| Description | |
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| Catalogue Number | S7830 |
| Brand Family | Chemicon® |
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| Description | CpG WIZ® BRCA1 -Methylation specific PCR assay |
| Overview | Principles of the Technique: Methylation-specific PCR (MSP), performed using the CpGenome™ DNA Modification Kit and the CpG WIZ® BRCA1 Amplification Kit, permits sensitive detection of altered DNA. Because this is a PCR-based assay, it is extremely sensitive, facilitating the detection of low numbers of methylated alleles and the study of samples containing small amounts of DNA. MSP also allows examination of all CpG sites, not just those with BRCA1 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 cytosines to uracils, making a region of the genome that is CG-rich less difficult to amplify by PCR. MSP employs an initial bisulfite reaction to modify the DNA, followed by a "hot start" PCR amplification with specific primers designed to distinguish methylated DNA from unmethylated DNA. As shown in Figure 1, in the bisulfite reaction, all unmethylated cytosines are converted to uracils while 5-methylcytosines remain unaltered. Thus, the sequence of the treated DNA will differ if the DNA is originally methylated vs. unmethylated. Primers contained in the CpG WIZ® BRCA1 Amplification Kit are designed to specifically amplify each of the sequences based upon these chemically-induced differences. If the sample DNA was originally unmethylated, a product will be generated after PCR using the U primer set. Conversely, a product will be generated using the M primer set if the sample was originally methylated. |
| Materials Required but Not Delivered | a. Microcentrifuge tubes for PCR amplification b. Aerosol-resistant pipette tips c. Thermocycler d. Gel electrophoresis apparatus (vertical or horizontal) e. Power Supply f. 302 nm UV transilluminator, camera and film Reagents a 2.5 mm dNTP mix (2.5 mm of each nucleotide) b. "Hot start" Taq polymerase c. "Hot start" PCR reagents (see Sec. II. Protocols). d. Reagents for gel electrophoresis (1X TBE and 2% agarose, 10% acrylamide, or suitable high resolution agarose) e. DNA markers (size range 100-300 bp) f. Ethidium bromide (10 mg/mL) g. Gel-loading solution / Loading Dye H. Bisulfite Modified DNA (CpGenome™ DNA Modification Kit, Cat. No. S7820) |
| Background Information | Methylation of cytosines located 5 to guanosine is known to have a profound effect on the expression of several eukaryotic genes (1). In normal cells, methylation occurs predominantly in CG-poor regions, while CG-rich areas, called CpG islands, remain unmethylated. Aberrant methylation of normally unmethylated CpG islands has been documented as a relatively frequent event in immortalized and transformed cells (2) and has been associated with transcriptional inactivation of defined tumor suppressor genes in human cancers (3). BRCA1 is a tumor suppressor gene in familial breast-ovarian carcinoma syndrome and in sporadic ovarian carcinomas. In normal breast epithelial cells, BRCA1 mRNA levels in tumors appear to be down-regulated by methylation, while BRCA2 shows significant over expression in sporadic breast cancers. Epigenetic hypermethylation of the BRCA1 gene plays an important role when it is expressed at reduced levels in breast and ovarian tumors as a result of hypermethylation of its promoter region (5,6,7). Previously developed methods to determine the methylation status of cytosine include digestion with methylation sensitive restriction enzymes and genomic DNA sequencing. Both techniques have limitations: restriction enzymes can only detect methylation sites within their recognition sequence and sequencing is time consuming. Increasing the detection sensitivity of CpG island methylation has the potential to define tumor suppressor gene function and provides a new strategy for early tumor detection. Methylation-specific PCR (MSP) is a new technology for sensitive detection of abnormal gene methylation utilizing small amounts of DNA (4). This process employs an initial bisulfite reaction to modify the DNA, followed by PCR amplification with specific primers designed to distinguish methylated from unmethylated DNA. The CpGenome™ DNA Modification Kit (Cat. No. S7820) contains the reagents necessary to perform the initial bisulfite reactions, while the CpG WIZ® BRCA1 Amplification Kit contains the reagents required for the PCR amplification reactions. |
