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S7811 CpG WIZ® hMLH1 - Methylation specific PCR assay

The components of the CpG WIZ hMLH1 Amplification Kit include those required for PCR amplification after bisulfite modification of DNA samples. Sufficient reagents are provided to analyze 25 samples.

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S7811
25 assays  
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Overview

Description
Catalogue NumberS7811
Brand Family Chemicon®
Trade Name
  • CpG Wiz
  • Chemicon
DescriptionCpG WIZ® hMLH1 - Methylation specific PCR assay
OverviewMethylation of cytosines located 5' to guanosine is known to have a profound effect on the expression of several eukaryotic genes (Bird, 1992). 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 (Antequera, 1990) and has been associated with transcriptional inactivation of defined tumor suppressor genes in human cancers (Merlo, 1995). The human mutL homolog 1 (hMLH1) gene exhibits characteristic hypermethylation.



Methylation-specific PCR (MSP) is a new technology for sensitive detection of abnormal gene methylation utilizing small amounts of DNA (Herman, 1996). 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.
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. Gel-loading solution / Loading Dye

h. Bisulfite Modified DNA (CpGenome™ DNA Modification Kit, S7820)
Product Information
Components
  • The components of the CpG WIZ® hMLH1 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 Set7.5 μM each primer (25X) 35 μL (neutral cap) 90563 -15°C to -25°C
  • M Primer Set7.5 μM each primer (25X) 35 μL (red cap) 90564 -15°C to -25°C
  • W Primer Set7.5 μM each primer (25X) 35 μL (green cap) 90565 -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 components of the CpG WIZ hMLH1 Amplification Kit include those required for PCR amplification after bisulfite modification of DNA samples. Sufficient reagents are provided to analyze 25 samples.
Key Applications
  • PCR
Application NotesPrinciples of the Technique

Use of either the CpGenome™ DNA Modification Kit (Cat. No. S7820) or the CpGENOME™ Fast DNA Modification Kit (Cat. No. S7824) facilitates the initial bisulfite reactions, while the CpG WIZ® HMLH1 Amplification Kit contains the reagents required for the gene-specific PCR amplification reactions.
Biological Information
Species Reactivity
  • Human
Entrez Gene Number
Entrez Gene SummaryThis gene was identified as a locus frequently mutated in hereditary nonpolyposis colon cancer (HNPCC). It is a human homolog of the E. coli DNA mismatch repair gene mutL, consistent with the characteristic alterations in microsatellite sequences (RER+ phenotype) found in HNPCC. Alternatively spliced transcript variants encoding different isoforms have been described, but their full-length natures have not been determined.
Gene Symbol
  • MLH1
  • HNPCC
  • COCA2
  • FCC2
  • hMLH1
  • MGC5172
  • HNPCC2
UniProt Number
UniProt SummaryFUNCTION: SwissProt: P40692 # Involved in the repair of mismatches in DNA.
SIZE: 756 amino acids; 84601 Da
SUBUNIT: Heterodimer of MLH1 and PMS2 or MLH1 and MLH3. Part of the BRCA1-associated genome surveillance complex (BASC), which contains BRCA1, MSH2, MSH6, MLH1, ATM, BLM, PMS2 and the RAD50- MRE11-NBS1 protein complex. This association could be a dynamic process changing throughout the cell cycle and within subnuclear domains. Interacts with MBD4. Interacts with EXO1.
SUBCELLULAR LOCATION: Nucleus.
TISSUE SPECIFICITY: Colon, lymphocytes, breast, lung, spleen, testis, prostate, thyroid, gall bladder and heart.
DISEASE: SwissProt: P40692 # Defects in MLH1 are the cause of hereditary non-polyposis colorectal cancer type 2 (HNPCC2) [MIM:609310]. Mutations in more than one gene locus can be involved alone or in combination in the production of the HNPCC phenotype (also called Lynch syndrome). Most families with clinically recognized HNPCC have mutations in either MLH1 or MSH2 genes. HNPCC is an autosomal, dominantly inherited disease associated with marked increase in cancer susceptibility. It is characterized by a familial predisposition to early onset colorectal carcinoma (CRC) and extra-colonic cancers of the gastrointestinal, urological and female reproductive tracts. HNPCC is reported to be the most common form of inherited colorectal cancer in the Western world, and accounts for 15% of all colon cancers. Cancers in HNPCC originate within benign neoplastic polyps termed adenomas. Clinically, HNPCC is often divided into two subgroups. Type I: hereditary predisposition to colorectal cancer, a young age of onset, and carcinoma observed in the proximal colon. Type II: patients have an increased risk for cancers in certain tissues such as the uterus, ovary, breast, stomach, small intestine, skin, and larynx in addition to the colon. Diagnosis of classical HNPCC is based on the Amsterdam criteria: 3 or more relatives affected by colorectal cancer, one a first degree relative of the other two; 2 or more generation affected; 1 or more colorectal cancers presenting before 50 years of age; exclusion of hereditary polyposis syndromes. The term suspected HNPCC or incomplete HNPCC can be used to describe families who do not or only partially fulfill the Amsterdam criteria, but in whom a genetic basis for colon cancer is strongly suspected. & Defects in MLH1 are a cause of Turcot syndrome [MIM:276300]. Turcot syndrome is an autosomal dominant disorder characterized by malignant tumors of the brain associated with multiple colorectal adenomas. Skin features include sebaceous cysts, hyperpigmented and cafe au lait spots. & Defects in MLH1 are a cause of Muir-Torre syndrome (MTS) [MIM:158320]. MTS is a rare autosomal dominant disorder characterized by sebaceous neoplasms and visceral malignancy. & Defects in MLH1 may contribute to lobular carcinoma in situ (LCIS), a non-invasive neoplastic disease of the breast. & Defects in MLH1 are a cause of susceptibility to endometrial cancer [MIM:608089].
SIMILARITY: SwissProt: P40692 ## Belongs to the DNA mismatch repair mutL/hexB family.
Product Usage Statements
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.
Packaging Information
Material Size25 assays
Global Trade Item Number
Catalogue Number GTIN
S7811 04053252727443