CpG Wiz® BRCA1 Amplification Kit

Description
Catalogue Number	S7830
Brand Family	Chemicon®
Trade Name	CpG Wiz Chemicon
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.

Description

Catalogue Number

S7830

Brand Family

Chemicon®

Trade Name

CpG Wiz
Chemicon

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
Components	The components of the CpG WIZ® BRCA1 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 (clear cap) / 90645 / -15°C to -25°C M Primer Set7.5 μM each primer (25X) / 35 μL (red cap) / 90646 / -15°C to -25°C W Primer Set7.5 μM each primer (25X) / 35 μL (green cap) / 90647 / -15°C to -25°C U control DNA0.1 μg/μL / 50 μL (clear 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

Product Information

Components

The components of the CpG WIZ® BRCA1 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 (clear cap) / 90645 / -15°C to -25°C
M Primer Set7.5 μM each primer (25X) / 35 μL (red cap) / 90646 / -15°C to -25°C
W Primer Set7.5 μM each primer (25X) / 35 μL (green cap) / 90647 / -15°C to -25°C
U control DNA0.1 μg/μL / 50 μL (clear 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

Applications
Application	Methylation-specific PCR (MSP), performed using the CpGenome DNA Modification Kit & the CpG WIZ BRCA1 Amplification Kit, permits sensitive detection of altered DNA.
Key Applications	PCR

Applications

Application

Methylation-specific PCR (MSP), performed using the CpGenome DNA Modification Kit & the CpG WIZ BRCA1 Amplification Kit, permits sensitive detection of altered DNA.

Key Applications

Biological Information
Species Reactivity	Human
Entrez Gene Number	NM_007294.2 NM_007295.2 NM_007296.2 NM_007297.2 NM_007298.2 NM_007299.2 NM_007300.2 NM_007302.2 NM_007303.2 NM_007304.2 NM_007305.2
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.
Gene Symbol	BRCA1 RNF53 BRCAI PSCP BRCC1 IRIS
UniProt Number	P38398
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.

Biological Information

Species Reactivity

Human

Entrez Gene Number

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.

Gene Symbol

BRCA1
RNF53
BRCAI
PSCP
BRCC1
IRIS

UniProt Number

P38398

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.

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.

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 Size	25 assays

Packaging Information

Material Size

25 assays

实验耗材

分子生物学试剂

蛋白与免疫学

生化试剂

细胞及检测试剂

色谱及层析

仪器设备

实验服务

办公用品

CpG Wiz® BRCA1 Amplification Kit

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