对微量或者珍贵样本进行高度均一的全基因组扩增
  • 扩增获得10 µg DNA,产量稳定
  • 同时扩增多个基因组位点
  • 使用多重置换扩增反应获得可靠结果
  • 扩增获得的DNA适用于多种下游应用
  • 长期储存不会导致DNA降解

REPLI-g Mini Kit提供优化的试剂,利用多重置换扩增(MDA)技术,对微量或珍贵样本进行高度均一的全基因组扩增(WGA)。50 μl反应体积的常规DNA产量为10 μg,产物的平均长度长于10 kb(在2 kb至100 kb之间)。独特的REPLI-g技术能够从多种类型的微量或珍贵样本中高度均一的进行全基因组扩增,起始样本包括纯化后基因组DNA、新鲜血液或或干血、口腔拭子、新鲜或冷冻组织或细胞。该方法可便利、准确的扩增基因组DNA,得到的DNA可即用于无需标记的多种下游应用,无需进行纯化或定量检测。与基于PCR的全基因组扩增技术相比,这种方法的无错配率提高了1000倍,避免出现假阴性或假阳性结果。

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REPLI g Human Control Kit 25
Human control DNA for 25 x 50 µl whole genome amplification reactions
150090
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REPLI-g Mini Kit (25)
DNA Polymerase, Buffers, and Reagents for 25 x 50 µl whole genome amplification reactions (typical yield 10 µg per reaction)
150023
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REPLI-g Mini Kit (100)
DNA Polymerase, Buffers, and Reagents for 100 x 50 µl whole genome amplification reactions (typical yield 10 µg per reaction)
150025
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The REPLI-g Mini Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.
Consistent DNA yields using any sample type.|Effect of heat and alkaline denaturation on loci representation.|Consistent long-term stability.|Comparable NGS (next-generation sequencing) results obtained using purified gDNA or REPLI-g amplified DNA.|REPLI-g Mini and Midi procedure.|Accurate genotyping.|Schematic representation of REPLI-g amplification.|Unbiased amplification with Phi29 polymerase.|Highly representative amplification.|Consistent and accurate whole genome amplification.|Uniform DNA yield from various amounts of template.|Reliable SNP genotyping.|
Various starting materials, including genomic DNA, and heparin- and EDTA-preserved whole blood, were amplified using REPLI-g Midi and Mini Kits. Typical yields of 40 µg (Midi Kit) and 8–10 µg (Mini Kit) were obtained.|Genomic DNA samples (10 ng) were denatured using heat (95°C) or the standard REPLI-g Kit alkaline lysis protocol. After amplification using REPLI-g DNA Polymerase, the CT values of 2 loci were compared between samples. The low CT values of loci amplified using the REPLI-g Kit alkaline lysis protocol indicate better locus representation, meaning there has been no loss of sequence information at these loci.|Real-time PCR of REPLI-g amplified DNA samples stored in 4 different formats at –20°C for the indicated time periods. Two loci, [A] locus A and [B] locus B, were assayed for each sample. gDNA: genomic DNA not amplified with REPLI-g. Storage formats: 50 µl REPLI-g reactions: 1) without further manipulation ("50 µl REPLI-g"); 2) aliquoted to 5 µl volumes ("5 µl REPLI-g"); 3) purified with QIAamp Mini Kit ("50 µl QIAamp purified REPLI-g"); and 4) diluted to a concentration of 50 ng/µl (50 µl diluted REPLI-g").|Whole genome sequencing of the Bacillus subtilis genome was performed. For analysis, 2 μg of genomic DNA or DNA amplified from 105 cells using the REPLI-g Midi Kit was sheared into 300 bp fragments. For library preparation, 1 μg of each was used. Sequencing was performed on the Illumina MiSeq instrument. [A] Comparable sequence coverage was observed for both gDNA and REPLI-g amplified DNA. [B] Alignment comparison of the genomic loci sequence demonstrates comparably high percentage of alignment for REPLI-g amplified DNA in comparison to the gDNA, which is an indication of minimized levels of junk DNA after WGA (whole genome amplification). Comparison of nonamplified and REPLI-g amplified DNA revealed error rates (mismatch, high-quality error, indels, or chimeras) in a similar percentage range.
(Alignment comparison performed using SMALT [Welcome Trust Sanger Institute]).|Amplification of genomic DNA using the REPLI-g Mini Kit involves 3 basic steps. First, the sample (10 ng purified genomic DNA, 0.5 µl whole blood or tissue culture cells) undergoes gentle alkaline denaturation, avoiding fragmentation and damage of template DNA. Next the sample is neutralized, and finally incubated with REPLI-g master mix at 30°C.|Twenty DNA samples amplified using REPLI-g technology, without subsequent DNA purification, were subjected to genotyping analysis using 3 STR loci (CSF1PO, TPOX, and THOI). Results were compared with those obtained for unamplified genomic DNA. The DNA was separated by polyacrylamide gel electrophoresis, and visualized by silver staining. A lane with one band represents a homozygote, while a lane with two bands represents a heterozygote for the specific STR locus.|Ph 29 polymerase moves along the DNA template strand displacing the complementary strand. The displaced strand becomes a template for replication, enabling high yields of high-molecular-weight DNA to be generated.|[A] Upon encountering secondary DNA structures, Taq polymerase may pause synthesis, slip, or dissociate from the template. This can result in inaccurate DNA amplification, incomplete loci coverage, and short fragment sizes. [B] REPLI-g Kits utilize Phi29 polymerase, which displaces secondary structures enabling accurate and highly uniform amplification of the entire genome.|The relative representation of eight loci was determined using real-time quantitative PCR for DNA amplified using [A] REPLI-g technology, [B] DOP-PCR, and [C] PEP. Locus representation was determined by comparison to 1 µg of unamplified control DNA. (Dean, F.B. et al. (2002). Proc. Natl. Acad. Sci. U.S.A. 99, 5261. © 2002 National Academy of Sciences, USA.)|Real-time PCR was performed on 47 human loci (2 loci on each autosomal pair, 2 loci on the X chromosome[s], and 1 locus on the Y chromosome) from 44 different samples amplified using REPLI-g technology. Each sample was amplified approximately 10,000-fold, with a maximum bias of representation between the loci being only 6-fold.|Various amounts of human genomic DNA were amplified in a standard REPLI-g Midi Kit reaction and aliquots taken at the indicated timepoints. The yield of amplified DNA from a 50 µl reaction was approximately 40 µg, regardless of the amount of starting material.|DNA amplified using REPLI-g technology, without subsequent purification, was subjected to SNP genotyping at 2 randomly selected loci (WIAF-1004 and WIAF-622) using a TaqMan analysis. Tight clusters of alleles allow reliable determination of genotyping of homo- and heterozygote genotypes.|
Performance

