REPLI-g Mini Kit

For highly uniform whole genome amplification from small or precious samples
  • Easy amplification with consistent yields of up to 10 µg
  • Unbiased amplification of genomic loci
  • Reliable results due to Multiple Displacement Amplification (MDA)
  • Amplified DNA highly suitable for most downstream applications
  • No risk of DNA degradation during long-term storage

The REPLI-g Mini Kit provides optimized reagents for whole genome amplification (WGA) from small samples using innovative Multiple Displacement Amplification (MDA) technology. The typical DNA yield of a 50 μl reaction is  up to 10 μg, with an average product length greater than 10 kb (ranging between 2 kb and 100 kb). Unique REPLI-g technology delivers highly uniform WGA from a variety of small or precious sample types, including purified genomic DNA, or directly from fresh or dried blood, buccal swabs, fresh or frozen tissue, and cells. This simple and reliable method is capable of accurate and unbiased amplification of genomes and generates DNA that can be applied without further purification or quantification for downstream applications that do not require labeling. In contrast to PCR-based WGA technologies, high fidelity rates are increased up to 1000-fold, avoiding costly false positive or negative results.

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产品 货号 目录价:
REPLI g Human Control Kit 25
Human control DNA for 25 x 50 µl whole genome amplification reactions
150090
询价
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
询价
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
询价

REPLI-g Mini Kit适用于分子生物学应用。该产品不适用于疾病的诊断、预防或治疗。


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任何类型样本都能获得高产量DNA。|高效的热变性和碱变性,位点序列信息不丢失。|长期保存具有稳定性。|基因组DNA和REPLI-g扩增获得的DNA,在二代测序中表现相当。|REPLI-g Mini and Midi实验流程。|准确的基因分型。|Schematic representation of REPLI-g amplification.|使用Phi29聚合酶进行无偏差的扩增。|REPLI-g技术的位点覆盖率出色。|稳定、准确的全基因组扩增。|多种样本量可获得稳定的DNA产量。|可靠的SNP基因分型。|
使用REPLI-g Midi and Mini Kits扩增各种起始材料,包括基因组DNA、肝素和EDTA保存的全血。常规产量为40 µg (Midi Kit) 和8–10 µg (Mini Kit)。|通过加热(95°C)或标准的REPLI-g Kit碱裂解法使基因组DNA样本(10 ng)变性。使用REPLI-g DNA Polymerase扩增后,比较样本2个位点的CT值。REPLI-g Kit碱裂解法扩增获得的位点CT值较低,表明这些位点的序列信息并未丢失。|对REPLI-g扩增的DNA样本进行real-time PCR,这些样本以4种不同形式在–20°C下保存了图示时间。检测每个样本的两个位点,[A]位点A和[B]位点B。gDNA:未用REPLI-g扩增的基因组DNA。保存形式:50 µl REPLI-g反应液:1) 未进一步处理("50 µl REPLI-g");2) 分装为5 µl体积("5 µl REPLI-g");3) 使用QIAamp Mini Kit纯化("50 µl QIAamp purified REPLI-g");4) 稀释至浓度为50 ng/µl ("50 µl diluted REPLI-g")。|对枯草芽孢杆菌的基因组进行全基因组测序。为进行分析,将2 μg基因组DNA、或REPLI-g Midi Kit从105个细胞扩增的DNA,剪切成300 bp大小的片段。每个取1 μg用于文库制备。在Illumina MiSeq仪器上进行测序。[A]基因组DNA和REPLI-g扩增的DNA得到的序列覆盖率相当。[B]基因组序列位点的比对表明,REPLI-g扩增的DNA的位点与基因组DNA相似度高,说明WGA(全基因组扩增)后DNA丢失水平较低。未扩增与REPLI-g扩增的DNA相比,错误率(错配、高级错误、缺失或嵌合体)在相同的比率范围。(使用SMALT [Welcome Trust Sanger Institute]进行比对。)|使用REPLI-g Mini Kit扩增基因组DNA包括3个基本步骤。首先,样品(10 ng纯化的基因组DNA,0.5 µl全血或组织培养细胞)需经过温和的碱变性,避免片段化和对模板DNA的损害。然后,样本被中和,最后再和REPLI-g预混液在30°C下孵育。|利用REPLI-g技术扩增20个DNA样本,后续不进行DNA纯化,直接对3个STR位点(CSF1PO、TPOX、和THOI)进行基因分型分析。将结果与为扩增的基因组DNA的基因型进行比对。通过聚丙烯酰胺凝胶电泳分离DNA,并用银染色。只有一条带的泳道代表纯合子,两条带的泳道表示该STR位点为杂合子。|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]当遇到DNA二级结构时,Taq聚合酶可能停止合成、跳过或从模板上解离。这可能导致不准确的DNA扩增、不完整的位点覆盖和DNA片段短。[B]REPLI-g Kits使用Phi29聚合酶,可替换二级结构,对整个基因组进行准确和高度统一的扩增。|使用定量real-time PCR对DNA样本的8个位点进行检测,DNA样本分别使用[A]REPLI-g技术[B]DOP-PCR和[C]PEP技术扩增而来。位点是否存在是与1 µg未扩增的对照DNA比较而确定的。(Dean, F.B. et al. (2002). Proc. Natl. Acad. Sci. U.S.A. 99, 5261. © 2002 National Academy of Sciences, USA.)|对使用REPLI-g技术扩增而来的44个不同样本的47个人类位点(每个常染色体对2个位点,X染色体两个位点,Y染色体1个位点)进行real-time PCR。每个样本被扩增约10,000倍,而覆盖位点的最大偏差只有6倍。|不同量的人类基因组DNA在标准REPLI-g Midi Kit反应中扩增,在图示时间点取样。无论起始材料量的大小,从50 µl反应中扩增的DNA产量约为40 µg。|使用REPLI-g技术扩增的DNA不经纯化,使用TaqMan分析对2个随机选取的位点(WIAF-1004和WIAF-622)进行SNP基因分型。等位基因的紧密集群能可靠确定纯合子和杂合子的基因型。|
性能
High yields from a variety of samples, suitable for numerous applications

