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cpg wiz? p16 amplification kit s7800 cpg wiz? p15 amplification kit S7802 cpg wiz? e-cadherin amplification kit s7804 cpg wiz? 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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i ____________________ ____________________ _________________ t able of c ontents i. i ntroduction .......................... .................... ................. ............... 1 using this manual ............ ................... .................... .................... .............. 1 background ............. ................. ................. ................. ................. .............. 1 principles of the techni que.............. .................... .................... ................. 2 fig 1: dna treatmen t with sod ium bisulfite ............. ............... ........... 3 ii. k it c omponents ................... ................... .................... ............... 4 materials required but not supplied . .................... .................... .............. 5 iii. p rotocols ................. .................. ................. ................. ............... 7 cpg wiz? p16 (s7800) , p15 (S7802) and e-cadherin (s 7804) amplificat ion kits ......... .............. ............... ........... 7 experimental design..... .................... .................... ................. .............. 7 fig. 2: specificity of the cpg wiz? p16 amplification kit ................. ................. ................. ................. .............. 9 amplification protocol.. .................... .................... ................. ............ 10 cpg wiz? prader-willi/angelman (s 7806) amplificati on kit ........... 12 experimental design..... .................... .................... ................. ............ 12 fig. 3: specificity of the cpg wiz? prader-willi/ angelman amplification kit ................. ................. ................. ............ 14 amplification protocol ................... .................... .................... ............ 15 iv. d ata a nalysis ................. ................. ............... ............... ........... 18 cpg wiz? p16 (s7800) , p15 (S7802) and e-cadherin (s7804) amplification ki ts ................ ................. ............ 18 cpg wiz? prader-willi/angelman (s 7806) amplificatio n kit ........... 19 v. t roubleshooting .................. .................. ................. ............... 20 cpg wiz? p16 (s7800) , p15 (S7802) and e-cadherin (s7804) amplification kits . .................... ................. ............ 20 cpg wiz? prader-willi/angelman (s 7806) amplificatio n kit ........... 21
ii vi. a ppendix ............................................................................ 23 laboratory setup and precautions ......... .................... ................. ............ 23 related products...... ................. ................. ................. ................. ............ 23 vii. r eferences .................. ................. ................. ............... ............. 24 references cited in this manual ......... .................... .................... ............ 24 disclaimers ......... .................... ................. ................. ................. ............ 25 warranty............. ................... .................... ................. ................. ............ 25
1 i. i ntroduction using this manual please read the entire instruction manual prior to using cpg wiz? amplification kits. note that in the pr ocedures and troubleshooting sections, instructions are separate for the cpg wiz? prader-willi/ angelman amplification kit (s7806). should additi onal questions aris e, assistance is available from chemicon technical se rvice 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 e ukaryotic genes (1). in normal cells, methylation occurs predominantly in cg-poor regions, while cg-rich areas, called cpg islands, remain unmeth ylated. 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) an d 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 transc riptional inactivation of defined tumor suppressor genes in human cancers (8, 9). e-cad herin, p16, and p15 are examples of genes that exhi bit characteristic hypermethylation. previously developed method s to determine the methyla tion 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 th eir recognition sequence and sequencing is time consuming. increasing the detection sensitivit y of cpg island methylation has the potent ial 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 de signed to distinguish methylated from unmethylated dna. the cpgenome? dna modification kit (s7820) contains the reagents necessary to perform the initial bisulfite re actions, while cpg wiz? amplification kits contain the reagents required for the pcr amplification reactions.
2 principles of the technique msp, performed using the cpgeno me? dna modification kit and cpg wiz? amplification kits, pe rmits sensitive detection of altered dna. due to the fact that it is a pcr-based assay, it is extremely sensit ive, facilitating the detection of low numbers of methylated alleles and the study of samples containing low amounts of dna. msp also allows exam ination of all cpg sites, not just those within sequ ences recognized by methylation sensitive restriction enzymes. increasing the number of su ch sites which can be assessed allows rapid, fine mapping of methylation patterns throughout cpg regions. in addition, the bisulfit e modification is idea lly suited for analys is of cpg islands since it converts the majority of cyto sines to uracils, making a region of the genome which is cg-rich less difficult to amplify by pcr. methylation-specific pcr employs an init ial bisulfite reactio n to modify the dna, followed by a "hot start" pcr amplification with specific primers designed to distinguish methylated dn a 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, th e sequence of the treated dna will differ if the dna is originally methylated vs. unmethylated. primers contained in cpg wiz? amplification kits are de signed to specifically amplify each of the sequences based upon these chem ically-induced differences. if the sample dna was originally unmethylated, a product will be generated after pcr using the u primer set. conver sely, a product will be generated using the m primer set if the sample was originally methylated.
