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REPLI-g FFPE Kit

直接对福尔马林固定、石蜡包埋组织(FFPE)中的DNA进行全基因组扩增

  • 直接从石蜡切片中进行全基因组扩增
  • 对FFPE组织中的DNA进行全基因组扩增
  • 可扩展的标准化DNA产量:每个组织切片可达40 µg
  • 简单快速的操作,快速获取结果

如果基因组DNA量不足以进行基因组分析,这一问题可通过对样本内全基因组DNA扩增解决。REPLI-g FFPE Kit包含DNA聚合酶、新型缓冲液和试剂,确保对福尔马林固定、石蜡包埋(FFPE)组织进行高效的全基因组扩增,无需预先纯化DNA。组织切片裂解后,对DNA进行处理后进行DNA片段连接,然后使用REPLI-g技术扩增长链DNA。

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Cat No./ID: 150243
REPLI-g FFPE Kit (25)
DNA Polymerase, Buffers, and Reagents for 25 x 50 μl whole genome amplification reactions
Cat No./ID: 150245
REPLI-g FFPE Kit (100)
DNA Polymerase, Buffers, and Reagents for 100 x 50 μl whole genome amplification reactions

REPLI-g FFPE 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。


Product Details

9
Reliable microsatellite analysis.

DNA from a 3.5-year-old FFPE carcinoma tissue sample was analyzed at a single microsatellite locus prior to and after DNA amplification using the REPLI-g FFPE Kit. Identical results were obtained for the original and REPLI-g amplified genomic DNA. (Data kindly provided by Hartmut Schmidt, Gerhard Domagk Institute for Pathology, Münster, Germany.)

9
High-molecular-weight ligated DNA.

DNA from different FFPE carcinoma tissues after (1a–5a) and before (1b–5b) amplification using the REPLI-g FFPE Kit was run on an agarose gel. The REPLI-g FFPE ligation procedure results in the formation of high-molecular-weight DNA. (Data kindly provided by Hartmut Schmidt, Gerhard Domagk Institute for Pathology, Münster, Germany.)

9
REPLI-g FFPE principle.

The REPLI-g FFPE procedure involves a novel DNA processing reaction that prepares and ligates fragmented DNA. This ligated DNA is then used in MDA-based whole genome amplification.

9
REPLI-g FFPE procedure.
Formalin fixation and paraffin embedding cause DNA fragmentation. During the REPLI-g FFPE procedure, these DNA fragments are randomly ligated and then amplified using multiple displacement amplification. As indicated, DNA fragments (containing different loci [L1–L4]) are not assembled in the original order. However, the different loci are equally amplified and can be detected in downstream genotyping applications.
Performance

提供两种实验方案,扩增时间不同。标准2小时扩增反应方案,每50 µl反应体系的常规产量为10 µg,高产量反应(8小时扩增)可得到40 µg产物。

Principle

用于基因分型研究的基因组DNA量通常不足。全基因组扩增(WGA)通过在样本内扩增全基因组,克服了这个难题,可扩增获得足量的来源于同一样本的DNA用于后续分析。

对福尔马林固定、石蜡包埋的组织样本(FFPE)进行基因分型,可将组织的形态变化直接与特定基因组变异联系起来。但是,福尔马林固定会导致DNA片段化,并与样本内的其他生物分子发生交联。此外,从FFPE样本中纯化得到的DNA量较少,只能用于少数几个分析。

REPLI-g FFPE Kit利用新型的DNA处理技术,将DNA片段连接起来(参见"REPLI-g FFPE principle")。然后通过经验证的REPLI-g技术对随机连接起来的DNA进行全基因组扩增。REPLI-g产品应与MDA以及独特的DNA聚合酶结合使用。REPLI-g DNA Polymerase凭借其活跃的进行性酶反应和单链置换反应,可很大程度上减少序列偏差和不均一位点的出现。该方法得到的结果比基于PCR的WGA方法更加可靠。新型的DNA处理技术可将MDA技术充分利用在FFPE组织样本中高度降解的DNA上。REPLI-g FFPE Kit在位点不变化的同时,实现对珍贵样本材料中DNA的扩增,产物可用于多种下游应用。

Procedure

REPLI-g FFPE可使用FFPE组织样本直接进行DNA扩增,无需预先纯化DNA。裂解组织切片后,使用新型缓冲液和酶处理DNA,将片段化的DNA连接起来(参见"REPLI-g FFPE procedure"和"High-molecular-weight ligated DNA")。反应后生成DNA长链,长链经REPLI-g技术扩增。扩增得到的DNA可即用于多数基因分型下游应用,无需进行纯化。

使用REPLI-g FFPE Kit扩增得到的DNA适用于real-time PCR(如使用QuantiFast Kits)和终点式PCR(如使用QIAGEN Fast Cycling PCR Kit)。PCR扩增子应比起始模板的平均片段长度短。还适用于微卫星分析(参见"Reliable microsatellite analysis")和SNP基因分型(REPLI-g FFPE扩增得到的DNA可能不适用于需要限制性内切进行DNA标记的基因分型应用)。

Applications

REPLI-g FFPE扩增的DNA适合以下应用:

  • Real-time PCR
  • 终点式PCR
  • SNP基因分型
  • 微卫星分析 

Specifications

Features
Specifications
扩增 Whole genomic DNA
应用 Genotyping, PCR, Real-time PCR
最大上样量 1 tissue section (10-40 µm thickness)
质量评估 No
反应时间 2-8h (2h: standard; 8h: high-yield reaction)
反应体积 50 µl
每次处理样本量;通量 medium
DNA起始量 100-300 ng*
起始材料 FFPE tissue sections or purified genomic DNA
技术 MDA
产量 10 µg (2h reaction)-40 µg (8h reaction)

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试剂盒操作手册 (1)
For direct whole genome amplification of DNA from formalin-fixed, paraffin-embedded (FFPE) tissue
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参考文献
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