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CpG WIZTM p16 Amplification Kit S7800 CpG WIZTM p15 Amplification Kit S7802 CpG WIZTM E-cadherin Amplification Kit S7804 CpG WIZTM Prader-Willi/Angelman Amplification Kit S7806
FOR RESEARCH USE ONLY Not for use in diagnostic procedures
USA & Canada Phone: +1(800) 437-7500 * Fax: +1 (909) 676-9209 * Europe +44 (0) 23 8026 2233 Australia +61 3 9839 2000 * Germany +49-6192-207300 * ISO Registered Worldwide www.chemicon.com * custserv@chemicon.com * techserv@chemicon.com
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TABLE OF CONTENTS
I. INTRODUCTION .............................................................................. 1
Using this Manual ..................................................................................... 1 Background ............................................................................................... 1 Principles of the Technique....................................................................... 2 Fig 1: DNA Treatment with Sodium Bisulfite....................................... 3
II. KIT COMPONENTS ......................................................................... 4
Materials Required But Not Supplied ....................................................... 5
III. PROTOCOLS.................................................................................... 7
CpG WIZTM p16 (S7800), p15 (S7802) and E-cadherin (S7804) Amplification Kits ................................................. 7 Experimental Design............................................................................ 7 Fig. 2: Specificity of the CpG WIZTM p16 Amplification Kit .................................................................................. 9 Amplification Protocol....................................................................... 10 CpG WIZTM Prader-Willi/Angelman (S7806) Amplification Kit ........... 12 Experimental Design.......................................................................... 12 Fig. 3: Specificity of the CpG WIZTM Prader-Willi/ Angelman Amplification Kit ............................................................... 14 Amplification Protocol....................................................................... 15
IV.
DATA ANALYSIS ........................................................................... 18
CpG WIZTM p16 (S7800), p15 (S7802) and E-cadherin (S7804) Amplification Kits ............................................. 18 CpG WIZTM Prader-Willi/Angelman (S7806) Amplification Kit ........... 19
V. TROUBLESHOOTING .................................................................... 20
CpG WIZTM p16 (S7800), p15 (S7802) and E-cadherin (S7804) Amplification Kits .................................................. 20 CpG WIZTM Prader-Willi/Angelman (S7806) Amplification Kit ........... 21 i
VI.
APPENDIX ............................................................................ 23
Laboratory Setup and Precautions .......................................................... 23 Related Products...................................................................................... 23
VII. REFERENCES ................................................................................ 24
References Cited in this Manual ............................................................. 24 Disclaimers ............................................................................................ 25 Warranty.................................................................................................. 25
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I. INTRODUCTION
Using This Manual
Please read the entire instruction manual prior to using CpG WIZTM Amplification Kits. Note that in the Procedures and Troubleshooting sections, instructions are separate for the CpG WIZTM Prader-Willi/ Angelman Amplification Kit (S7806). Should additional questions arise, assistance is available from Chemicon Technical Service at techserv@chemicon.com or (800) 437-7500.
Background
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. The exception is extensive methylation of CpG islands associated with transcriptional inactivation of regulatory regions of imprinted genes (2, 3) such as those associated with Prader-Willi/Angelman Syndrome (4) and genes on the inactive X-chromosome of females (5, 6). Aberrant methylation of normally unmethylated CpG islands has been documented as a relatively frequent event in immortalized and transformed cells (7) and has been associated with transcriptional inactivation of defined tumor suppressor genes in human cancers (8, 9). E-cadherin, p16, and p15 are examples of genes that exhibit 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 (10). 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 CpGenomeTM DNA Modification Kit (S7820) contains the reagents necessary to perform the initial bisulfite reactions, while CpG WIZTM Amplification Kits contain the reagents required for the PCR amplification reactions. 1
Principles of the Technique
MSP, performed using the CpGenomeTM DNA Modification Kit and CpG WIZTM Amplification Kits, permits sensitive detection of altered DNA. Due to the fact that it is a PCR-based assay, it is extremely sensitive, facilitating the detection of low numbers of methylated alleles and the study of samples containing low 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 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 CpG WIZTM Amplification Kits 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.