3 figure 1: dna treatment with sodium bisulfite. unmethylated dna methylated dna
4 ii. k it c omponents the components of cpg wiz? amplificat ion kits include those required for pcr amplification after bisulfite modifi cation of dna samples. sufficient reagents are provided to analyze 25 samples with appropriate controls. table 1: cpg wiz? p16 (s7800), p1 5 (S7802) and e-cadherin (s7804) amplification kit components (color -coded microcentrifuge tube caps) description amount storage conditions u primer set 5 m each primer (25x) 35 l (white cap) -15c to -25c m primer set 5 m each primer (25x) 35 l (red cap) -15c to -25c w primer set 5 m each primer (25x) 35 l (green cap) -15c to -25c u control dna 0.1 g ? l 50 l (white cap) -15c to -25c m control dna 0.1 g/l 50 l (red cap) -15c to -25c w control dna 0.05 g/l 50 l (green cap) -15c to -25c universal 10 x pcr buffer 265 l (blue cap) -15c to -25c
5 table 2: cpg wiz? pr ader-willi/angelman (s7806) amplification kit components (color-coded microcentrifuge tubes) description amount storage conditions p (paternal) primer set 5 m each primer (25x) 70 l (white cap) -15c to -25c m (maternal) primer set 5 m each primer (25x) 70 l (red cap) -15c to -25c normal control dna 0.1 g/l 20 l (green cap) -15c to -25c 10x pcr buffer 175 l (blue cap) -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-will i/angelman amp lification 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 ).
6 d. reagents for gel electrophoresis (2% agarose, 10% acrylamide, or suitable high resolution agarose) e. dna markers (size range 100-300 bp) f. ethidium brom ide (10 mg/ml) g. gel-loading solution h. bisulfite modified dna (cpgen ome? dna modification kit, s7820)
7 iii. p rotocols cpg wiz? p16 (s7800), p15 (s 7802) and e-cadherin (s7804) amplification kits experimental design primer sets cpg wiz? amplification kits contain primers that can be used for analysis of dna samples by msp. however, the sa mples must first undergo bisulfite modification prior to pcr amplification. cpgenome ? dna modifi cation kit, s7820, contains the reagents necessary to perform the modification. chemical modification creates the sequence diff erences between the methylated and unmethylated dna. the primer sets in th e 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 dn a 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 methylat ed) that has not undergone chemical modification, hence, the "wild type", or w. data interpretation can still proceed in th e case of incomp lete chemical modification (up to 50%). amplification regions the amplified region is defi ned as the sequence between the 3' nucleotide of the sense primer and the compleme nt of the 3' nucleotide of the anti-sense primer for each gene promoter. the nucleotide nu mbering systems are those used in the genbank submissions identified by the following accession numbers: p16 , x94154; p15 , s75756; e-cadherin , l34545.
8 ?hot start? pcr the three sets of primers used in the cpg wiz? methylation assay are derived from sequences closely relate d to each other, which in troduces the possibility of mispriming. in order to avoid this and ot her pcr-related artifacts, "hot start" pcr is recommended. "hot start" pcr pe rmits the taq polyme rase to begin the reaction only after the te mplate and primers are in single-stranded form. there are several modifications of the st andard pcr protocol which allow a "hot start" to occur. in one scenario, the pcr reaction mixt ure excluding the polymerase can be overlaid with mineral oi l prior to heating to 95c. at the end of the incubation, the enzyme is pipett ed 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 wa x bead. the enzyme combines with the rest of the reaction mixtur e only after the wa x melts. in another variation, an anti-taq antibody inhibits the polymera se during reaction setup by forming a complex with the taq enzyme. taq po lymerase becomes active when the complex is abolished due to antibody denaturation duri ng 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 polymera se with 3'-5' exonuc lease activity (i.e. proofreading).do not use a wax bead that contains mg 2+ . any extra mg 2+ 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 prio r to pcr amplification using the cpgenome? dna modification kit (s7820). when used with their respective u and m primer sets, a single pcr product of an expect ed size is obtained in each case. w control genomic dna is used in th e 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.
