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Nucleic Acids Research Advance Access originally published online on January 21, 2008
Nucleic Acids Research 2008 36(3):e19; doi:10.1093/nar/gkm1159
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Nucleic Acids Research, 2008, Vol. 36, No. 3 e19
© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Methods Online

PAP-LMPCR for improved, allele-specific footprinting and automated chromatin fine structure analysis

R. Ingram1, C. Gao2, J. LeBon2, Q. Liu3, R. J. Mayoral1, S. S. Sommer3, M. Hoogenkamp1, A. D. Riggs2 and C. Bonifer1,*

1Section of Experimental Haematology, University of Leeds, St James's University Hospital, Leeds LS9 7TF, UK, 2Biology Division, Beckman Research Institute of City of Hope and 3Department of Molecular Genetics and Department of Molecular Diagnosis, City of Hope National Medical Center, Duarte, CA 91010, USA

*To whom correspondence should be addressed. Tel: +44 113 3438525; Fax: +44 113 3438502; Email: c.bonifer{at}leeds.ac.uk

Received November 1, 2007. Revised December 4, 2007. Accepted December 14, 2007.

The analysis of chromatin fine structure and transcription factor occupancy of differentially expressed genes by in vivo footprinting and ligation-mediated-PCR (LMPCR) is a powerful tool to understand the impact of chromatin on gene expression. However, as with all PCR-based techniques, the accuracy of the experiments has often been reduced by sequence similarities and the presence of GC-rich or repeat sequences, and some sequences are completely refractory to analysis. Here we describe a novel method, pyrophosphorolysis activated polymerization LMPCR or PAP-LMPCR, which is capable of generating accurate and reproducible footprints specific for individual alleles and can read through sequences previously not accessible for analysis. In addition, we have adapted this technique for automation, thus enabling the simultaneous and rapid analysis of chromatin structure at many different genes.


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