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Figure 1: DNA Treatment with Sodium Bisulfite. Unmethylated DNA
Methylated DNA
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II. KIT COMPONENTS
The components of CpG WIZTM Amplification Kits include those required for PCR amplification after bisulfite modification of DNA samples. Sufficient reagents are provided to analyze 25 samples with appropriate controls. Table 1: CpG WIZTM p16 (S7800), p15 (S7802) and E-cadherin (S7804) Amplification Kit Components (color-coded microcentrifuge tube caps) Description U Primer Set 5 M each primer (25X) M Primer Set 5 M each primer (25X) W Primer Set 5 M each primer (25X) U control DNA 0.1 gL M control DNA 0.1 g/L W control DNA 0.05 g/L Universal 10X PCR Buffer Amount 35 L (white cap) 35 L (red cap) 35 L (green cap) 50 L (white cap) 50 L (red cap) 50 L (green cap) 265 L (blue cap) Storage Conditions -15C to -25C -15C to -25C -15C to -25C -15C to -25C -15C to -25C -15C to -25C -15C to -25C
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Table 2: CpG WIZTM Prader-Willi/Angelman (S7806) Amplification Kit Components (color-coded microcentrifuge tubes) Description P (paternal) primer set 5 M each primer (25X) M (maternal) primer set 5 M each primer (25X) Normal control DNA 0.1 g/L 10X PCR buffer Amount 70 L (white cap) 70 L (red cap) 20 L (green cap) 175 L (blue cap) Storage Conditions -15C to -25C -15C to -25C -15C to -25C -15C to -25C
Materials Required But Not Supplied
Equipment and Supplies
a. Thermocycler b. Gel electrophoresis apparatus (vertical or horizontal) c. Power Supply d. Screw-cap tubes for PCR amplification e. Aerosol-resistant pipette tips f. Microcentrifuge (to 12,000 X g) g. 302 nm UV transilluminator, camera and film
Reagents
a 2.5 mM dNTP mix (2.5 mM of each nucleotide) b. Taq polymerase Note: For S7806 (CpG WIZTM Prader-Willi/Angelman Amplification Kit), the use of a "hot start" enzyme is strongly recommended. See Sec. III. Protocols. c. "Hot start" PCR reagents for S7800, S7802, and S7804 (see Sec. III. Protocols).
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d. Reagents for gel electrophoresis (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 h. Bisulfite Modified DNA (CpGenomeTM DNA Modification Kit, S7820)
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III.
PROTOCOLS
CpG WIZTM p16 (S7800), p15 (S7802) and E-cadherin (S7804) Amplification Kits
Experimental Design
Primer Sets CpG WIZTM Amplification Kits contain primers that can be used for analysis of DNA samples by MSP. However, the samples must first undergo bisulfite modification prior to PCR amplification. CpGenomeTM DNA Modification Kit, S7820, contains the reagents necessary to perform the modification. Chemical modification creates the sequence differences between the methylated and unmethylated DNA. The primer sets in the kit are engineered to anneal to the DNA, based upon the sequence differences. U Primer Set will anneal to unmethylated DNA that has undergone a chemical modification M Primer Set will anneal to methylated DNA that has undergone a chemical modification W Primer Set serves as a control for the efficiency of chemical modification. It will anneal to any DNA (unmethylated or methylated) that has NOT undergone chemical modification, hence, the "wild type", or W. Data interpretation can still proceed in the case of incomplete chemical modification (up to 50%). Amplification Regions The amplified region is defined as the sequence between the 3' nucleotide of the sense primer and the complement of the 3' nucleotide of the anti-sense primer for each gene promoter. The nucleotide numbering systems are those used in the GenBank submissions identified by the following accession numbers: p16, X94154; p15, S75756; E-cadherin, L34545.