With the REPLI-g Mini Kit, various clinical and non-clinical research samples can be used, including genomic DNA, fresh or dried blood, fresh or frozen tissue, and cells. Typical DNA yields per 50 µl reaction consistently reach 10 µg (see figure "Consistent DNA yields using any sample type"), while a uniform yield of amplified DNA is usually achieved regardless of the quantity of template DNA (see figure "Uniform DNA yield from various amounts of template"). Obtaining uniform DNA yields from varying template concentrations is always important, but particularly essential for high-throughput applications, which require subsequent genetic analyses to be possible without additional measurement or adjustment of DNA concentration.

The average product length of REPLI-g amplified DNA is typically more than 10 kb, with a range between 2 kb and 100 kb, enabling downstream applications such as complex restriction enzyme analysis and long-range PCR to be carried out. REPLI-g amplified DNA is highly suited for genotyping applications, such as SNP genotyping with TaqMan® primer/probe sets (see figure "Reliable SNP genotyping "), sequencing, and STR/microsatellite analysis (see figure "Accurate genotyping").

Successfully used in next-generation sequencing

Numerous publications have demonstrated the successful utilization of REPLI-g amplified DNA for next-generation sequencing (NGS) applications that range from exome and whole genome sequencing of tumor cells, to metagenomics research, to single cell analysis (for a range of recent publications that successfully used REPLI-g in NGS, please see our WGA resource page). Since the use of whole genome amplified DNA for NGS and array applications has been debated, we detected potential factors that could influence the success of using amplified DNA for these downstream applications. We determined that the quality of input material strongly influences the success of downstream NGS experiments. If working with low quality DNA (e.g., degraded DNA) or aged tissue material, the resulting amplified DNA may not give reliable results (data not shown). However, WGA, using REPLI-g technology, on intact cells or non-degraded purified DNA shows that NGS results are comparable to those obtained with purified gDNA. Sequence coverage and alignment comparison of the genomic loci sequence indicates minimized levels of junk DNA after WGA, whereas error rates are in a similar percentage range for both amplified and genomic DNA(see figure “Comparable NGS (next-generation sequencing) results obtained using purified gDNA or REPLI-g amplified DNA”).

