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S7805 CpG WIZ® VHL - Methylation specific PCR assay

The CpG WIZ VHL Amplification Kit contains the reagents required for PCR amplification reactions.

S7805
25 assays  
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      Overview

      Replacement Information
      Description
      Catalogue NumberS7805
      Brand Family Chemicon®
      Trade Name
      • CpG Wiz
      • Chemicon
      DescriptionCpG WIZ® VHL - Methylation specific PCR assay
      Materials Required but Not DeliveredEquipment and Supplies

      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. Loading dye

      h. Bisulfite Modified DNA (CpGenome™ DNA Modification Kit, S7820)
      Background InformationMethylation 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, 4). The von Hippel-Lindau (VHL) gene exhibits characteristic hypermethylation.

      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 (5). 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 (S7820) contains the reagents necessary to perform the initial bisulfite reactions, while the CpG WIZ® VHL Amplification Kit contains the reagents required for the PCR amplification reactions.
      References
      Product Information
      Components
      • The components of the CpG WIZ® VHL Amplification Kit include those required for PCR amplification after bisulfite modification of DNA samples. Sufficient reagents are provided to analyze 25 samples with appropriate controls.
      • U Primer Set5 μM each primer (25X) 35 μL (neutral cap) 90516 -15°C to -25°C
      • M Primer Set5 μM each primer (25X) 35 μL (red cap) 90517 -15°C to -25°C
      • W Primer Set5 μM each primer (25X) 35 μL (green cap) 90518 -15°C to -25°C
      • U control DNA0.1 μg/μL 50 μL (white cap) 90393 -15°C to -25°C
      • M control DNA0.1 μg/μL 50 μL (red cap) 90394 -15°C to -25°C
      • W control DNA0.05 μg/μL 50 μL (green cap) 90395 -15°C to -25°C
      • Universal 10X PCR Buffer 265 μL (blue cap) 90396 -15°C to -25°C
      Quality LevelMQ100
      Applications
      ApplicationThe CpG WIZ VHL Amplification Kit contains the reagents required for PCR amplification reactions.
      Key Applications
      • PCR
      Application NotesINTRODUCTION

      Using This Manual

      Please read the entire instruction manual prior to using the CpG WIZ® VHL Amplification Kit. Should additional questions arise, assistance is available from Chemicon Technical Service at techserv@chemicon.com or (800) 437-7500.

      Principles of the Technique

      MSP, performed using the CpGenome™ DNA Modification Kit and the CpG WIZ® VHL 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 within sequences recognized by methylation sensitive restriction enzymes. Increasing the number of such sites which 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 which is CG-rich less difficult to amplify by PCR.