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"Hot Start" PCR The three sets of primers used in the CpG WIZTM Methylation Assay are derived from sequences closely related to each other, which introduces the possibility of mispriming. In order to avoid this and other PCR-related artifacts, "hot start" PCR is recommended. "Hot start" PCR permits the Taq polymerase to begin the reaction only after the template and primers are in single-stranded form. There are several modifications of the standard PCR protocol which allow a "hot start" to occur. In one scenario, the PCR reaction mixture excluding the polymerase can be overlaid with mineral oil prior to heating to 95C. At the end of the incubation, the enzyme is pipetted directly into the mixture under the mineral oil. A second method involves the physical separation of the polymerase and the rest of the PCR mix with a wax bead. The enzyme combines with the rest of the reaction mixture only after the wax melts. In another variation, an anti-Taq antibody inhibits the polymerase during reaction setup by forming a complex with the Taq enzyme. Taq polymerase becomes active when the complex is abolished due to antibody denaturation during the 95C incubation. Alternatively, a "hot start" enzyme can be used. Refer to the manufacturer's instructions for enzyme activation protocol. Note: Do not use a polymerase with 3'-5' exonuclease activity (i.e. proofreading).Do not use a wax bead that contains Mg2+. Any extra Mg2+ added to the reaction mixture produces suboptimal results. Genomic Control DNAs The methylated (red cap) and unmethylated (white cap) control DNAs must undergo bisulfite modification prior to PCR amplification using the CpGenomeTM DNA Modification Kit (S7820). When used with their respective U and M primer sets, a single PCR product of an expected size is obtained in each case. W control genomic DNA is used in the PCR and is NOT to be used in the chemical modification step. When used with the W primer set, it is a positive control for PCR amplification. Failure to generate a PCR product indicates a general failure in the PCR reaction.
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Specificity of the Assay The specificity of the CpG WIZTM p16 Amplification Kit is shown in Figure 2. With a complete chemical modification reaction, U primers amplify only unmethylated DNA (154 bp, lane 1) and M primers amplify only methylated DNA (145 bp, lane 5). W primers amplify only DNA which is not chemically modified, or "wild type W" (142 bp, lane 9). Figure 2: Specificity of the CpG WIZTM p16 Amplification Kit. M 1 2 3 4 5 6 7 8 9 10 11 12 M
Polyacrylamide gel analysis using the primers and the control DNA samples included in the kit is shown. (Lanes 1-3: U control DNA; lanes 4-6: M control DNA; lanes 7-9: W control DNA; lanes 10-12: Minus DNA control. Lanes 1, 4, 7, 10: U primers; lanes 2, 5, 8, 11: M primers; lanes 3, 6, 9, 12: W primers. M lane: 100 base-pair marker.) Experiment Setup CpG WIZTM Amplification Kits (S7800, S7802, and S7804) include sufficient reagents to analyze 25 samples with appropriate controls (105 PCR reactions). Each experiment will include chemical modification of seven DNAs: 5 experimental DNA and 2 control DNA samples, (M and U). In the subsequent PCR reactions, each chemically modified experimental DNA sample is amplified with each of three oligonucleotide primer sets U, M and W. The chemically modified genomic control DNAs, U and M, are amplified with their corresponding primer set. Untreated W genomic control DNA is amplified with the W primer set. Lastly, a negative PCR control (i.e. no DNA) is performed for each set of primers. 9
For example, a typical gel for the analysis of five experimental DNA samples includes a total of 21 lanes: Lanes 1-3 Lanes 4-6 Lanes 7-9 Lanes 10-12 Lanes 13-15 Lane 16 Lane 17 Lane 18 Lanes 19-21 Experimental sample 1 with U, M and W primers Experimental sample 2 with U, M and W primers Experimental sample 3 with U, M and W primers Experimental sample 4 with U, M and W primers Experimental sample 5 with U, M and W primers Chemically modified control U DNA with U primers Chemically modified control M DNA with M primers Untreated control W DNA with W primers No DNA control with U, M, and W primers
Amplification Protocol
