扩增。该试剂盒十分适用于分离的肿瘤细胞或细菌细胞等单一细胞，能够扩增获得高产量DNA（参见表1）。此外，该试剂盒还配有专用于鲜血或干血、新鲜或冷冻组织的实验方案，可用于多种研究样本。常规DNA产量可达40 µg，对模板的起始量要求低，因此进行后续遗传分析时无需检测DNA浓度。REPLI-g Single Cell Kit的扩增产物平均长度超过10 kb，且覆盖所有位点，适合于多种应用，包括二代测序、基于芯片技术的比较基因组杂交、焦磷酸测序和real-time PCR分析（参见表2）。

表2. 下游应用和仪器
应用	仪器
全基因组测序	二代测序平台^†
外显子组测序	二代测序平台^†
SNP基因分型芯片	芯片平台^†
Array CGH	芯片平台^†
qPCR/PCR技术	Real-time PCR/PCR仪^†
Sanger测序	毛细管测序仪^†
焦磷酸测序	PyroMark (QIAGEN)

扩增原理

REPLI-g Single Cell Kit采用等温基因组扩增技术，即多重置换扩增（MDA）。六聚体与变性DNA随机结合，然后在等温条件下，在优化的Phi 29聚合酶作用下，发生链置换合成反应。每个置换后的单链作为模板，与引物结合，可扩增获得高产量DNA（参见" Multiple Displacement Amplification (MDA) technology"）。Phi 29聚合酶为噬菌体衍生酶，具有3'→5'引物外切酶活性（校正活性），其保真性是Taq DNA聚合酶的1000倍。Phi 29聚合酶在优化的REPLI-g Single Cell缓冲液体系中，能够轻松的打开发卡结构等二级结构，因此可促进扩增时聚合酶正常工作。可扩增获得长达100 kb的DNA片段（参见" Unbiased amplification with Phi 29 polymerase"）。

细胞裂解和DNA的碱变性

基因组DNA在用于酶促扩增反应前必须经过变性，而这一变性过程通常使用高温或高pH值（强碱）孵育。REPLI-g Single Cell Kit采用温和的碱孵育，在细胞裂解、DNA变性的同时，确保DNA片段化程度小，不产生碱基突变。因此扩增获得的DNA完整度高，产物长度长，覆盖位点多（参见" Effect of heat and alkaline denaturation on loci representation"）。

高效去除可检测到的DNA污染

REPLI-g Single Cell Kit中的所有组分都经过了独特的去污染流程，避免扩增产物被污染。在标准化流程中，对缓冲液和试剂进行紫外线照射，确保其不会污染DNA（参见" Innovative decontamination procedure"）。紫外线照射后，对试剂盒的所有组分进行严格的质量控制，确保其性能。

查看图表

Multiple Displacement Amplification (MDA) technology.

Unbiased amplification with Phi29 polymerase.

Effect of heat and alkaline denaturation on loci representation.

Innovative decontamination procedure.

程序

简单的单管操作

REPLI-g Single Cell Kit可对单一细胞或有限的样本进行简便、可靠、准确的全基因组扩增。反应体系构建十分便利、可靠，仅需15分钟左右的手动操作（参见" REPLI-g Single Cell Kit procedure"）。专用的缓冲液和试剂可对单一细胞、有限的组织材料、或纯化的DNA进行扩增，产量高、位点覆盖率大（参见表3）。REPLI-g技术扩增获得的DNA可在–20°C长期储存（参见" Consistent long-term stability"）。请参见表4，了解REPLI-g产品线的更多信息。

表3. REPLI-g Single Cell Kit的成分
试剂盒成分	优势
REPLI-g sc Polymerase	最长可达70 kb的长片段
	保真性是Taq聚合酶的1000倍
	覆盖所有位点
	均一扩增所有位点
REPLI-g sc Reaction Buffer^‡	对所有位点进行无偏差的扩增
Buffer DLB （裂解和变性）	高效扩增
Buffer DLB （裂解和变性）	不损伤DNA
紫外线照射去除污染	减少可检测到的残留DNA污染

