Synthesis of universal unmethylated control DNA by nested whole genome amplification with φ29 DNA polymerase

Naoyuki Umetani, Michiel F G De Maat, Takuji Mori, Hiroya Takeuchi, Dave S B Hoon

Research output: Contribution to journalArticle

32 Citations (Scopus)

Abstract

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 φ29 DNA polymerase. Contamination of the template genomic DNA in UUC was only 3.1 × 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 φ29 WGA achieves highly accurate and homogeneous amplification with very low amounts of genomic DNA as an initial template. The UUC DNA created by nested φ29 WGA is practically very useful for methylation analysis.

Original languageEnglish
Pages (from-to)219-223
Number of pages5
JournalBiochemical and Biophysical Research Communications
Volume329
Issue number1
DOIs
Publication statusPublished - 2005 Apr 1
Externally publishedYes

Fingerprint

DNA-Directed DNA Polymerase
Amplification
Genes
Genome
Methylation
DNA
Cytosine
CpG Islands
Genetic Promoter Regions
Microsatellite Repeats
Limit of Detection
Contamination
Polymerase Chain Reaction

Keywords

  • φ29 DNA polymerase
  • Bisulfite modification
  • Methylation analysis
  • Universal unmethylated control
  • Whole genome amplification

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

Synthesis of universal unmethylated control DNA by nested whole genome amplification with φ29 DNA polymerase. / Umetani, Naoyuki; De Maat, Michiel F G; Mori, Takuji; Takeuchi, Hiroya; Hoon, Dave S B.

In: Biochemical and Biophysical Research Communications, Vol. 329, No. 1, 01.04.2005, p. 219-223.

Research output: Contribution to journalArticle

Umetani, Naoyuki ; De Maat, Michiel F G ; Mori, Takuji ; Takeuchi, Hiroya ; Hoon, Dave S B. / Synthesis of universal unmethylated control DNA by nested whole genome amplification with φ29 DNA polymerase. In: Biochemical and Biophysical Research Communications. 2005 ; Vol. 329, No. 1. pp. 219-223.
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