EpiTect Fast Bisulfite Conversion Kits

对来自基因组DNA、福尔马林固定石蜡包埋(FFPE)样本、细胞或组织样本的DNA进行超快速、高效的亚硫酸氢盐转化

  • 优化的操作流程对胞嘧啶转化率超过99%
  • 新型Bisulfite Solution可在少至30分钟内完成有效地亚硫酸氢盐转化
  • 可用于基因组DNA、全血、培养细胞、组织样本和福尔马林固定石蜡包埋(FFPE)切片
  • 快速、简洁的操作流程,有效转化和纯化DNA
  • 独特的DNA Protect技术可防止DNA降解

EpiTect Fast Bisulfite Conversion Kits可在少至30分钟内将未甲基化的胞嘧啶高效转化成尿嘧啶,起始材料选择也有出色的灵活性。简化的样本裂解操作流程和直接的DNA亚硫酸氢盐转化使转化过程快速、省力,样本包括全血、培养细胞、组织样本、基因组DNA和福尔马林固定石蜡包埋(FFPE)切片。创新的DNA Protect技术确保有效的DNA变性而不会降解,得到高产量DNA和可重复性结果。此外,转化DNA可在少至10 µl洗脱缓冲液中洗脱,得到高浓度DNA。

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EpiTect Fast Bisulfite Kit (10)
Trial kit for 10 preps: Deparaffinization Solution, Lysis Buffer, Proteinase K, Bisulfite Solution, DNA Protect Buffer, MinElute DNA Spin Columns, Carrier RNA, and Buffers
59802
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EpiTect Fast DNA Bisulfite Kit (50)
For 50 preps: Bisulfite Solution, DNA Protect Buffer, MinElute DNA Spin Columns, Carrier RNA, and Buffers
59824
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EpiTect Fast DNA Bisulfite Kit (200)
For 200 preps: Bisulfite Solution, DNA Protect Buffer, MinElute DNA Spin Columns, Carrier RNA, and Buffers
59826
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EpiTect Fast LyseAll Bisulfite Kit (50)
For 50 preps: Lysis Buffer, Proteinase K, Bisulfite Solution, DNA Protect Buffer, MinElute DNA Spin Columns, Carrier RNA, and Buffers
59864
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EpiTect Fast LyseAll Bisulfite Kit (200)
For 200 preps: Lysis Buffer, Proteinase K, Bisulfite Solution, DNA Protect Buffer, MinElute DNA Spin Columns, Carrier RNA, and Buffers
59866
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EpiTect Fast Bisulfite Conversion Kits are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease.


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Highly efficient cytosine conversion of X-chromosome DNA.|EpiTect Fast DNA Bisufite Kit procedure.|EpiTect Fast FFPE Bisulfite Kit procedure.|EpiTect Fast LyseAll Bisulfite Kit procedure.|Analysis of DNA fragment sizes after bisulfite conversion.|Superior bisulfite conversion.|DNA Protect Buffer.|EpiTect Fast Bisufite Conversion Kit.|Interconnected protocols of the EpiTect Fast Bisulfite Kits.|
K562 genomic DNA was bisulfite converted using the EpiTect Fast DNA Bisulfite Kit, and a 100 bp amplicon from the X-chromosome was amplified using cytosine-free primers to allow amplification of both unconverted and bisulfite converted DNA. PCR products were cloned into a vector and Sanger sequencing was done to identify converted cytosines. 1252 individual cytosines were analyzed, revealing a highly efficient cytosine conversion rate of 99.8%. This high conversion rate is especially significant as the genomic DNA was derived from a female; therefore, one of the 2 X-chromosomes is highly methylated.|Streamlined and straightforward, the EpiTect Fast DNA Bisulfite Kit enables complete bisulfite conversion and cleanup of genomic DNA in approximately 1 hour.|Upon completing deparaffinization, lysis, and decrosslinking steps, the bisulfite conversion is completed using the included EpiTect Fast DNA Bisulfite Kit.|Upon completion of the sample lysis steps, bisulfite conversion is completed using the included EpiTect Fast DNA Bisulfite Kit.|Following bisulfite conversion with the EpiTect Fast DNA Bisulfite Kit, 1 µg of genomic DNA showed the lowest level of fragmentation, with a size distribution ranging from several hundred base pairs to over 6 kb, compared to other methods with peaks at 500 bp and 1 kb, respectively. Samples were analyzed on an Agilent RNA 6000 Nano Chip.|Following bisulfite conversion of genomic DNA using the EpiTect Fast DNA Bisulfite Kit or a kit from Supplier Z, unconverted genomic DNA and bisulfite converted DNA were coamplified and then sequenced by Pyrosequencing to quantify the rate of conversion of individual cytosines. [A] The EpiTect Fast DNA Bisulfite Kit gave comparable results to a kit from Supplier Z when 2 µg of DNA was analyzed. [B] However, when a limited amount of DNA was analyzed (10 ng), the 4 cytosine residues shown in this Pyrogram demonstrate that the EpiTect Fast DNA Bisulfite Kit delivered significantly better results than the kit from Supplier Z. (Percentage of each cytosine residue that did not undergo bisulfite conversion using the kit from Supplier Z vs. the EpiTect Fast DNA Bisulfite Kit: 12% vs. 2%, 5% vs. 1%, 37% vs. 9% and 11% vs. 2%, respectively.)|Upon addition to the bisulfite conversion reaction, the DNA Protect Buffer should turn from green to blue, indicating sufficient mixing and correct pH.||Depending on sample type, the bisulfite conversion of DNA calls for the use of a specific kit and protocol. Kits contain buffers optimized for DNA extraction and all reagents for the DNA bisulfite conversion.|
Performance