9 specificity of the assay the specificity of the cpg wiz? p16 amplification ki t is shown in figure 2. with a complete chemical modificati on reaction, u primers amplify only unmethylated dna (154 bp, lane 1) an d m primers amplify only methylated dna (145 bp, lane 5). w primers amplif y only dna which is not chemically modified, or "wild type w" (142 bp, lane 9). figure 2: specificity of the cp g wiz? p16 ampl ification 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 pr imers; lanes 3, 6, 9, 12: w primers. m lane: 100 base-pair marker.) experiment setup cpg wiz? amplification kits (s7800, s7 802, and s7804) in clude sufficient reagents to analyze 25 samples with appr opriate controls (105 pcr reactions). each experiment will include chem ical modification of seven dnas: 5 experimental dna and 2 control dna samp les, (m and u). in the subsequent pcr reactions, each chemically modifi ed experimental dna sample is amplified with each of three oligonucleo tide primer sets u, m and w. the chemically modified genomic control d nas, 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 p cr control (i.e. no dna) is performed for each set of primers.
10 for example, a typical gel for the analys is of five experimental dna samples includes a total of 21 lanes: lanes 1-3 experime ntal sample 1 with u, m and w primers lanes 4-6 experime ntal sample 2 with u, m and w primers lanes 7-9 experime ntal sample 3 with u, m and w primers lanes 10-12 experimental sample 4 with u, m and w primers lanes 13-15 experimental sample 5 with u, m and w primers lane 16 chemically modified control u dna wi th u primers lane 17 chemically modified control m dna with m primers lane 18 untreated contro l w dna with w primers lanes 19-21 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 pr imer sets provid ed in cpg wiz? amplification kits, one microgram of purified dna must undergo bisulfite modification with the reagents cont ained in cpgenome? 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 fo r 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" whic h correspond to the 3 possible primer sets u, m and w (primer cap colors-w hite, red and green) by mixing all the reagents ou tlined below except for the template dna.
11 to analyze five experime ntal samples with approp riate controls, use the following amount of "master mixes" of each color: 7 tubes x 23.0 l = 161 l, plus 10% of that vol ume to adjust for pi petting error. mult iply the volume of each reagent listed below by 7 and add 10% for each "master mix" (white, red and green). thaw all reagents and stor e on ice while creating the "master mixes". the amount of reagents required in each reaction is: 10x universal pcr buffer 2.5 l 2.5 mm dntp mix** 2.5 l u, m or w primers (white, red and green) 1.0 l ? ? takara? taq or "hot start" enzyme (5 u/l) 0.2 l (1 unit) dh 2 o 16.8 l 23.0 l template dna (50 ng/l) 2.0 l total volume 25.0 l ** upon first use, make aliquots of 2.5 mm dntps, which should be freeze- thawed 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 (m odified u and m, un modified w) to each control tube. e. place tubes in the thermocycler bloc k, 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
12 f. remove the tubes from the thermocycler block. from this point on, it is important to designate separate pipettes a nd 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 reso lution 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 ca rcinogen. exercise appropriate caution and good lab practice when using this reagent. cpg wiz? prader-willi/angelman (s7806) amplification kit experimental design primer sets the cpg wiz? prader-wil li/angelman amplification kit contains primers that can be used for analys is of dna samples by msp. however, the first step of msp is the bisulfit e modification of the dna samples. cpgenome? 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 unmethylat ed dna that has undergone a chemical modification. m (maternal) primer set will anneal to methylated dna that has undergone a chemical modification.
13 amplified regions the p and m primer sets are designed to amplify a differentially methylated site present at the cpg island of the smal l nuclear ribonucleoprotein-associated polypeptide n (snrpn), a candidate gene for prader-willi syndrome. the amplified region is defined as the sequ ence between the 3' nucleotide of the sense primer and the compleme nt of the 3' nucleotide of the anti-sense primer for each primer pair. the nucleotide nu mbering 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 wiz? prader-willi/angelman amplification kit are derive d from sequences closely related to each other, which introduces the possibility of mispri ming. in order to av oid 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 bo th maternal and pa ternal homologs which means that both methylated and unmethylated snrpn sequences are represented. therefore, pcr products wi ll 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 genera te a 174 bp product while the p primer set produces a 100 bp frag ment. multiplex amplificatio n is feasible since the fragment sizes are suff iciently dissimilar. specificity of the assay the specificity of the cpg wiz? prader -willi/angelman amp lification kit is shown in figure 3. with a complete ch emical modification reaction, p primers amplify only unmethylated dna (100 bp , lane 1) and m primers amplify only methylated dna (174 bp, lane 2). the sm aller bands present in the lanes where the m primer set was used in the pcr reaction represent excess primers.