High fidelity whole genome amplification

REPLI-g technology provides highly uniform DNA amplification across the entire genome. Phi29 polymerase can replicate up to 70 kb without dissociating from the genomic DNA template (see figure "Schematic representation of REPLI-g amplification"). In contrast to PCR-based whole genome amplification (WGA) technologies, Phi29 polymerase has 3'→5' exonuclease proofreading activity and maintains up to 1000-fold higher fidelity compared to Taq DNA polymerase during replication. Exonuclease-resistant primers provided in the kit ensure high yields of DNA product, and the WGA buffer system is optimized for very long read length and unbiased locus representation.

REPLI-g outperforms PCR-based WGA methods
 

Traditional methods of genomic DNA amplification include the time-consuming process of creating EBV-transformed cell lines followed by whole genome amplification using random or degenerate oligonucleotide-primed PCR. Also, PCR-based methods (e.g., DOP-PCR and PEP), as generally used by other suppliers, can produce nonspecific amplification artifacts and give incomplete coverage of loci. In several cases, DNA less than 1 kb long may be generated that cannot be used in many downstream applications. In general, the resulting DNA is generated with a much higher mutation rate due to the use of the low-fidelity enzyme Taq DNA polymerase, which can lead to error-prone amplification that results in, for example, single base-pair mutations, STR contractions, and expansions. In contrast to these disadvantages, REPLI-g provides highly uniform amplification across the entire genome, with minimal locus bias and minimized mutation rates during amplification (see figures "Highly representative amplification using REPLI-g technology" and "Consistent and accurate whole genome amplification").

原理

Unique REPLI-g technology uses the innovative, high-fidelity enzyme Phi 29 polymerase to amplify complex genomic DNA using Multiple Displacement Amplification (MDA) combined with a gentle alkaline denaturation step to amplify genomic loci uniformly. The typical yield of the REPLI-g Mini Kit is up to 10 µg, and can be easily scaled down according to your needs with the REPLI-g Midi Kit, since both kits are based on the same protocol and use the same reaction volumes. The easy reaction set-up and very low handling time of approximately 15 minutes makes REPLI-g an easy and reliable method to use when complete and unbiased locus representation is needed from limited or precious samples.

Amplification principle

REPLI-g uses isothermal genome amplification, termed Multiple Displacement Amplification (MDA), which involves the binding of random hexamers to denatured DNA followed by strand displacement synthesis at a constant temperature with the enzyme Phi29 polymerase. Additional priming events occur on each displaced strand that serve as a template, enabling generation of high yields of amplified DNA (see figure “Schematic representation of REPLI-g amplification”). Phi 29 polymerase, a phage derived enzyme, is a DNA polymerase with 3'→5' prime exonuclease activity (proofreading activity) that delivers up to 1000-fold higher fidelity compared to Taq DNA polymerase. Supported by the unique, optimized REPLI-g buffer system, Phi 29 polymerase easily solves secondary structures such as hairpin loops, thereby preventing slipping, stoppage, and dissociation of the polymerase during amplification. This enables the generation of DNA fragments up to 100 kb without sequence bias (see figure "Unbiased amplification with Phi 29 polymerase").

Alkaline denaturation of DNA

Genomic DNA must be denatured before use in enzymatic amplification procedures, which is often accomplished using harsh methods such as incubation at elevated temperatures (heat incubation) or increased pH (chemical alkaline incubation). The REPLI-g Midi Kit uses gentle alkaline incubation, allowing uniform DNA denaturation with very low DNA fragmentation or generation of abasic sites. This results in amplified DNA with very high integrity, and maximizes the length of amplified fragments so that genomic loci and sequences are uniformly represented. With the REPLI-g Mini Kit, reliable results without false positive or negative data are ensured in subsequent downstream applications, unlike with other WGA technologies that use heat-induced denaturation that can damage template DNA, leading to biased and underrepresented loci (see figure "Effect of heat and alkaline denaturation on loci representation").