To prevent PCR contamination, read Sec. VI. Appendix, Laboratory Setup and Precautions before beginning. STEP 1. Modification Prior to performing PCR with the primer sets provided in CpG WIZTM Amplification Kits, one microgram of purified DNA must undergo bisulfite modification with the reagents contained in CpGenomeTM DNA Modification Kit (S7820). STEP 2. Amplification "Hot start" PCR is recommended for this assay (refer to Sec. III. Protocols, Experimental Design). This is accomplished by several mechanisms, including a wax barrier or anti-Taq antibody. Refer to the instructions specified by the manufacturer of the "hot start" PCR reagents, and modify the amplification "master mix" and reaction conditions accordingly in steps b-f, below. a. Determine the number of assays to be run in the experiment: run three amplification reactions for each experimental DNA sample plus six control reactions per each set of methylation assays (refer to Sec. III. Protocols, Experimental Design). b. Prepare three (3) "master mixes" which correspond to the 3 possible primer sets U, M and W (primer cap colors-white, red and green) by mixing all the reagents outlined below except for the template DNA. 10
To analyze five experimental samples with appropriate controls, use the following amount of "master mixes" of each color: 7 tubes X 23.0 L = 161 L, plus 10% of that volume to adjust for pipetting error. Multiply the volume of each reagent listed below by 7 and add 10% for each "master mix" (white, red and green). Thaw all reagents and store on ice while creating the "master mixes". The amount of reagents required in each reaction is: 10X Universal PCR buffer 2.5 mM dNTP Mix** U, M or W primers (white, red and green) TaKaRaTM Taq or "hot start" enzyme (5 U/L) dH2O Template DNA (50 ng/L) TOTAL VOLUME 2.5 L 2.5 L 1.0 L 0.2 L (1 Unit) 16.8 L 23.0 L 2.0 L 25.0 L
** Upon first use, make aliquots of 2.5 mM dNTPs, which should be freezethawed no more than 5 times. c. Aliquot 23 L of each "master mix" (white, red and green) into corresponding PCR tubes. d. Add: 2 L of water to the no DNA control tube. 2 L of modified sample DNA to each of the sample tubes. 2 L of corresponding DNA controls (modified U and M, unmodified W) to each control tube. e. Place tubes in the thermocycler block, and perform PCR under the following conditions: Denature: for Taq Polymerase 95C / 5 minutes for "hot start" enzyme check manufacturer's specifications Then, perform 35 cycles of the following conditions: denature 95C / 45 seconds anneal 60C / 45 seconds extend 72C / 60 seconds
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f. Remove the tubes from the thermocycler block. From this point on, it is important to designate separate pipettes and work areas for amplified vs. unamplified samples. This prevents carry-over contamination of future DNA samples with the amplified product. STEP 3. Gel Electrophoresis a. After the completion of PCR, add an appropriate amount of loading dye to the sample and analyze 5 L of the reaction on a 2% agarose, or a 10% native acrylamide or other high resolution agarose gel. Use DNA markers (100-300 bp range) to determine the size of PCR products. b. After electrophoresis, stain the gel with ethidium bromide. Dilute the 10 mg/mL stock solution 1:10,000 in deionized water. Stain for 10-30 minutes and destain for 10-30 minutes in deionized water at room temperature. Note: Ethidium bromide is a known carcinogen. Exercise appropriate caution and good lab practice when using this reagent.
CpG WIZTM Prader-Willi/Angelman (S7806) Amplification Kit
Experimental Design
Primer Sets The CpG WIZTM Prader-Willi/Angelman Amplification Kit contains primers that can be used for analysis of DNA samples by MSP. However, the first step of MSP is the bisulfite modification of the DNA samples. CpGenomeTM DNA Modification Kit (S7820), contains the reagents necessary to perform the modification. This chemical modification creates the sequence differences between the methylated and unmethylated DNA. The primer sets in the kit are engineered to anneal to the DNA, based upon the sequence differences. P (Paternal) Primer Set will anneal to unmethylated DNA that has undergone a chemical modification. M (Maternal) Primer Set will anneal to methylated DNA that has undergone a chemical modification.