14 figure 3: specificity of the cpg wiz? 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 se t with modified control dna; lane 2: m primer set with modified control dna; l ane 3: p primer set with unmodified control dna; lane 4: m primer set with unmodified co ntrol dna. marker lane: 100 bp ladder) experiment setup the cpg wiz? prader-willi/angelman am plification kit includes sufficient reagents to analyze 25 samples with a ppropriate controls (70 pcr reactions). for each batch of experimental dna samp les to be analyzed, the experimental samples and the normal cont rol dna must first underg o bisulfite modification. in the subsequent pcr reactions, the ch emically 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 anal ysis of five experimental dna samples and proper controls includes a total of 14 lanes: lanes 1-2 experimental sample 1 with p and m primers lanes 3-4 experimental sample 2 with p and m primers lanes 5-6 experimental sample 3 with p and m primers
15 lanes 7-8 experimental sample 4 with p and m primers lanes 9-10 experimental sa mple 5 with p and m primers lane 11 chemically modified no rmal control dna with p primers lane 12 chemically modified no rmal control dna with m primers lanes 13-14 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 pr imer sets in the cpg wiz? prader- willi/angelman amplification kit, th e dna samples must undergo bisulfite modification. cpgenome? dna modifi cation 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 experi mental dna sample plus four control reactions per each set of methylation assays (see sec. iii. protocols, experimental design ). b. prepare two (2) "master mixes" whic h correspond to the 2 possible primer sets p and m (primer cap co lors-white and red) by mixing all the reagents outlined below except for the template dna. to analyze five experime ntal samples with approp riate controls, use the following amount of "master mixes" of each color: 7 tubes x 23.0 l = 161 l, plus 10% of that vol ume to adjust for pi petting error. mult iply 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".
16 the amount of reagents required in each reaction is: 10x universal pcr buffer 2.5 l 2.5 mm dntp mix** 2.0 l p or m primers (white, red) 2.0 l "hot start" enzyme (5 u/l) 0.1 l (0.5 u) dh 2 o 16.4 l 23.0 l template dna (50 ng/l) 2.0 l total volume 25.0 l ** upon first use, make aliquots of 2.5 mm dntps, which should be freeze- thawed 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 cont rol 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 bloc k, 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. fr om this point on, it is important to designa te separate pipettors and work areas for amplified vs. unamplified samples. this prevents carry-over contamination of future dna samples with the amplified product.
17 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 react ion on a 2% agarose or a 10% native acrylamide or other high resolution ag arose gel. use dna markers (100-300 bp range) to determine th e 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 dei onized water at room temperature. note: ethidium bromide is a known ca rcinogen. exercise appropriate caution and good lab practice when using this reagent.
18 iv. d ata a nalysis cpg wiz? p16 (s7800), p15 (s 7802) and e-cadherin (s7804) amplification kits table 3: sizes of expected products from controls controls p16 p15 e-cadherin u primer/u control dna 154 162 212 m primer set/m control dna 145 154 206 w primer set/w control dna 142 137 194 in the three no dna contro l lanes, no pcr products should be generated. table 4: sizes of expected products from samples experimental samples p16 p15 e-cadherin u primer set/ unmethylated dna 154 162 212 m primer set/methylated dna 145 154 206 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 produc t with an experime ntal sample, it is an indication of incomplete chemical modification.
19 cpg wiz? prader-willi/angelman (s7806) amplification kit table 5: sizes of ex pected products template primer set p m normal control dna 100 174 experimental methylated dna ? 174 experimental unmethylated dna 100 ? in the two no dna control lanes, no pcr products should be generated. in a clinical research sample obtained from a normal individu al, a 100 bp and a 174 bp fragment will be obtained when using the p primer se t and m primer set, respectively, in pcr reactions. msp perf ormed on a research sample from an individual with prader-wil li syndrome will produce th e 174 bp fragment with the m primer set but will generate no pro duct with the p primer set. conversely, a research sample from an individual wi th angelman syndrome will produce the 100 bp fragment with the p primer set, but will generate no product with the m primer set.
20 v. t roubleshooting cpg wiz? p16 (s7800), p15 (s 7802) 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 th e reaction tube. b. confirm that the time and temperature settings on the thermocycler match those described in this manual. c. if performing "hot star t" pcr using a "hot start" enzyme, verify the initial denaturation/activ ation time of 12 minutes at 95c. d. for all other "hot start" methods, co nfirm the proper use of the reagents. e. confirm that the pcr po lymerase is still active. f. confirm that no additional mg 2+ 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 c onditions to suit yo ur amplification instrument. no amplification prod uct 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 th an two months prior to pcr. recommendation: repeat the chemical modificati on on new genomic dna samples. u or m primer sets are producing bands in all samples, including the "no dna" controls. potential problem: pcr reagents are contaminated with amplification products. ? ? ?
