Synthesis of universal unmethylated control DNA by nested whole genome amplification with phi29 DNA polymerase.
Optimization of highly sensitive methods to detect methylation of CpG islands in gene promoter regions requires adequate methylated and unmethylated control DNA. Whereas universal methylated control DNA is available, universal unmethylated control (UUC) DNA has not been made because demethylase is not available to remove methyl groups from all methylated cytosines. On the basis that DNA synthesized by DNA polymerase does not contain methylated cytosines, we developed a method to create UUC DNA by nested whole genome amplification (WGA) with phi29 DNA polymerase. Contamination of the template genomic DNA in UUC was only 3.1 x 10(-7), below the detection limit of sensitive methods used for methylation studies such as methylation-specific PCR. Assessment of microsatellite markers demonstrated that even nested phi29 WGA achieves highly accurate and homogeneous amplification with very low amounts of genomic DNA as an initial template. The UUC DNA created by nested phi29 WGA is practically very useful for methylation analysis.
Umetani
Naoyuki
N
Department of Molecular Oncology, Martin H. Well Laboratory, John Wayne Cancer Institute, Santa Monica, CA 90404, USA.
de Maat
Michiel F G
MF
Mori
Takuji
T
Takeuchi
Hiroya
H
Hoon
Dave S B
DS
D017103:Bacillus Phages / Q000201:enzymology*; D001483:Base Sequence; D018899:CpG Islands; D003596:Cytosine / Q000378:metabolism; D019175:DNA Methylation; D015257:DNA-Cytosine Methylases / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry*; D015894:Genome, Human; D006801:Humans; D008214:Lymphocytes / Q000378:metabolism; D018895:Microsatellite Repeats; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D020033:Protein Isoforms; D017422:Sequence Analysis, DNA / Q000379:methods*; D013447:Sulfites / Q000494:pharmacology
D020033:Protein Isoforms; D013447:Sulfites; D003596:Cytosine; C081573:DNA modification methylase SssI; D015257:DNA-Cytosine Methylases; D004259:DNA-Directed DNA Polymerase