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Amplified Regions The P and M primer sets are designed to amplify a differentially methylated site present at the CpG island of the small nuclear ribonucleoprotein-associated polypeptide N (SNRPN), a candidate gene for Prader-Willi Syndrome. The amplified region is defined as the sequence between the 3' nucleotide of the sense primer and the complement of the 3' nucleotide of the anti-sense primer for each primer pair. The nucleotide numbering system is that used in the GenBank submission identified by the accession number L32702. "Hot Start" PCR The two sets of primers used in the CpG WIZTM Prader-Willi/Angelman Amplification Kit are derived from sequences closely related to each other, which introduces the possibility of mispriming. In order to avoid this and other PCR-related artifacts, it is recommended that a "hot start" enzyme be used. Normal Control DNA The normal control contains DNA from both maternal and paternal homologs which means that both methylated and unmethylated SNRPN sequences are represented. Therefore, PCR products will be obtained after the bisulfite modification of the normal control DNA when using either the M or the P primer sets. The M primer set will generate a 174 bp product while the P primer set produces a 100 bp fragment. Multiplex amplification is feasible since the fragment sizes are sufficiently dissimilar. Specificity of the Assay The specificity of the CpG WIZTM Prader-Willi/Angelman Amplification Kit is shown in Figure 3. With a complete chemical modification reaction, P primers amplify only unmethylated DNA (100 bp, lane 1) and M primers amplify only methylated DNA (174 bp, lane 2). The smaller bands present in the lanes where the M primer set was used in the PCR reaction represent excess primers.
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Figure 3: Specificity of the CpG WIZTM Prader-Willi/Angleman Amplification Kit. M 1 2 3 4
Polyacrylamide gel analysis using the primers and the control DNA samples included in the kit is shown. (Lane 1: P primer set with modified control DNA; Lane 2: M primer set with modified control DNA; Lane 3: P primer set with unmodified control DNA; Lane 4: M primer set with unmodified control DNA. Marker Lane: 100 bp ladder) Experiment Setup The CpG WIZTM Prader-Willi/Angelman Amplification Kit includes sufficient reagents to analyze 25 samples with appropriate controls (70 PCR reactions). For each batch of experimental DNA samples to be analyzed, the experimental samples and the normal control DNA must first undergo bisulfite modification. In the subsequent PCR reactions, the chemically modified DNA samples, both experimental and control, are amplified using both the maternal (M) and paternal (P) primer sets. In addition, a negative PCR control (i.e. no DNA) is performed for each set of primers. For example, a typical gel for the analysis of five experimental DNA samples and proper controls includes a total of 14 lanes: Lanes 1-2 Lanes 3-4 Lanes 5-6 Experimental sample 1 with P and M primers Experimental sample 2 with P and M primers Experimental sample 3 with P and M primers 14
Lanes 7-8 Lanes 9-10 Lane 11 Lane 12 Lanes 13-14
Experimental sample 4 with P and M primers Experimental sample 5 with P and M primers Chemically modified normal control DNA with P primers Chemically modified normal control DNA with M primers No DNA Control with P and M primers
Amplification Protocol
To prevent PCR contamination, read Sec. VI. Appendix, Laboratory Setup and Precautions before beginning. STEP 1. Modification Prior to performing PCR with the primer sets in the CpG WIZTM PraderWilli/Angelman Amplification Kit, the DNA samples must undergo bisulfite modification. CpGenomeTM DNA Modification Kit (S7820), contains the reagents necessary to perform the modification. STEP 2. Amplification a. Determine the number of assays to be run in the experiment: run two amplification reactions for each experimental DNA sample plus four control reactions per each set of methylation assays (see Sec. III. Protocols, Experimental Design). b. Prepare two (2) "master mixes" which correspond to the 2 possible primer sets P and M (primer cap colors-white and red) by mixing all the reagents outlined below except for the template DNA. To analyze five experimental samples with appropriate controls, use the following amount of "master mixes" of each color: 7 tubes X 23.0 L = 161 L, plus 10% of that volume to adjust for pipetting error. Multiply the volume of each reagent listed below by 7 and add 10% for each "master mix" (white and red). Thaw all reagents and store on ice while creating the "master mixes".