21 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 fo r 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 amplif ication product in some or all experimental samples, in a ddition 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 lo ng 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 cpgenome? dna modifi cation kit (s7820), check the troubleshooting sect ion of the manual. cpg wiz? 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 p cr components were added to the reaction tube. b. confirm that the time and temperature settings on the thermocycler match those described in this manual. ? ? ?
22 c. if performing "hot start" pcr using a "hot start" enzyme, verify the initial denaturation/activation time of 12 minutes at 95c. d. confirm that the pcr po lymerase is still active. e. confirm that no additional mg 2+ was added to the pcr reaction mix. f. the optimal annealing temperature is 62c. if items #a-e have not remedied the problem, re-optimize annealing c onditions to suit yo ur amplification instrument. no amplification prod uct 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 re peat the chemical modification. potential problem #2: chemically modified experimental dna samples were stored for more th an two months prior to pcr. recommendation: repeat the chemical modificati on 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 fo r 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 clea n labcoat and gloves. ? ?
23 vi. a ppendix laboratory setup and precautions one of the most important considerations when performing msp using cpg wiz? amplification kits is the environmen t 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, tu be racks, no tebooks, lab coats and any other item expose d to amplified pcr products. the following precautions should be follow ed in all steps of the assay protocol: a. always wear gloves. b. use sterile water for all solutions, a liquot the solutions in small amounts, and use fresh aliquots as working solutions . discard working so lutions after use. c. keep the assay solutions (10x pc r 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 catalog number cpgenome? dna modifi cation kit s7820 cpgenome? universal methylated dna (human male genomic dna) s7821 dna extraction kit, non-organic s4520 ex-wax? dna ex traction kit for paraffin-embedded tissue s4530
24 vii. r eferences references cited in this manual 1. bird, a. (1992) the essentials of dna methylation. cell 70: 5-8. 2. li, e., c. beard and r. jaenisch. (1993) role for dna methylation in genomic imprinting. nature 366: 362-365. 3. tremblay, k.d., j.r. saam, r.s. ingram, s.m. tilghman and m.s. bartolomei. (1995) a paternal-speci fic methylation imprint marks the alleles of the mouse h19 gene. nat. genet. 9: 407-413. 4. kubota, t., s. das, s.l. christian, s.b. baylin , j.g. herman , and d.h. ledbetter. (1997) methylation-specific pcr simplifies impr inting analysis. nat. genet. 16: 16-17. 5. pfeifer, g. p., s.d. steigerwald, p. r. mueller, b. wold and a.d. riggs. (1989) genome sequenci ng and methylation analysis by ligation mediated pcr. science 246: 810-813. 6. riggs, a. d. and g.p. pfeifer. (19 92) x-chromosome inactivation and cell memory. trends genet. 8: 169-174. 7. antequera, f., j. boyes and a.bi rd. (1990) high levels of de novo methylation and altered chromatin stru cture at cpg islands in cell lines. cell 62: 503-514. 8. 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-supp ressor gene by dna methylation in renal carcinoma. proc. natl. acad. sci. usa 91: 9700-9704. 9. merlo, a., j.g. herman, l. mao, d.j. lee, e. gabr ielson, p.c. burger, s.b. baylin and d. sidransky. (1995) 5' cpg island methylation is associated with transcriptional silencing of th e tumour suppressor p16/cdkn2/mts1 in human cancers. nature medicine 1: 686-692. 10. herman, j.g., j.r. graff, s. myoha nen, b.d. nelkin and s.b. baylin. (1996) methylation-specific pcr: a no vel assay for methylation status of cpg islands. proc. natl. acad. sci. usa 93: 9821-9826.
25 disclaimers takara is a trademark of takara biomedicals. the polymerase chain reaction ("pcr") is covered by one or more of the following u.s. patent s: nos. 4,683,202; 4,683,19 5; and 4,899,818 issued to cetus corporation and owned and licens ed by hoffmann-laroche molecular systems, inc. purchase of a chemicon pcr-related pr oduct does no t convey a license to use the pcr pro cess covered by these patent s. purchasers of these products must obtain a license to use the pcr process befo re performing pcr. the cpg wiz? methylation products appl y technologies excl usively licensed from the johns hopkins univ ersity 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 accord ance with their instructions. there are no warranties, which extend beyond this expressed warranty and chemicon ? disclaims any implied warranty of merchan tability 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, in cidental or consequential damages in connection with the products. ? 2003: chemicon ? international, inc. - by chemicon ? international, inc. all rights reserved. no pa rt of these works may be reproduced in any form without permissions in writing.

cat no. s7800 august 2003 revision a: 41423

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