| 中文名称 | CpGenome 快速DNA修饰试剂盒 |
| 英文名称 | CpGenome Fast DNA Modification Kit |
| 货号 | S7824 |
| 包装 | 25 units |
| 价格 | 2,058.03元 |
| 产地 | Millipore |
| 说明 | Description: CpGenome Fast DNA Modification Kit
Trade Name:
Product Overview: Principles of the Technique
Bisulfite Modification
The CpGenome™ Fast DNA Modification Kit contains reagents required to perform a bisulfite modification on a DNA sample. As shown in Figure 1, in the bisulfite reaction, all unmethylated cytosines are deaminated and sulfonated, converting them to uracils, while 5-methylcytosines remain unaltered. Thus, the sequence of the treated DNA will differ depending on whether the DNA is originally methylated or unmethylated. Also, the initially complementary DNA strands will no longer be complementary after cytosine conversion. Primers for use in MSP can be designed to specifically amplify either a bisulfite-sensitive, unmethylated strand or a bisulfite-resistant, methylated strand, based upon these chemically-induced differences. MSP The PCR primers are designed to specifically amplify the promoter regions of the gene of interest. If the sample DNA was originally unmethylated, an MSP reaction product will be detectable when using the primer set (labeled as 'U') designed to be complementary to the unmethylated bisulfite converted DNA sequence. No product will be generated using a primer set (labeled as 'M') designed to be complementary to the derivative methylated DNA sequence. Conversely, an MSP product will be generated only using the M primer set if the sample was originally methylated, and the U primers will not amplify such a template. Methylation specific PCR permits sensitive detection of altered DNA. Because it is a PCR-based assay, it is extremely sensitive, facilitating the detection of low numbers of methylated alleles and the evaluation of DNA from small samples, including paraffin-embedded, formalin fixed materials. MSP also allows examination of all CpG sites, not just those within sequences recognized by methylation sensitive restriction enzymes. Increasing the number of such sites that can be assessed allows rapid, fine mapping of methylation patterns throughout CpG regions. In addition, the bisulfite modification is ideally suited for analysis of CpG islands since it converts the majority of unmethylated cytosines to uracils, making a region of the genome which is CG rich more easily amplified by PCR. Background Information: Methylation of cytosines located 5' to guanosine is known to have a profound effect on the expression of many eukaryotic genes (1). In normal cells methylation occurs predominantly in CG-poor regions, while CG-rich areas, called CpG-islands remain unmethylated. The exceptions are the extensive methylation of CpG islands associated with transcriptional inactivation of regulatory regions of imprinted genes (2, 3) and genes on the inactive X-chromosome of females (4, 5). Aberrant methylation of normally unmethylated CpG islands has been documented as a relatively frequent event in immortalized and transformed cells (6) and has been associated with transcriptional inactivation of defined tumor suppresser genes in human cancers (7, 8). Hundreds of CpG islands are now known to exhibit the characteristic of hypermethylation in tumors (9).
Several methods have been developed to determine the methylation status of cytosine. These include digestion with methylation sensitive restriction enzymes as in restriction landmark genomic scanning, oligonucleotide arrays, bisulfite genomic DNA sequencing and Methylation Specific PCR (MSP). Some techniques are more useful for discovery while others are better used for monitoring of known methylated cytosines. Genomic DNA sequencing, although time consuming and labor intensive, offers a more universal detection method (10, 11). MSP is now an established technology for the monitoring of abnormal gene methylation in selected gene sequences (12). Utilizing small amounts of DNA, this procedure offers sensitive and specific detection of 5-methylcytosine in promoters. It is being exploited to define tumor suppresser gene function, and to provide a new strategy for early tumor detection. The initial step of both bisulfite genomic sequencing and MSP is to perform a bisulfite modification of the DNA sample. MSP then involves PCR amplification with specific primers designed to distinguish methylated from unmethylated DNA. The CpGenome™ Fast DNA Modification Kit contains the reagents for the initial bisulfite modification of the DNA required for both methodologies. Application Notes: For MSP primer design, please use the MethPrime software package. Click here
Species Reactivity: All
Usage Statement: Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.
Kit or Assay Type:
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Packaging: 25 samples
Materials Required but Not Delivered: Equipment and Supplies a. Water bath incubator or heat block at 37°C and 50°C. b. Microcentrifuge (to 12,000X G) c. Screw-cap microcentrifuge tubes, 1.5-2.0 mL d. Narrow range pH indicator paper (pH 0-6, three different indicators per strip, and/or pH 4-7, one indicator per strip) or a dedicated pH electrode. Reagents a. NaOH pellets b. 100% EtOH c. Nuclease-free water |