CpG Wiz® RB1 Amplification Kit

Description
Catalogue Number	S7810
Brand Family	Chemicon®
Trade Name	CpG Wiz Chemicon
Description	CpG WIZ® RB1 - Methylation specific PCR assay
Overview	MSP, performed using the CpGenome™ DNA Modification Kit and the CpG WIZ® RB1 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® RB1 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	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)
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, 4). The retinoblastoma 1 (RB1) gene exhibits characteristic hypermethylation im many maligninant tumors. 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® RB1 Amplification Kit contains the reagents required for the PCR amplification reactions.

Description

Catalogue Number

S7810

Brand Family

Chemicon®

Trade Name

CpG Wiz
Chemicon

Description

CpG WIZ® RB1 - Methylation specific PCR assay

Overview

MSP, performed using the CpGenome™ DNA Modification Kit and the CpG WIZ® RB1 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® RB1 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

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)

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, 4). The retinoblastoma 1 (RB1) gene exhibits characteristic hypermethylation im many maligninant tumors.

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® RB1 Amplification Kit contains the reagents required for the PCR amplification reactions.

Product Information
Components	The components of the CpG WIZ® RB1 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) 90552 -15°C to -25°C M Primer Set7.5 μM each primer (25X) 35 μL (red cap) 90553 -15°C to -25°C W Primer Set7.5 μM each primer (25X) 35 μL (green cap) 90554 -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

Product Information

Components

The components of the CpG WIZ® RB1 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) 90552 -15°C to -25°C
M Primer Set7.5 μM each primer (25X) 35 μL (red cap) 90553 -15°C to -25°C
W Primer Set7.5 μM each primer (25X) 35 μL (green cap) 90554 -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

Applications
Application	MSP, performed using the CpGenome DNA Modification Kit & the CpG WIZ RB1 Amplification Kit, permits sensitive detection of altered DNA.
Key Applications	PCR

Applications

Application

MSP, performed using the CpGenome DNA Modification Kit & the CpG WIZ RB1 Amplification Kit, permits sensitive detection of altered DNA.

Key Applications

Biological Information
Species Reactivity	Human
Entrez Gene Number	NM_000321.2
Entrez Gene Summary	Retinoblastoma (RB) is an embryonic malignant neoplasm of retinal origin. It almost always presents in early childhood and is often bilateral. Spontaneous regression ('cure') occurs in some cases. The retinoblastoma gene RB was the first tumor suppressor gene cloned, and is a negative regulator of the cell cycle through its ability to bind the transcription factor E2F (MIM 189971) and repress transcription of genes required for S phase (Hanahan and Weinberg, 2000 [PubMed 10647931]).[supplied by OMIM]
Gene Symbol	RB1 P105-RB retinoblastoma-1 RB PP110 OSRC
UniProt Number	P06400
UniProt Summary	FUNCTION: SwissProt: P06400 # Key regulator of entry into cell division that acts as a tumor suppressor. Directly involved in heterochromatin formation by maintaining overall chromatin structure and, in particular, that of constitutive heterochromatin by stabilizing histone methylation. Recruits and targets histone methyltransferases SUV39H1, SUV420H1 and SUV420H2, leading to epigenetic transcriptional repression. Controls histone H4 'Lys-20' trimethylation. Also acts as a transcription repressor of E2F target genes by recruiting chromatin-modifying enzymes to promoters. Inhibits the intrinsic kinase activity of TAF1. Forms a complex with adenovirus E1A and with SV40 large T antigen. May bind and modulate functionally certain cellular proteins with which T and E1A compete for pocket binding. SIZE: 928 amino acids; 106159 Da SUBUNIT: Interacts preferentially with transcription factor E2F1. The unphosphorylated form interacts with ARID3B, JARID1A, SUV39H1, MJD2A/JHDM3A and THOC1. Interacts with the N-terminal domain of TAF1. Interacts with AATF, DNMT1, LIN9, LMNA, SUV420H1, SUV420H2, PELP1 and TMPO-alpha. May interact with KNTC2. Interacts with EID1 and ZUBR1. Interacts with ARID4A and JARID1B. SUBCELLULAR LOCATION: Nucleus. TISSUE SPECIFICITY: Expressed in the retina. PTM: Phosphorylated from S to M phase of the cell cycle and is dephosphorylated in G1. T, but not E1A, binds only to the unphosphorylated form. DISEASE: SwissProt: P06400 # Defects in RB1 are the cause of childhood cancer retinoblastoma (RB) [MIM:180200]. RB is a congenital malignant tumor that arises from the nuclear layers of the retina. It occurs in about 1:20'000 live births and represents about 2% of childhood malignancies. It is bilateral in about 30% of cases. Although most RB appear sporadically, about 20% are transmitted as an autosomal dominant trait with incomplete penetrance. The diagnosis is usually made before the age of 2 years when strabismus or a gray to yellow reflex from pupil (cat eye) is investigated. & Defects in RB1 are a cause of bladder cancer [MIM:109800]. & Defects in RB1 are a cause of osteogenic sarcoma [MIM:259500]. SIMILARITY: SwissProt: P06400 ## Belongs to the retinoblastoma protein (RB) family.

