Sensitive and selective amplification of methylated DNA sequences using helper-dependent chain reaction in combination with a methylation-dependent restriction enzyme
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Date
2012-09-10
Authors
Rand, Keith N
Young, Graeme Paul
Ho, Thu
Molloy, Peter L
Journal Title
Journal ISSN
Volume Title
Publisher
Oxford University Press
Rights
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Rights Holder
Authors retain copyright.
Abstract
We have developed a novel technique for specific
amplification of rare methylated DNA fragments in
a high background of unmethylated sequences that
avoids the need of bisulphite conversion. The
methylation-dependent restriction enzyme GlaI is
used to selectively cut methylated DNA. Then
targeted fragments are tagged using specially
designed ‘helper’ oligonucleotides that are also
used to maintain selection in subsequent amplification
cycles in a process called ‘helper-dependent
chain reaction’. The process uses disabled primers
called ‘drivers’ that can only prime on each cycle if
the helpers recognize specific sequences within
the target amplicon. In this way, selection for the
sequence of interest is maintained throughout the
amplification, preventing amplification of unwanted
sequences. Here we show how the method can be
applied to methylated Septin 9, a promising biomarker
for early diagnosis of colorectal cancer.
The GlaI digestion and subsequent amplification
can all be done in a single tube. A detection sensitivity
of 0.1% methylated DNA in a background of
unmethylated DNA was achieved, which was
similar to the well-established Heavy Methyl
method that requires bisulphite-treated DNA.
Description
Keywords
Public health, Cancer research, Colorectal cancer
Citation
Rand, K.N., Young, G.P., Ho, T. and Molloy, P.L., 2012. Sensitive and selective amplification of methylated DNA sequences using helper-dependent chain reaction in combination with a methylation-dependent restriction enzyme. Nucleic Acids Research, 41(1), e15.