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The amount of reagents required in each reaction is: 10X Universal PCR buffer 2.5 mM dNTP Mix** P or M primers (white, red) "Hot start" enzyme (5 U/L) dH2O Template DNA (50 ng/L) TOTAL VOLUME 2.5 L 2.0 L 2.0 L 0.1 L (0.5 U) 16.4 L 23.0 L 2.0 L 25.0 L
** Upon first use, make aliquots of 2.5 mM dNTPs, which should be freezethawed no more than 5 times. c. Aliquot 23 L of each "master mix" (white and red) into corresponding PCR tubes. d. Add: 2 L of water to the no DNA control tube. 2 L of modified sample DNA to each of the sample tubes. 2 L of modified normal DNA control to each control tube. e. Place tubes in the thermocycler block, and perform PCR under the following conditions: Denature: for "hot start" enzyme check manufacturer's specifications Then, perform 35 cycles of the following conditions: denature 95C / 30 seconds anneal 62C / 30 seconds extend 72C / 30 seconds Final Extension: 72C /10 minutes
f. Remove the tubes from the thermocycler block. From this point on, it is important to designate separate pipettors and work areas for amplified vs. unamplified samples. This prevents carry-over contamination of future DNA samples with the amplified product.
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STEP 3. Gel Electrophoresis a. After the completion of PCR, add an appropriate amount of loading dye to the sample and analyze 5 L of the reaction on a 2% agarose or a 10% native acrylamide or other high resolution agarose gel. Use DNA markers (100-300 bp range) to determine the size of PCR products. b. After electrophoresis, stain the gel with ethidium bromide. Dilute the 10 mg/mL stock solution 1:10,000 in deionized water. Stain for 10-30 minutes and destain for 10-30 minutes in deionized water at room temperature. Note: Ethidium bromide is a known carcinogen. Exercise appropriate caution and good lab practice when using this reagent.
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IV.
DATA ANALYSIS
CpG WIZTM p16 (S7800), p15 (S7802) and E-cadherin (S7804) Amplification Kits
Table 3: Sizes of Expected Products from Controls Controls U primer/U control DNA M primer set/M control DNA W primer set/W control DNA p16 154 145 142 p15 162 154 137 E-cadherin 212 206 194
In the three no DNA control lanes, no PCR products should be generated. Table 4: Sizes of Expected Products from Samples Experimental Samples U primer set/ unmethylated DNA M primer set/methylated DNA p16 154 145 p15 162 154 E-cadherin 212 206
If the sample is a mixture of unmethylated and methylated DNA, both the U and M primer will produce a PCR product. If the W primer set produces a PCR product with an experimental sample, it is an indication of incomplete chemical modification.
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CpG WIZTM Prader-Willi/Angelman (S7806) Amplification Kit
Table 5: Sizes of Expected Products Template Normal Control DNA Experimental Methylated DNA Experimental Unmethylated DNA Primer Set P 100 -- 100 M 174 174 --
In the two no DNA control lanes, no PCR products should be generated. In a clinical research sample obtained from a normal individual, a 100 bp and a 174 bp fragment will be obtained when using the P primer set and M primer set, respectively, in PCR reactions. MSP performed on a research sample from an individual with Prader-Willi Syndrome will produce the 174 bp fragment with the M primer set but will generate no product with the P primer set. Conversely, a research sample from an individual with Angelman Syndrome will produce the 100 bp fragment with the P primer set, but will generate no product with the M primer set.
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V.
TROUBLESHOOTING
CpG WIZTM p16 (S7800), p15 (S7802) and E-cadherin (S7804) Amplification Kits
?
There is no visual evidence of products in any lane.
Potential Problem: PCR amplification is not initiated.
Recommendations: a. Confirm that all PCR components were added to the reaction tube. b. Confirm that the time and temperature settings on the thermocycler match those described in this manual. c. If performing "hot start" PCR using a "hot start" enzyme, verify the initial denaturation/activation time of 12 minutes at 95C. d. For all other "hot start" methods, confirm the proper use of the reagents. e. Confirm that the PCR polymerase is still active. f. Confirm that no additional Mg2+ was added to the PCR reaction mix. g. The optimal annealing temperature is 60C. If items #a-f have not remedied the problem, re-optimize annealing conditions to suit your amplification instrument.
?
No amplification product is generated in the experimental samples using U, M and W primer sets, but products of the correct size are observed with the control samples.
Potential Problem #1: Experimental DNA samples were degraded prior to chemical modification.
Recommendation: Purify the genomic DNA again and repeat the chemical modification. Potential Problem #2: Chemically modified experimental DNA samples were stored for more than two months prior to PCR. Recommendation: Repeat the chemical modification on new genomic DNA samples.