Biological Information

Species Reactivity

Human

Entrez Gene Number

NM_000321.2

Entrez Gene Summary

Retinoblastoma (RB) is an embryonic malignant neoplasm of retinal origin. It almost always presents in early childhood and is often bilateral. Spontaneous regression ('cure') occurs in some cases. The retinoblastoma gene RB was the first tumor suppressor gene cloned, and is a negative regulator of the cell cycle through its ability to bind the transcription factor E2F (MIM 189971) and repress transcription of genes required for S phase (Hanahan and Weinberg, 2000 [PubMed 10647931]).[supplied by OMIM]

Gene Symbol

RB1
P105-RB
retinoblastoma-1
RB
PP110
OSRC

UniProt Number

P06400

UniProt Summary

FUNCTION: SwissProt: P06400 # Key regulator of entry into cell division that acts as a tumor suppressor. Directly involved in heterochromatin formation by maintaining overall chromatin structure and, in particular, that of constitutive heterochromatin by stabilizing histone methylation. Recruits and targets histone methyltransferases SUV39H1, SUV420H1 and SUV420H2, leading to epigenetic transcriptional repression. Controls histone H4 'Lys-20' trimethylation. Also acts as a transcription repressor of E2F target genes by recruiting chromatin-modifying enzymes to promoters. Inhibits the intrinsic kinase activity of TAF1. Forms a complex with adenovirus E1A and with SV40 large T antigen. May bind and modulate functionally certain cellular proteins with which T and E1A compete for pocket binding.
SIZE: 928 amino acids; 106159 Da
SUBUNIT: Interacts preferentially with transcription factor E2F1. The unphosphorylated form interacts with ARID3B, JARID1A, SUV39H1, MJD2A/JHDM3A and THOC1. Interacts with the N-terminal domain of TAF1. Interacts with AATF, DNMT1, LIN9, LMNA, SUV420H1, SUV420H2, PELP1 and TMPO-alpha. May interact with KNTC2. Interacts with EID1 and ZUBR1. Interacts with ARID4A and JARID1B.
SUBCELLULAR LOCATION: Nucleus.
TISSUE SPECIFICITY: Expressed in the retina.
PTM: Phosphorylated from S to M phase of the cell cycle and is dephosphorylated in G1. T, but not E1A, binds only to the unphosphorylated form.
DISEASE: SwissProt: P06400 # Defects in RB1 are the cause of childhood cancer retinoblastoma (RB) [MIM:180200]. RB is a congenital malignant tumor that arises from the nuclear layers of the retina. It occurs in about 1:20'000 live births and represents about 2% of childhood malignancies. It is bilateral in about 30% of cases. Although most RB appear sporadically, about 20% are transmitted as an autosomal dominant trait with incomplete penetrance. The diagnosis is usually made before the age of 2 years when strabismus or a gray to yellow reflex from pupil (cat eye) is investigated. & Defects in RB1 are a cause of bladder cancer [MIM:109800]. & Defects in RB1 are a cause of osteogenic sarcoma [MIM:259500].
SIMILARITY: SwissProt: P06400 ## Belongs to the retinoblastoma protein (RB) 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.

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® RB1 Amplification Kit

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