      Methylation-specific 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® VHL 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.
      Biological Information
      Species Reactivity
      • Human
      Entrez Gene Number
      Entrez Gene SummaryVon Hippel-Lindau syndrome (VHL) is a dominantly inherited familial cancer syndrome predisposing to a variety of malignant and benign tumors. A germline mutation of this gene is the basis of familial inheritance of VHL syndrome. The protein encoded by this gene is a component of the protein complex that includes elongin B, elongin C, and cullin-2, and possesses ubiquitin ligase E3 activity. This protein is involved in the ubiquitination and degradation of hypoxia-inducible-factor (HIF), which is a transcription factor that plays a central role in the regulation of gene expression by oxygen. RNA polymerase II subunit POLR2G/RPB7 is also reported to be a target of this protein. Alternatively spliced transcript variants encoding distinct isoforms have been observed.
      Gene Symbol
      • RASGRP1
      • V
      • CALDAG-GEFI
      • CALDAG-GEFII
      • MGC129999
      • RASGRP
      • pVHL
      • MGC129998
      • CalDAG-GEFII
      • PP2CB
      • hRasGRP1
      • PP2C-beta
      • EC 3.1.3.16
      UniProt Number
      UniProt SummaryFUNCTION: SwissProt: P40337 # Involved in the ubiquitination and subsequent proteasomal degradation via the von Hippel-Lindau ubiquitination complex. Seems to act as target recruitment subunit in the E3 ubiquitin ligase complex and recruits hydroxylated hypoxia- inducible factor (HIF) under normoxic conditions. Involved in transcriptional repression through interaction with HIF1A, HIF1AN and histone deacetylases.
      SIZE: 213 amino acids; 24153 Da
      SUBUNIT: Part of a complex with Elongin BC complex and hydroxylated HIF1A. Interacts with CUL2. Part of multisubunit CBC(VHL) E3 ubiquitin ligase complexes with Elongin BC complex, CUL2 or CUL5 and RBX1. Interacts with chaperone CCT/TRiC. The interaction with CCT is required for the interaction with the Elongin BC complex. Part of a complex with CCT and members of the Hsp70 chaperone family. Interacts with HIF1AN. Part of a complex with HIF1A, HIF1AN and HDAC1 or HDAC2 or HDAC3. The C-terminus interacts with VBP1. Interacts with RNF139 and UBP33. Interacts with PHF17.
      SUBCELLULAR LOCATION: Isoform 1: Cytoplasm. Membrane; Peripheral membrane protein. Nucleus. Note=Found predominantly in the cytoplasm and with less amounts nuclear or membrane-associated. & Isoform 3: Cytoplasm. Nucleus. Note=Equally distributed between the nucleus and the cytoplasm but not membrane-associated.
      TISSUE SPECIFICITY: Expressed in the adult and fetal brain and kidney.
      DOMAIN: SwissProt: P40337 The Elongin BC complex binding domain is also known as BC- box with the consensus [APST]-L-x(3)-C-x(3)-[AILV].
      DISEASE: SwissProt: P40337 # Defects in VHL are a cause of pheochromocytoma [MIM:171300]. The pheochromocytomas are catecholamine-producing, chromaffin tumors that arise in the adrenal medulla in 90% of cases. In the remaining 10% of cases, they develop in extra- adrenal sympathetic ganglia and may be referred to as paraganglioma. Pheochromocytoma usually presents with hypertension. Approximately 10% of pheochromocytoma is hereditary. The genetic basis for most cases of non-syndromic familial pheochromocytoma is unknown. & Defects in VHL are the cause of von Hippel-Lindau disease (VHLD) [MIM:193300]. VHLD is a dominantly inherited familial cancer syndrome characterized by the development of retinal angiomatosis, cerebellar and spinal hemangioblastoma, renal cell carcinoma (RCC), phaeochromocytoma and pancreatic tumors. VHL type 1 is without pheochromocytoma, type 2 is with pheochromocytoma. VHL type 2 is further subdivided into types 2A (pheochromocytoma, retinal angioma, and hemangioblastomas without renal cell carcinoma and pancreatic cyst) and 2B (pheochromocytoma, retinal angioma, and hemangioblastomas with renal cell carcinoma and pancreatic cyst). VHL type 2C refers to patients with isolated pheochromocytoma without hemangioblastoma or renal cell carcinoma. The estimated incidence is 3/100000 births per year and penetrance is 97% by age 60 years. & Defects in VHL are the cause of familial erythrocytosis type 2 (ECYT2) [MIM:263400]; also called VHL-dependent polycythemia or Chuvash type polycythemia (CP). ECYT2 is an autosomal recessive disorder characterized by increased red blood cell mass, increased serum levels of erythropoietin and normal oxygen affinity. Patients with ECYT2 carry a high risk for peripheral thrombosis and cerebrovascular events. & Defects in VHL are a cause of renal cell carcinoma type 1 (RCC1) [MIM:144700]; also called hypernephroma or adenocarcinoma of kidney. Familial renal cell carcinoma syndromes form a group of diseases characterized by a predisposition to development of renal cell carcinomas (RCCs) with various histological subtypes.
      SIMILARITY:
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      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.
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      Material Size25 assays
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      Supplemental Information
      Specifications
      Global Trade Item Number
      Catalogue Number GTIN
      S7805 04053252724817

      Documentation