表 4. 多种REPLI-g Kits的规格
	REPLI-g Single Cell	REPLI-g Mini	REPLI-g UltraFast Mini	REPLI-g Midi	REPLI-g Screening	REPLI-g FFPE	REPLI-g Mitochondrial DNA
起始材料	单一细胞、gDNA	纯化的基因组DNA、血液、细胞			纯化的基因组DNA、血液、细胞	FFPE组织、FFPE组织中纯化的基因组DNA	纯化的基因组DNA
起始材料	另外提供其他起始材料的实验方案				纯化的基因组DNA、血液、细胞	FFPE组织、FFPE组织中纯化的基因组DNA	纯化的基因组DNA
起始量	单一细胞、2–1000各细胞、组织、纯化的gDNA （1–10 ng）	>10 ng gDNA、0.5 µl血液或细胞（>600个细胞/µl）			>10 ng gDNA、0.5 µl血液或细胞（>600个细胞/µl）	组织切片（直径1 cm，厚度10–40 µm）；>100 ng gDNA	>1 ng纯化的gDNA
产量（µg/反应）	40	10	7–10	40	8	标准产量：≤10; 高产量：≤40	3–5
反应时间	8–16小时	10–16 小时	1.5小时	8–16小时	12–16小时	标准产量：4小时；高产量：10小时	8小时
手动操作时间	15分钟	15分钟	15分钟	15分钟	15分钟	40分钟	15分钟
规格	反应管	反应管	反应管	反应管	孔板

查看图表

REPLI-g Single Cell Kit procedure.

Consistent long-term stability.

应用

使用REPLI-g Single Cell Kit扩增获得的DNA可用于多种下游应用，包括：

二代测序
采用TaqMan引物/探针对进行的SNP基因分型
基于qPCR和PCR的突变检测
STR/微卫星分析
Sanger测序
焦磷酸测序
aCGH等芯片技术

辅助数据和图表

Effect of heat and alkaline denaturation on loci representation.

资源

Accelerate your NGS performance through Sample to Insight solutions

This protocol is optimized for whole genome amplification from biopsies using the REPLI-g Single Cell Kit.

This protocol is optimized for whole genome amplification from flash-frozen tissue samples using the REPLI-g Single Cell Kit.

This protocol is optimized for whole genome amplification from buccal cells using the REPLI-g Single Cell Kit.

Purification of DNA after whole Genome amplification using the REPLI-g Single Cell Kit

For whole genome amplification from single cells, limited samples, or purified genomic DNA

Download Safety Data Sheets for QIAGEN product components.

FAQ

The REPLI-g Whole Genome Amplification (WGA) method is a rapid and reliable method of generating unlimited DNA from a few cells or a few nanograms of genomic DNA. This technology amplifies the genome with comprehensive loci coverage and minimal bias between any loci, yielding 12+ kb fragments in a simple, scalable reaction.

FAQ ID -654

The REPLI-g amplified products can be used directly for downstream analysis such as PCR, PCR-based applications, restriction enzyme digestion, cycle sequencing, and more, after appropriate dilution to adjust to work concentrations.

However, to determine DNA concentration by absorbance, the MDA product should be run through a spin column to eliminate the random hexamers, as they will contribute to the absorbance reading and give an artificially high concentration. For this reason, we recommend determining DNA concentration by PicoGreen analysis, which preferentially binds double-stranded DNA. As a result, single-stranded random hexamers will not contribute to the apparent DNA concentration in the quantitation assay. When using this method, the concentration of the MDA product can be determined directly, without any purification.

A Protocol for the use of PicoGreen to quantitate REPLI-g WGA product can be found in the REPLI-g Mini/Midi Handbook. Please follow this link .

FAQ ID -713

These regions are not amplified because only a subset of the random primers in the REPLI-g amplification mix can prime within these extensively repeated regions. Despite the high processivity of the Phi29 DNA Polymerase, centromeres and telomeres are at a competitive disadvantage during amplification, and drop out. In experiments we have done, single copy sequences within approximately 5000 bases of a poorly amplified region can be affected during amplification. If your gene is further than 5000 bases apart from a centromere or telomere, it should be amplified just fine.

FAQ ID -702

Yes. Chromosomes are equally amplified. We and our customers use amplified DNA for SNP genotyping on a regular basis, using Illumina, TaqMan®, Sequenom, PCR, gel-based sequencing, and other techniques. Tzvetkov et al. (2005) used Affymetrix’ GeneChip Human Mapping 10K Arrays to investigate the accuracy and allele amplification bias in DNA samples subjected to MD-WGA with REPLI-g. They observed an excellent concordance (99.95%) between single-nucleotide polymorphisms (SNPs) called both in the nonamplified and the corresponding amplified DNA. Genomic DNA for this study was extracted from blood samples of four unrelated donors using the QIAamp DNA Blood Kit. High-throughput microarray genotyping of 11 555 different SNPs was performed using GeneChip Human Mapping 10K Arrays version Xba131 (Affymetrix).