从多种样本中得到高产量DNA,可用于多种应用

REPLI-g Mini Kit可用于多种样本,包括基因组DNA、新鲜或干血、新鲜或冰冻组织、细胞。50 μl反应体系的常规DNA产量可以达10 μg(参见"Consistent DNA yields using any sample type")。不同的模板质量均可扩增得到同样的DNA产量(参见"Uniform DNA yield from various amounts of template")。从不同的模板浓度获得一致的DNA产量十分要,对于高通量应用而言尤其如此,在下游的基因分析中不需要纯化或定量。

产物平均长度大于10 kb,在2 kb到100 kb之间,因此能够用于复杂的限制性酶切和长片段PCR等下游应用。REPLI-g扩增的DNA十分适用于基因分型,如使用TagMan引物/探针对的SNP基因分型(参见"Reliable SNP genotyping "),测序和STR/微卫星分析(参见"Accurate genotyping")。

成功应用于二代测序

许多文献已展示了REPLI-g技术扩增的DNA在二代测序中的成功应用,包括肿瘤细胞的外显子组测序、全基因组测序、宏基因组学的研究、以及单细胞分析。对于将全基因组扩增DNA用于二代测序一直有一些争论,因此我们检测了可能影响全基因组扩增DNA在下游应用中表现的影响因素。我们发现:起始材料的质量对二代测序实验有很大影响。如果使用的是低质量DNA(如降解的DNA),或长时间保存的组织材料,用扩增获得的DNA进行二代测序则无法获得可靠结果。但是,对完整细胞或未降解的纯化后DNA使用REPLI-g技术进行全基因组扩增,然后用于二代测序,结果可与纯化的基因组DNA的测序结果相媲美。分析测序结果的各基因组位点和错配率得出结论,全基因组扩增后垃圾DNA水平低,扩增获得的DNA和纯化获得的DNA的错配率基本相同(参见"Comparable NGS (next-generation sequencing) results obtained using purified gDNA or REPLI-g amplified DNA")。

高度可靠的全基因组扩增

REPLI-g技术提供高度均一的全基因组扩增。Phi29聚合酶可复制长达70 kb的DNA,而不会从DNA模板上脱离(参见"Schematic representation of REPLI-g amplification")。与基于PCR的全基因组扩增技术不同,Phi29聚合酶具有3'→5'外切酶校对活性,在复制过程中的高保真性是Taq DNA聚合酶的1000倍。试剂盒中提供的抗外切酶的引物可确保DNA的高产量,优化的全基因组扩增缓冲液体系适用于长片段DNA,对各位点进行均一的扩增。

REPLI-g技术的性能优于基于PCR的全基因组扩增技术

传统方法扩增基因组DNA需要进行耗时的EBV转化细胞系步骤,并用随机或变性的寡聚核苷酸引物进行全基因组扩增。基于PCR的方法(如:DOP-PCR和PEP)会产生非特异性的扩增产物,不能完全覆盖所有位点。有时,产生小于1 kb的DNA,在许多下游应用中都无法使用。由于使用低保真酶Taq DNA聚合酶,产物DNA突变率较高,扩增易出错,会导致碱基对突变。REPLI-g技术没有这些缺陷,并能够高度均一的扩增全基因组,位点偏差小、错配率小(参见"Highly representative amplification using REPLI-g technology" and "Consistent and accurate whole genome amplification")。