?
U or M primer sets are producing bands in all samples, including the "no DNA" controls.
Potential Problem: PCR amplification products. 20 reagents are contaminated with
Recommendations: see Sec. VI. Appendix, Laboratory Setup and Precautions. a. Use fresh aliquots of every PCR component (i.e. dNTPs, buffer, etc.) b. Use separate sets of pipettors for pre- vs. post-amplification liquid dispensing. c. Devote a work area to pre- and post-amplification procedures. d. Always use aerosol-resistant pipette tips. e. Always use a clean labcoat and gloves.
?
W primer set produces an amplification product in some or all experimental samples, in addition to an amplification product from the U or M primer set.
Potential Problem: Chemical modification of the experimental DNA sample(s) is incomplete.
Recommendation: This will not jeopardize the validity of the assay as long as a product is also produced using the U or M primer set. The PCR product produced with the W primer set is always smaller than that produced with either U or M.
?
The only lanes containing a PCR product are from those DNA samples (experimental and control) amplified with the W primer set.
Potential Problem: Chemical modification of the experimental DNA samples did not work.
Recommendation: If using the CpGenomeTM DNA Modification Kit (S7820), check the troubleshooting section of the manual.
CpG WIZTM Prader-Willi/Angelman (S7806) Amplification Kit
?
There is no visual evidence of products in any lane.
Potential Problem: PCR amplification is not initiated.
Recommendations: a. Confirm that all PCR components were added to the reaction tube. b. Confirm that the time and temperature settings on the thermocycler match those described in this manual.
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c. d. e. f.
If performing "hot start" PCR using a "hot start" enzyme, verify the initial denaturation/activation time of 12 minutes at 95C. Confirm that the PCR polymerase is still active. Confirm that no additional Mg2+ was added to the PCR reaction mix. The optimal annealing temperature is 62C. If items #a-e have not remedied the problem, re-optimize annealing conditions to suit your amplification instrument.
?
No amplification product is generated in the experimental samples using the P and M primer sets, but products of the correct size are observed with the control samples.
Potential Problem #1: Experimental DNA samples were degraded prior to chemical modification.
Recommendation: Purify the genomic DNA again and repeat the chemical modification. Potential Problem #2: Chemically modified experimental DNA samples were stored for more than two months prior to PCR. Recommendation: Repeat the chemical modification on new genomic DNA samples.
?
P or M primer sets are producing bands in all samples, including the "no DNA" controls.
Potential Problem: PCR reagents are contaminated with amplification products.
Recommendations: see Sec. VI. Appendix, Laboratory Setup and Precautions a. Use fresh aliquots of every PCR component (i.e. dNTPs, buffer, etc.) b. Use separate sets of pipettors for pre- vs. post-amplification liquid dispensing. c. Devote a work area to pre- and post-amplification procedures. d. Always use aerosol-resistant pipette tips. e. Always use a clean labcoat and gloves.
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VI.