For additional references, please visit our continuously expanding Citation Data Base online.

See trademarks.

FAQ ID -700

These are DNA primers of random sequence, six nucleotides long, with two thiophosphate linkages at the 3' terminus to prevent digestion of the oligos by the 3' to 5' exonuclease activity of Phi29 Polymerase.

FAQ ID -710

The reaction works at 30^oC and will not work efficiently at higher temperatures. This is because the Phi29 DNA polymerase is not a thermostable enzyme and the random hexamer primers bind less efficiently as temperature is increased.

FAQ ID -656

Since REPLI-g amplification products contain single-stranded DNA as well as residual primers, it is important to utilize a DNA quantification method that is specific for double-stranded DNA. PicoGreen is a fluorescence-based nucleic acid quantitation method that is specific for double-stranded DNA and may be used to quantify the double-stranded REPLI-g products. For best results, the sample should be diluted with 2 volumes of water and thoroughly mixed prior to addition of PicoGreen.
before taking an OD reading on a spectrophotometer the reaction product must be purified as it contains unused primers and dNTPs

FAQ ID -694

A DNA decontamination process is performed on the REPLI-g sc polymerase and REPLI-g buffers.

Paez et al. 2004 have shown in direct sequencing experiments sampling 500 000 bp, that the estimated error rate (9.5 x 10^-6) was the same in WGA generated samples as in paired unamplified samples.

FAQ ID -701

The REPLI-g kit is designed for whole genome amplification using random hexamers. Addition of specific primers instead of random hexamers may introduce amplification bias, or the preferential amplification of specific DNA fragments at the expense of others. Currently, specific primers alone cannot be used to amplify a specific region of the genome with REPLI-g.

FAQ ID -712

After the REPLI-g reactions are completed, 1 ug of the WGA reaction product can be analyzed by electrophoresis through a 1.0% agarose gel in TBE buffer (90 mM Tris-borate, pH 8.0, 2 mM EDTA), stained with GelStar, ethidium bromide, or SYBR Green, and imaged with a UV-box or a Phosphor-Imager. The majority of product should be above 10 Kb in length, and generate a trail of smear by electrophoresis down to about 2 Kb.

FAQ ID -695

Phi29 DNA polymerase is a high fidelity proofreading enzyme and assures a very low replication error rate. It has an error rate of 1 x 10^-6 - 10^-7 nucleotides both in its intrinsic enzymatic activity and during the amplification reaction.

In contrast, Taq DNA polymerase has an intrinsic error rate of approximately 2 x 10^-5 nucleotides, with an accumulation of about one mutation per 900 bases after 20 PCR cycles.

FAQ ID -707

We have been conducting an ongoing stability study for more than a year without observing breakdown of the amplified product. There is nothing in the amplification product that indicates that it would not be stable for a number of years.

FAQ ID -693

The enzyme used in the REPLI-g reaction is Phi29 DNA Polymerase.

FAQ ID -704

DOP (Degenerate Oligonucleotide-primed PCR) and PEP (Primer Extension Preamplification) are PCR-based whole genome amplification (WGA) methods. REPLI-g amplification uses MDA (Multiple Displacement Amplification) which is not a PCR-based method. MDA is scalable with yields adjustable from ug to mg quantities, whereas DOP typically yields 2-3 ug of DNA per reaction. DOP also generates a shorter product which is not suitable for certain downstream applications (e.g. Southern blot and sub-cloning).

DOP and PEP products are different from REPLI-g MDA products for a number of reasons. First, after amplification is complete, PEP products have active thermostable polymerase that will degrade the amplification product over time, because the polymerase cannot be inactivated. Second, PEP reactions consist of PCR amplicons which have the potential to contaminate other reactions as 'runaway amplicons' (e.g., amplicons in the aerosol that may be co-amplified if they accidentally get into other reactions).

REPLI-g product has neither of these issues. The polymerase is heat-inactivated after the REPLI-g reaction is complete, so it cannot digest the amplified product. There are no issues with 'runaway amplicons', because the reaction is performed at constant temperature by a hyper-branching amplification mechanism, amplifying the genome randomly without generating specific amplicons.

FAQ ID -665