Methylated CpG Island Amplification
Protocol written by Minoru Toyota
2.Materials
2.1.MCA
Restriction enzymes SmaI,XmaI
T4 DNA ligase
Taq DNA polymerase
10X PCR reaction buffer:
670mM Tris-HCl,pH 8.8
40mM MgCl2
160 mM NH4 (SO4)2
100 mM b -Mercaptoethanol
1 mg/ml bovine serum albumin.
Tris-EDTA (TE)pH 8.0
DNA precipitation reagents:
Phenol/Chloroform pH 8-9
3M NaOAc (for general precipitation)
5M NH4 Oac (for precipitation and quantitation when dNTPs are present)
100% ETOH
Agarose gel electrophoresis reagents
Filter hybridization reagents:
96 pin replicator system (Nunc)
Nylon membranes
DNA hybridization solution (e.g.BLOTTO)
Random-primed DNA labeling kit
Wash solutions (Wash1 2xSSC,0.1%SDS; Wash2 0.1XSSC,0.1%SDS)
2.2.RDA and Cloning PCR Products
3 X EE buffer : 30 mM EPPS (SIGMA)pH 8.0,3 mM EDTA pH 8.0.
5 M NaCl
cDNA spun column (Amersham)
Mung bean nuclease (NEB)
pBluescript (Stratagene)
Methylated CpG Island Amplification
Protocol written by Minoru Toyota
2.Materials
2.1.MCA
Restriction enzymes SmaI,XmaI
T4 DNA ligase
Taq DNA polymerase
10X PCR reaction buffer:
670mM Tris-HCl,pH 8.8
40mM MgCl2
160 mM NH4 (SO4)2
100 mM b -Mercaptoethanol
1 mg/ml bovine serum albumin.
Tris-EDTA (TE)pH 8.0
DNA precipitation reagents:
Phenol/Chloroform pH 8-9
3M NaOAc (for general precipitation)
5M NH4 Oac (for precipitation and quantitation when dNTPs are present)
100% ETOH
Agarose gel electrophoresis reagents
Filter hybridization reagents:
96 pin replicator system (Nunc)
Nylon membranes
DNA hybridization solution (e.g.BLOTTO)
Random-primed DNA labeling kit
Wash solutions (Wash1 2xSSC,0.1%SDS; Wash2 0.1XSSC,0.1%SDS)
2.2.RDA and Cloning PCR Products
3 X EE buffer : 30 mM EPPS (SIGMA)pH 8.0,3 mM EDTA pH 8.0.
5 M NaCl
cDNA spun column (Amersham)
Mung bean nuclease (NEB)
pBluescript (Stratagene)
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2.3 Oligonucleotides
RXMA Primers
RXMA24 : 5’-AGCACTCTCCAGCCTCTCACCGAC-3’
RXMA12 : 5’-CCGGGTCGGTGA-3’
JXMA24 : 5’-ACCGACGTCGACTATCCATGAACC-3’
JXMA12 : 5’-CCGGGGTTCATG-3’
NXMA24 : 5’-AGGCAACTGTGCTATCCGAGTGAC-3’
NXMA12 : 5’-CCGGGTCACTCG-3’
RMCA Primers
RMCA24 : 5’-CCACCGCCATCCGAGCCTTTCTGC-3’
RMCA12 : 5’-CCGGGCAGAAAG-3’
JMCA24 : 5’-GTGAGGGTCGGATCTGGCTGGCTC-3’
JMCA12 : 5’-CCGGGAGCCAGC-3’
NMCA24 : 5’-GTTAGCGGACACAGGGCGGGTCAC-3’
NMCA12 : 5’-CCGGGTGACCCG-3’
3.Methods
3.1.Preparation of MCA Amplicons
3.1.1 Digestion of Genomic DNA
Digest 5 m g of genomic DNA using 100 units of SmaI over night.
Add 20 units of XmaI and incubate at 37℃ for 6 hours.
Add one volume PC9,vortex,spin and extract the supernatant.
Precipitate the DNA: Add 1/10th volume 3M NaOAc and 2 volumes 100% ETOH.Store at
3.1.3 PCR Amplification
Prepare tubes containing 10 ml of 10 X PCR buffer,100 pmol of RXMA24 (or RMCA24)primers,15 Units of Taq DNA polymerase,1.2 ml dNTP mix (25mM),0 ml (RXMA)or 5 ml (RMCA)DMSO,H2O to a total volume of 97 ml.
Add 3 ml of ligation mixture.Cover with mineral oil.
To fill the 3’-recessed ends of the ligated fragments,incubate at 72℃ for 5 min.
Perform 25 cycles of PCR (95℃ for 1 min and 72℃ (for RXMA24)or 77℃ (for RMCA24)for 3min),with a final extension time of 10 min.
After the reaction,electrophorese 10 ml of the PCR products in a 1.5% agarose gel to check the quality of the amplification.You should see a relatively strong smear,ranging from 300 bp to 2 kb.
3.2.Detection of Aberrant Methylation by Dot Blot Analysis
3.2.1 Preparation of Filters
Transfer the PCR products to a new tube and add 2/3 volume of 5M NH4OAc and 350 ml of 100% ethanol.
Chill at
3.3 MCA Coupled with RDA
3.3.1 Outline
For detection of differentially methylated sequences,you need to generate MCA amplicons from the tester samples (e.g.,a cancer sample)and relatively large quantities of MCA amplicons from the driver samples (e.g.,DNA from normal tissues)with the adaptors removed.The tester’s adaptors will then be changed and the DNA hybridized with driver DNA,followed by PCR amplification using the second set of adaptors.The subtraction is then repeated once,and the resulting amplicons are further cloned and characterized.
3.3.2 Removal of Adaptors from the Driver Amplicon
Perform MCA on multiple aliquots of driver DNA.We typically run 10 reactions in parallel.Verify and pool the aliquots.Quantitate in a spectrophotometer.
Digest the driver MCA amplicons using 2 Units/m g SmaI to remove the RMCA/RXMA adaptor.Inactivate SmaI by phenol/chloroform extraction.
Remove the adaptors using a cDNA Spun column (Amersham).
Electrophorese an aliquot of DNA before and after column filtration to check for complete elimination of adaptors.
Add 1/30th volume of 3M Sodium acetate,two volumes of ethanol,chill at
3.3.5 Selective Amplification
Heat 100 ml of 1 M NaCl at 67℃ for 5 min.,and add 45 ml to the competitive hybridization solution.
Prepare PCR mixture as follows: 10 ml of 10X PCR buffer,5 ml (1/10th)of the hybridization mix,1.2 ml of 25 mM dNTP mix,100 pmol JXMA24 (or JMCA24)primers,15 Units of Taq DNA polymerase,0 ml (RXMA)or 5 ml (RMCA)DMSO,H2O to a total volume of 100 ml.Cover with mineral oil.
Fill the ends at 72℃ for 5 min.PCR amplification is then done at 10 cycles of 95℃ for 1 min and 72℃ (JXMA24)or 77℃ (JMCA24)for 3 min.Final extension is at 72℃ for 10 min.
Transfer the PCR products to a clean microcentrifuge tube.
To digest the single stranded amplified MCA products,add 10 ml of 10X Mung bean nuclease buffer and 100 units of Mung bean nuclease,and incubate at 30℃ for 30 min.
Purify DNA with phenol/chloroform extraction,add 2/3 volume of NH4 OAc,and two volumes of ethanol,chill at
