CpG WIZ® APC Amplification Kit

CpG WIZ® APC Amplification Kit

• CpG Wiz
• Chemicon (Millipore)

25 assays

Methylation 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 (Herman, 1994; Merlo, 1995). The Adenomatous Polyposis Coli (APC) 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.

PCR

Principles 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 The CpG WIZ® APC Amplification Kit contains the reagents required for the gene-specific PCR amplification reactions.

Human

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This gene encodes a tumor suppressor protein that includes among its many intracellular functions one of nuclear export. Defects in this gene cause familial adenomatous polyposis (FAP), an autosomal dominant pre-malignant disease that usually progresses to malignancy. Disease-associated mutations tend to be clustered in a small region designated the mutation cluster region (MCR) and result in a truncated protein product.

FUNCTION: SwissProt: P25054 # Tumor suppressor. Promotes rapid degradation of CTNNB1 and participates in Wnt signaling. APC activity is correlated with its phosphorylation state.
SIZE: 2843 amino acids; 311646 Da
SUBUNIT: Forms homooligomers. Interacts with DIAPH1 and DIAPH2 (By similarity). Interacts with PDZ domains of DLG1 and DLG3. Associates with catenins. Binds axin. Interacts with the N- terminus of ARHGEF4, and the C-terminus of MAPRE1, MAPRE2 and MAPRE3. Found in a complex consisting of ARHGEF4, APC and CTNNB1.
TISSUE SPECIFICITY: Expressed in a variety of tissues.
PTM: Phosphorylated by GSK3B.
DISEASE: SwissProt: P25054 # Defects in APC are a cause of familial adenomatous polyposis (FAP) [MIM:175100]; which includes also Gardner syndrome (GS). FAP and GS contribute to tumor development in patients with uninherited forms of colorectal cancer. FAP is characterized by adenomatous polyps of the colon and rectum, but also of upper gastrointestinal tract (ampullary, duodenal and gastric adenomas). This is a viciously premalignant disease with one or more polyps progressing through dysplasia to malignancy in untreated gene carriers with a median age at diagnosis of 40 years. & APC mutations have led to some interesting observations. (1) the great majority of the mutations found to date would result in truncation of the APC product. (2) almost all the mutations have occurred within the first half of the coding sequence, and somatic mutations in colorectal tumors are further clustered in a particular region, called MCR (mutation cluster region). (3) most identified point mutations in the APC gene are transitions from cytosine to other nucleotides. (4) the location of germline mutations tends to correlate with the number of colorectal polyps in FAP patients. Inactivation of both alleles of the APC gene seems to be required as an early event to develop most adenomas and carcinomas in the colon and rectum as well as some of those in the stomach. & Defects in APC are a cause of hereditary desmoid disease (HDD) [MIM:135290]; also called familial infiltrative fibromatosis (FIF). It is an autosomal dominant trait with 100% penetrance and possible variable expression among affected relatives. HDD patients show multifocal fibromatosis of the paraspinal muscles, breast, occiput, arms, lower ribs, abdominal wall, and mesentery. Desmoid tumors appears also as a complication of familial adenomatous polyposis. & Defects in APC are a cause of medulloblastoma (MDB) [MIM:155255]. MDB is a malignant, invasive embryonal tumor of the cerebellum with a preferential manifestation in children. Although the majority of medulloblastomas occur sporadically, some manifest within familial cancer syndromes such as Turcot syndrome and basal cell nevus syndrome (Gorlin syndrome). & Defects in APC 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.
SIMILARITY: SwissProt: P25054 ## Contains 7 ARM repeats.

• The components of the CpG WIZ® APC 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) 90560 -15°C to -25°C
• M Primer Set7.5 μM each primer (25X) 35 μL (red cap) 90561 -15°C to -25°C
• W Primer Set7.5 μM each primer (25X) 35 μL (green cap) 90562 -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

Chemicon

P25054

The protein encoded by APC (adenomatous polyposis coli) is a tumor suppressor that is involved in the degradation of b-catenin when phosphorylated by GSK-3. Loss of APC results in the accumulation of b-catenin, which is free to localize to the nucleus and activate TCF transcription. One of the critical genes activated by the TCFs is cyclin D1 (also cyclin A, cdc2, and cdc25C), driving cell cycle progression. This loss is seen in a large proportion of colon cancers.

• APC
• DP2.5
• FAP
• GS
• DP3
• DP2
• FPC

CpG WIZ® APC Amplification Kit

NM_000038.3

Equipment 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)