APPENDIX
Laboratory Setup and Precautions
One of the most important considerations when performing MSP using CpG WIZTM Amplification Kits is the environment where the initial reaction mixtures are set up. The ideal environment is free of amplified DNA products, which can cause false-positive results. Potential sources of PCR product contamination are: contaminated pipettors and tips, gel box and buffer, tube racks, notebooks, lab coats and any other item exposed to amplified PCR products. The following precautions should be followed in all steps of the assay protocol: a. Always wear gloves. b. Use sterile water for all solutions, aliquot the solutions in small amounts, and use fresh aliquots as working solutions. Discard working solutions after use. c. Keep the assay solutions (10X PCR buffers, dNTPs, polymerase, etc.) separate from the amplified DNA. d. Always use aerosol resistant pipette tips. e. Separate micropipettors and work areas are recommended for the following three steps of the assay: 1. DNA modification and purification 2. Amplification setup 3. Post-amplification analysis
Related Products
Product CpGenomeTM DNA Modification Kit CpGenomeTM Universal Methylated DNA (human male genomic DNA) DNA Extraction Kit, Non-Organic EX-WAXTM DNA Extraction Kit for paraffin-embedded tissue Catalog Number S7820 S7821 S4520 S4530
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VII. REFERENCES
References Cited in this Manual
1. 2. 3. Bird, A. (1992) The essentials of DNA methylation. Cell 70: 5-8. Li, E., C. Beard and R. Jaenisch. (1993) Role for DNA methylation in genomic imprinting. Nature 366: 362-365. Tremblay, K.D., J.R. Saam, R.S. Ingram, S.M. Tilghman and M.S. Bartolomei. (1995) A paternal-specific methylation imprint marks the alleles of the mouse H19 gene. Nat. Genet. 9: 407-413. Kubota, T., S. Das, S.L. Christian, S.B. Baylin, J.G. Herman, and D.H. Ledbetter. (1997) Methylation-specific PCR simplifies imprinting analysis. Nat. Genet. 16: 16-17. Pfeifer, G. P., S.D. Steigerwald, P.R. Mueller, B. Wold and A.D. Riggs. (1989) Genome Sequencing and Methylation Analysis by Ligation Mediated PCR. Science 246: 810-813. Riggs, A. D. and G.P. Pfeifer. (1992) X-chromosome inactivation and cell memory. Trends Genet. 8: 169-174. Antequera, F., J. Boyes and A.Bird. (1990) High levels of De Novo Methylation and Altered Chromatin Structure at CpG Islands in Cell Lines. Cell 62: 503-514. Herman, J.G., F. Latif, Y. Weng, M.I. Lerman, B. Zbar, S. Liu, D. Samid, D.S. Duan, J.R. Gnarra, W.M. Linehan and S.B. Baylin. (1994) Silencing of the VHL tumor-suppressor gene by DNA methylation in renal carcinoma. Proc. Natl. Acad. Sci. USA 91: 9700-9704. Merlo, A., J.G. Herman, L. Mao, D.J. Lee, E. Gabrielson, P.C. Burger, S.B. Baylin and D. Sidransky. (1995) 5' CpG island methylation is associated with transcriptional silencing of the tumour suppressor p16/CDKN2/MTS1 in human cancers. Nature Medicine 1: 686-692.
4.
5.
6. 7.
8.
9.
10. Herman, J.G., J.R. Graff, S. Myohanen, B.D. Nelkin and S.B. Baylin. (1996) Methylation-specific PCR: A novel assay for methylation status of CpG islands. Proc. Natl. Acad. Sci. USA 93: 9821-9826.
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Disclaimers
Takara is a trademark of Takara Biomedicals. The polymerase chain reaction ("PCR") is covered by one or more of the following U.S. Patents: nos. 4,683,202; 4,683,195; and 4,899,818 issued to Cetus Corporation and owned and licensed by Hoffmann-LaRoche Molecular Systems, Inc. Purchase of a Chemicon PCR-related product does not convey a license to use the PCR process covered by these patents. Purchasers of these products must obtain a license to use the PCR process before performing PCR. The CpG WIZTM Methylation Products apply technologies exclusively licensed from The Johns Hopkins University School of Medicine. Methylation-specific PCR (MSP) technology is covered by U.S. Patent # 5,786,146. All trademarks, unless otherwise noted, are the property of Serologicals Royalty Company or Chemicon International, Inc.
Warranty
These products are warranted to perform as described in their labeling and in CHEMICON literature when used in accordance with their instructions. THERE ARE NO WARRANTIES, WHICH EXTEND BEYOND THIS EXPRESSED WARRANTY AND CHEMICON DISCLAIMS ANY IMPLIED WARRANTY OF MERCHANTABILITY OR WARRANTY OF FITNESS FOR PARTICULAR PURPOSE. CHEMICON's sole obligation and purchaser's exclusive remedy for breach of this warranty shall be, at the option of CHEMICON, to repair or replace the products. In no event shall CHEMICON be liable for any proximate, incidental or consequential damages in connection with the products.
2003: CHEMICON International, Inc. - By CHEMICON International, Inc. All rights reserved. No part of these works may be reproduced in any form without permissions in writing.
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Cat No. S7800 August 2003 Revision A: 41423

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