操作流程
Simple, one tube procedure

The REPLI-g Mini Kit uses a simple and reliable method to achieve accurate genome amplification from small quantities of isolated target genomic DNA, or directly from whole blood, dried blood cards, buffy coat, and tissue culture cells (see figure "REPLI-g Mini and Midi procedure"). The addition of lysis buffer, which both lysis the sample material and denatures the DNA, is followed by a short minute incubation (see figure "REPLI-g Mini and Midi procedure"). After neutralization, master mix (including REPLI-g Mini DNA Polymerase) is added and the isothermal amplification reaction proceeds overnight at 30°C. REPLI-g amplified DNA can be stored long-term at –20°C with no negative effects (see figure "Consistent long-term stability").

Select the REPLI-g Kit most suited to your specific requirements from our complete range of dedicated REPLI-g products (see table).

Specifications for the wide range of REPLI-g Kits
REPLI-g Single Cell REPLI-g Mini REPLI-g UltraFast Mini REPLI-g Midi REPLI-g Screening REPLI-g FFPE REPLI-g Mitochondrial DNA
Starting material Single cells, gDNA Purifed gDNA, blood, cells Purifed gDNA, blood, cells FFPE tissue, purified gDNA from FFPE tissue Purified gDNA
(Protocols for other starting materials available from www qiagen.com)
Input amount Single cells, 2–1000 cells, tissue, purified gDNA (1–10 ng) >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 diamter, 10–40 µm thick); >100 ng gDNA >1 ng purified gDNA
Yield (µg/reaction) 40 10 7–10 40 8 Standard yield: ≤10; High yield: ≤40 3–5
Reaction time 8–16 h 10–16 h 1.5 h 8–16 h 12–16 h Standard yield: 4 h; High yield: 10 h 8 h
Hands-on time 15 min 15 min 15 min 15 min 15 min 40 min 15 min
Format Tube Tube Tube Tube Plate Tube Tube
应用

REPLI-g amplified genomic can be used in a variety of downstream applications, including:

  • SNP genotyping with TaqMan® primer/probe sets
  • qPCR- and PCR-based mutation detection
  • Next-generation sequencing
  • STR/microsatellite analysis
  • Sanger sequencing
  • RFLP and Southern blot analysis
  • Array technologies, such as comparative genomic hybridization 
特点
参数
扩增 Whole genomic DNA
应用 Genotyping, hybridization, RFLP
变性步骤 Alkaline
最大上样量 >10 ng DNA, 0.1– 0.5 µl whole blood, >600 cells/µl
所需最小吸液量 0.5 µl
质量评估 No
反应时间 8–16 hours (overnight)
反应体积 50 µl
每次处理样本量;通量 Mid
DNA起始量 >10 ng purified genomic DNA
起始材料 Genomic DNA, blood, cells, tissue
技术 Multiple Displacement Amplification (MDA)
产量 10–40 µg