Principle

独特的REPLI-g技术使用创新的高保真酶Phi29聚合酶,利用多重置换扩增(MDA)技术扩增复杂的基因组DNA,并结合温和的碱变性,均一的扩增基因组位点。REPLI-g Mini Kit的常规产量为10 µg,如需更多产量,可使用REPLI-g Midi Kit;因为这两个试剂盒的实验方案相同,反应体积也相同。反应体系构建十分简便,仅需约15分钟的手动操作时间,因此REPLI-g技术使用简便、可靠,适用于对少量或珍贵的样本进行完全、均一的扩增。

扩增原理

REPLI-g利用等温基因组扩增,即多重置换扩增(MDA)技术:随机引物与变性DNA结合,在等温环境中,在Phi29聚合酶的作用下,进行置换合成反应。每个置换的单链作为模板,与引物结合,扩增生成高产量的DNA(参见"Schematic representation of REPLI-g amplification")。Phi 29聚合酶为噬菌体衍生酶,具有3'→5'外切酶活性,准确性为Taq DNA聚合酶的1000倍。Phi29聚合酶与独特的REPLI-g缓冲液体系相配合,能够轻松的打开DNA的二级结构,如发卡结构,所以可确保在扩增过程中,聚合酶正常发挥作用。因此该技术生成的DNA片段长度可达100 kb,没有序列偏差(参见"Unbiased amplification with Phi29 polymerase")。

DNA的碱变性

在酶扩增前,必须使基因组DNA变性,这通常是通过高温孵育、或在强碱性条件下进行的。REPLI-g Mini Kit采用温和的碱性环境孵育,使DNA变性,而片段化程度低,突变少。由此扩增获得的DNA十分完整,扩增的片段长,覆盖所有基因组位点和序列。使用REPLI-g Mini Kit能够获得可靠的结果,确保在下游应用中不出现假阳性或假阴性结果。采用热变性的WGA技术会损伤模板DNA,导致扩增结果不准确(参见"Effect of heat and alkaline denaturation on loci representation")。

Procedure

便利的单管操作

REPLI-g Mini Kit通过简便、高效的方法从纯化的少量基因组DNA样本或直接从全血、干血卡、白膜层、组织、培养的细胞中进行准确的全基因组扩增(参见"REPLI-g Mini and Midi procedure")。加入裂解缓冲液使样本DNA裂解并变性,之后进行短暂孵育(参见"REPLI-g Mini and Midi procedure")。中和后加入预混液(包括REPLI-g Mini DNA Polymerase),在30°C过夜进行等温扩增。REPLI-g扩增获得的DNA刻在–20°C长期储存(参见"Consistent long-term stability")。

选择适合您实验需求的REPLI-g Kit(参见下表)。

表1. 种类丰富的REPLI-g Kits

REPLI-g Mini REPLI-g UltraFast Mini REPLI-g Midi REPLI-g Screening REPLI-g FFPE REPLI-g Mitochondrial DNA
起始样本 纯化后gDNA、血液、细胞 纯化后gDNA、血液、细胞 FFPE组织、FFPE组织中纯化得到的gDNA 纯化后gDNA
起始样本量 >10 ng gDNA, 0.5 µl blood or cells (>600 cells/µl) >10 ng gDNA, 0.5 µl blood or cells (>600 cells/µl) Section (1 cm diameter, 10-40 µm thick); >100 ng gDNA >1 ng纯化后gDNA
产量(µg/反应) 10 7–10 40 8 标准产量:≤10,高产量≤40 3–5
反应时间(小时) 10–16 1.5 8–16 12–16 标准产量:4,高产量:10 8
手动操作时间(分钟) 15 15 15 15 40 15
规格 反应管 反应管 反应管 孔板 反应管 反应管
Applications

REPLI-g扩增得到的DNA可用于多种下游应用,包括:

  • 使用TagMan引物/探针对的SNP基因分型
  • 基于定量PCR或PCR的突变检测
  • 二代测序
  • STR/微卫星分析
  • Sanger测序
  • RFLP和Southern印迹分析
  • 芯片技术,如比较基因组杂交
Feature
Specifications
Amplification Whole genomic DNA
Applications Genotyping, hybridization, RFLP
Denaturation step Alkaline
Maximum input volume >10 ng DNA, 0.1– 0.5 µl whole blood, >600 cells/µl
Minimal pipetting volume needed 0.5 µl
Quality assessment No
Reaction time 8–16 hours (overnight)
Reaction volume 50 µl
Samples per run; throughput Mid
Starting amount of DNA >10 ng purified genomic DNA
Starting material Genomic DNA, blood, cells, tissue
Technology Multiple Displacement Amplification (MDA)
Yield 10–40 µg

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For whole genome amplification from purified genomic DNA, blood, and cells
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