高效亚硫酸氢盐转化 

在甲基化分析中,对DNA亚硫酸氢盐处理,将未甲基化的胞嘧啶转化成胞嘧啶是最关键的步骤,因为反应效率会对下游分析的可靠性造成很大影响。EpiTect Fast Bisulfite Conversion Kits含有的创新型Bisulfite Solution可进行超快速、高效的亚硫酸氢盐转化,甚至可处理难分析的样本,如来自女性样本的X染色体基因组DNA,因为2个X染色体中有一个是高度甲基化的(参见"Highly efficient cytosine conversion of X chromosome DNA")。焦磷酸测序对胞嘧啶转化定量分析表明,与其他供应商的试剂盒相比,EpiTect Fast Bisulfite Conversion Kit的转化效果明显更好,特别是当起始材料的量很有限时(参见"Superior bisulfite conversion")。

DNA片段化减少

完全亚硫酸氢盐转化所需的剧烈条件通常会造成高度DNA片段化和样本损失,导致DNA产量低、高度片段化DNA以及不可重复的转化率。QIAGEN独特的DNA Protect Buffer可防止DNA降解,与其他供应商的试剂盒转化的DNA相比,可获得更大片段大小的完全转化DNA(参见"Analysis of DNA fragment sizes after bisulfite conversion")。

Principle

DNA样本的快速、完全的亚硫酸氢盐转化对于准确的DNA甲基化分析是必需的。EpiTect Fast Bisulfite Conversion Kits含有的创新型Bisulfite Solution可在少至30分钟内完成高效的亚硫酸氢盐转化。简化的样本裂解和转化操作流程可对DNA进行有效纯化和亚硫酸氢盐转化,而独特的DNA Protect技术可最大程度减少亚硫酸氢盐处理时的DNA片段化。3种EpiTect Fast Bisulfite Conversion Kits相互联系,形成的完整系统可对一系列起始材料的DNA进行直接亚硫酸氢盐转化(参见"Interconnected protocols of the EpiTect Fast Bisulfite Conversion Kits")。但每种试剂盒特有其独特之处,重点处理特定样本类型,各自含有需要的所有缓冲液和试剂。每个试剂盒的组分可有效裂解样本,减少DNA损失,进行完全亚硫酸氢盐转化,并在转化及DNA回收过程中形成有效保护。

快速亚硫酸氢盐转化

EpiTect Fast Bisulfite Conversion Kits提供非常快速、简洁的操作流程,可有效转化并纯化来自基因组DNA、福尔马林固定石蜡包埋(FFPE)切片、血液、细胞或组织样本中的DNA。试剂盒含有高浓度的Bisulfite Solution,将未甲基化胞嘧啶转化为尿嘧啶所需的时间从几个小时减少到了30分钟,含有的制备缓冲液无需在亚硫酸氢盐处理前分离DNA。而且,亚硫酸氢盐热循环程序提供优化的孵育步骤,可使DNA热变性、磺基化以及胞嘧啶脱氨,使胞嘧啶转化率超过99%。胞嘧啶转化的最后一个步骤是脱磺基化,此过程可在纯化流程时简单柱上完成。

DNA Protect Buffer

样本DNA的快速、完全的亚硫酸氢盐转化对于正确确定甲基化模式是最关键的。这个过程需要将DNA在高浓度亚硫酸氢盐、高温、低pH条件下孵育。这些剧烈的条件经常造成高度DNA片段化和纯化时的DNA损失。普通亚硫酸氢盐转化需要大量的起始DNA,以弥补转化时的DNA降解和纯化时的DNA损失,这常常导致DNA产量低、片段化高和不可重复的转化率。使用EpiTect Fast Bisulfite Conversion Kits时,独特的DNA Protect Buffer可在亚硫酸氢盐转化时防止由于高温、低pH值造成的DNA片段化。此缓冲液还可使DNA有效变性,得到完全胞嘧啶转化所需的单链DNA。DNA Protect Buffer含有方便的pH指示剂,可确保胞嘧啶转化时具有正确的pH(参见"DNA Protect Buffer")。

