PAP-LMPCR for improved, allele-specific footprinting and automated chromatin fine structure analysis.
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.
Ingram
R
R
Section of Experimental Haematology, University of Leeds, St James's University Hospital, Leeds LS9 7TF, UK.
Gao
C
C
Lebon
J
J
Liu
Q
Q
Mayoral
R J
RJ
Sommer
S S
SS
Hoogenkamp
M
M
Riggs
A D
AD
Bonifer
C
C
D000483:Alleles; D000818:Animals; D001483:Base Sequence; D002843:Chromatin / Q000737:chemistry* / Q000378:metabolism; D018983:DNA Footprinting / Q000379:methods*; D011756:Diphosphates / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D041681:NIH 3T3 Cells; D016133:Polymerase Chain Reaction / Q000379:methods*; D011401:Promoter Regions, Genetic; D016186:Receptor, Macrophage Colony-Stimulating Factor / Q000235:genetics; D012091:Repetitive Sequences, Nucleic Acid; D012371:Robotics; D014157:Transcription Factors / Q000378:metabolism*
D002843:Chromatin; D011756:Diphosphates; D014157:Transcription Factors; D016186:Receptor, Macrophage Colony-Stimulating Factor