| Product Information | |
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| Quality Level | MQ100 |
| Applications | |
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| Application | Methylation-specific PCR (MSP), performed using the CpGenome DNA Modification Kit & the CpG WIZ BRCA1 Amplification Kit, permits sensitive detection of altered DNA. |
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| Biological Information | |
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| Species Reactivity |
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| Entrez Gene Summary | This gene encodes a nuclear phosphoprotein that plays a role in maintaining genomic stability and acts as a tumor suppressor. The encoded protein combines with other tumor suppressors, DNA damage sensors, and signal transducers to form a large multi-subunit protein complex known as BASC for BRCA1-associated genome surveillance complex. This gene product associates with RNA polymerase II, and through the C-terminal domain, also interacts with histone deacetylase complex. This protein thus plays a role in transcription, DNA repair of double-stranded breaks, and recombination. Mutations in this gene are responsible for approximately 40% of inherited breast cancers and more than 80% of inherited breast and ovarian cancers. Alternative splicing plays a role in modulating the subcellular localization and physiological function of this gene. Many alternatively spliced transcript variants have been described for this gene but only some have had their full-length natures identified. |
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| UniProt Summary | FUNCTION: SwissProt: P38398 # Plays a central role in DNA repair by facilitating cellular response to DNA repair. Required for appropriate cell cycle arrests after ionizing irradiation in both the S-phase and the G2 phase of the cell cycle. Involved in transcriptional regulation of P21 in response to DNA damage. Required for FANCD2 targeting to sites of DNA damage. May function as a transcriptional regulator. Mediates E2-dependent ubiquitination. Inhibits lipid synthesis by binding to inactive phosphorylated ACACA and preventing its dephosphorylation. SIZE: 1863 amino acids; 207721 Da SUBUNIT: Part of the BRCA1-associated genome surveillance complex (BASC), which contains BRCA1, MSH2, MSH6, MLH1, ATM, BLM, PMS2 and the RAD50-MRE11-NBN protein complex. This association could be a dynamic process changing throughout the cell cycle and within subnuclear domains. CTIP interacts specifically with the BRCT domains. Interacts with RNA polymerase II holoenzyme. Interacts with SMC1A and COBRA1/NELFB. Binds BRIP1 through the BRCT domains. Interacts with ubiquitinated FANCD2. Interacts with BAP1. Interacts with DCLRE1C/Artemis and CLSPN. Interacts with histone H2AFX and this requires phosphorylation of H2AFX on Ser-139. Interacts with CHEK1/CHK1. Interacts with BRCC3. Interacts through its BRCT domains with phosphorylated ACACA and prevents its dephosphorylation. SUBCELLULAR LOCATION: Nucleus. DOMAIN: SwissProt: P38398 The RING-type zinc finger domain interacts with BAP1. PTM: Phosphorylated in response to IR, UV, and various stimuli that cause checkpoint activation, probably by ATM or ATR. DISEASE: "SwissProt: P38398 # Defects in BRCA1 are a cause of genetic susceptibility to breast cancer (BC) [MIM:113705, 114480]. BC is an extremely common malignancy, affecting one in eight women during their lifetime. A positive family history has been identified as major contributor to risk of development of the disease, and this link is striking for early-onset breast cancer. Mutations in BRCA1 are thought to be responsible for 45% of inherited breast cancer. Moreover, BRCA1 carriers have a 4-fold increased risk of colon cancer, whereas male carriers face a 3-fold increased risk of prostate cancer. Cells lacking BRCA1 show defects in DNA repair by homologous recombination. & Defects in BRCA1 are a cause of genetic susceptibility to breast-ovarian cancer (BOC) [MIM:113705]. Mutations in BRCA1 are thought to be responsible for more than 80% of inherited breast- ovarian cancer. & Defects in BRCA1 are a cause of genetic susceptibility to ovarian cancer [MIM:113705]." SIMILARITY: Contains 2 BRCT domains. & Contains 1 RING-type zinc finger. |
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| Material Size | 25 assays |