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产品介绍与指南
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试剂盒操作手册
1
For whole genome amplification from purified genomic DNA, blood, and cells
显示更多
图片
Consistent DNA Yields Using any Sample Type.
任何类型样本都能获得高产量DNA。
使用REPLI-g Midi and Mini Kits扩增各种起始材料,包括基因组DNA、肝素和EDTA保存的全血。常规产量为40 µg (Midi Kit) 和8–10 µg (Mini Kit)。
Effect of Heat and Alkaline Denaturation on Loci Representation.
高效的热变性和碱变性,位点序列信息不丢失。
通过加热(95°C)或标准的REPLI-g Kit碱裂解法使基因组DNA样本(10 ng)变性。使用REPLI-g DNA Polymerase扩增后,比较样本2个位点的CT值。REPLI-g Kit碱裂解法扩增获得的位点CT值较低,表明这些位点的序列信息并未丢失。
Consistent long-term stability
长期保存具有稳定性。
对REPLI-g扩增的DNA样本进行real-time PCR,这些样本以4种不同形式在–20°C下保存了图示时间。检测每个样本的两个位点,[A]位点A和[B]位点B。gDNA:未用REPLI-g扩增的基因组DNA。保存形式:50 µl REPLI-g反应液:1) 未进一步处理("50 µl REPLI-g");2) 分装为5 µl体积("5 µl REPLI-g");3) 使用QIAamp Mini Kit纯化("50 µl QIAamp purified REPLI-g");4) 稀释至浓度为50 ng/µl ("50 µl diluted REPLI-g")。
Comparable NGS results obtained using gDNA or REPLI-g amplified DNA
基因组DNA和REPLI-g扩增获得的DNA,在二代测序中表现相当。
对枯草芽孢杆菌的基因组进行全基因组测序。为进行分析,将2 μg基因组DNA、或REPLI-g Midi Kit从105个细胞扩增的DNA,剪切成300 bp大小的片段。每个取1 μg用于文库制备。在Illumina MiSeq仪器上进行测序。[A]基因组DNA和REPLI-g扩增的DNA得到的序列覆盖率相当。[B]基因组序列位点的比对表明,REPLI-g扩增的DNA的位点与基因组DNA相似度高,说明WGA(全基因组扩增)后DNA丢失水平较低。未扩增与REPLI-g扩增的DNA相比,错误率(错配、高级错误、缺失或嵌合体)在相同的比率范围。(使用SMALT [Welcome Trust Sanger Institute]进行比对。)
REPLI-g Mini and Midi procedure
REPLI-g Mini and Midi实验流程。
使用REPLI-g Mini Kit扩增基因组DNA包括3个基本步骤。首先,样品(10 ng纯化的基因组DNA,0.5 µl全血或组织培养细胞)需经过温和的碱变性,避免片段化和对模板DNA的损害。然后,样本被中和,最后再和REPLI-g预混液在30°C下孵育。
Accurate Genotyping.
准确的基因分型。
利用REPLI-g技术扩增20个DNA样本,后续不进行DNA纯化,直接对3个STR位点(CSF1PO、TPOX、和THOI)进行基因分型分析。将结果与为扩增的基因组DNA的基因型进行比对。通过聚丙烯酰胺凝胶电泳分离DNA,并用银染色。只有一条带的泳道代表纯合子,两条带的泳道表示该STR位点为杂合子。
Schematic representation of REPLI-g amplification
Schematic representation of REPLI-g amplification.

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.

Unbiased amplification with Phi29 polymerase
使用Phi29聚合酶进行无偏差的扩增。
[A]当遇到DNA二级结构时,Taq聚合酶可能停止合成、跳过或从模板上解离。这可能导致不准确的DNA扩增、不完整的位点覆盖和DNA片段短。[B]REPLI-g Kits使用Phi29聚合酶,可替换二级结构,对整个基因组进行准确和高度统一的扩增。
Highly representative amplification using REPLI-g technology
REPLI-g技术的位点覆盖率出色。
使用定量real-time PCR对DNA样本的8个位点进行检测,DNA样本分别使用[A]REPLI-g技术[B]DOP-PCR和[C]PEP技术扩增而来。位点是否存在是与1 µg未扩增的对照DNA比较而确定的。(Dean, F.B. et al. (2002). Proc. Natl. Acad. Sci. U.S.A. 99, 5261. © 2002 National Academy of Sciences, USA.)
Consistent and accurate whole genome amplification
稳定、准确的全基因组扩增。
对使用REPLI-g技术扩增而来的44个不同样本的47个人类位点(每个常染色体对2个位点,X染色体两个位点,Y染色体1个位点)进行real-time PCR。每个样本被扩增约10,000倍,而覆盖位点的最大偏差只有6倍。
Uniform DNA Yield from Various Amounts of Template.
多种样本量可获得稳定的DNA产量。
不同量的人类基因组DNA在标准REPLI-g Midi Kit反应中扩增,在图示时间点取样。无论起始材料量的大小,从50 µl反应中扩增的DNA产量约为40 µg。
Reliable SNP genotyping
可靠的SNP基因分型。
使用REPLI-g技术扩增的DNA不经纯化,使用TaqMan分析对2个随机选取的位点(WIAF-1004和WIAF-622)进行SNP基因分型。等位基因的紧密集群能可靠确定纯合子和杂合子的基因型。