福尔马林固定石蜡包埋(FFPE)样本的脱蜡

从福尔马林固定石蜡包埋(FFPE)样本中抽提DNA的第一步是脱蜡,石蜡先是被熔解再被去除,暴露出的样本进行后续的裂解液和蛋白酶K处理。EpiTect Fast FFPE Bisulfite Kit中含有的新型化学试剂Deparaffinization Solution可优化福尔马林固定石蜡包埋(FFPE)切片的去石蜡、裂解和解交联过程。Deparaffinization Solution可获得高DNA回收率,因为溶液在样本上时就可进行蛋白酶K消化。由于无需沉淀福尔马林固定石蜡包埋(FFPE)样本或去除含有石蜡的上清,可避免脱蜡时的样本损失风险。

Procedure

EpiTect Fast Bisulfite Conversion Kit操作流程包括几个简单的步骤:从样本中制备DNA,未甲基化胞嘧啶的亚硫酸氢盐转化;转化的单链DNA结合到MinElute DNA离心柱膜上;洗涤;膜上结合DNA的去磺基化;漂洗膜上结合DNA,去除去磺基化试剂;从离心柱上洗脱纯化的转化DNA。福尔马林固定石蜡包埋(FFPE)切片、全血、细胞培养基或组织样本的制备是不同的,但是提取DNA的亚硫酸氢盐转化流程对所有样本类型都是相同的(参见"Interconnected protocols of the EpiTect Fast DNA Conversion Kits")。所有实验方案可获得相同的胞嘧啶转化率,转化DNA的纯化也能获得同等DNA回收,与DNA起始量无关。

试剂盒描述

EpiTect Fast DNA Bisulfite Kit

EpiTect Fast DNA Bisulfite Kit包括精心配制的缓冲液和试剂,可对基因组DNA进行简洁、有效的亚硫酸氢盐转化和对转化DNA的回收(参见"EpiTect Fast DNA Bisulfite Kit procedure")。新型Bisulfite Solution可在少至30分钟内进行完全亚硫酸氢盐转化,运用DNA Protect技术和QIAGEN离心柱可获得很少降解的完全转化DNA,在10 µl洗脱液中洗脱,用于需要高浓度的下游应用。EpiTect Fast DNA Bisulfite Kit在20 µl体系中可转化1 ng–2 µg DNA,在最大40 µl体系中可转化1–500 ng DNA。

EpiTect Fast FFPE Bisulfite Kit

EpiTect Fast FFPE Bisulfite Kit可处理福尔马林固定石蜡包埋(FFPE)组织,EpiTect Fast FFPE Lysis Kit含有的专门缓冲液可有效去石蜡并裂解福尔马林固定石蜡包埋(FFPE)切片(参见"EpiTect Fast FFPE Bisulfite Kit procedure"),EpiTect Fast DNA Bisulfite Kit可对提取的DNA进行快速、有效的亚硫酸氢盐转化。实验方案包括的优化步骤便于DNA结合,可用于单个福尔马林固定石蜡包埋(FFPE)组织切片(厚度为10 µm)。从福尔马林固定石蜡包埋(FFPE)组织中提取DNA很难避免DNA片段化,后续亚硫酸氢盐处理时的剧烈条件更会加重片段化。QIAGEN提供可靠地DNA Protect技术可最大程度减少转化反应造成的片段化,获得更高产量的亚硫酸氢盐转化DNA。

EpiTect Fast LyseAll Bisulfite Kit

EpiTect Fast LyseAll Bisulfite Kit可处理全血、培养细胞或组织样本,EpiTect Fast LyseAll Lysis Kit(参见"EpiTect Fast 96 LyseAll Bisulfite Kit procedure")含有新型裂解缓冲液,EpiTect Fast DNA Bisulfite Kit可对提取DNA进行亚硫酸氢盐转化。实验方案包括的优化步骤便于DNA结合,独特配制的裂解缓冲液使DNA从样本中释放更为简洁。此试剂盒可用于0.5–20 µl血液或10–105个细胞(少至60 pg DNA)。

Applications
EpiTect Fast Bisulfite Conversion Kits可对各种起始材料的DNA进行快速、有效的亚硫酸氢盐转化,非常适合DNA甲基化分析的技术,包括:
  • PCR:终点、real-time和甲基化特异性PCR
  • 亚硫酸氢盐测序(直接和克隆)
  • COBRA (Combined Bisulfite Restriction Analysis)
  • 焦磷酸测序

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Kit Handbooks
1
For sample lysis and complete bisulfite conversion/cleanup of DNA from FFPE, blood, cultured cells, or tissue samples, optimized for methylation analysis
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