Seeing genetic and epigenetic information without DNA denaturation using sequence-enabled reassembly (SEER).
Virtually all methods for reading the sequence of bases in DNA rely on the ability to denature double-stranded DNA into single strands and then use Watson-Crick base-pairing rules to hybridize the strands with high specificity to another DNA primer or probe. However, nature frequently uses an alternative method, reading the sequence information directly from double-stranded DNA using sequence-specific DNA-binding proteins. Here we describe methods for the construction and testing of sequence probes based on engineered zinc finger DNA-binding proteins. Background is reduced using split-reporter molecules, and signal is amplified using enzymatic reporters. The resulting sequence-enabled reassembly (SEER) probes can be configured to detect DNA sequence (genetic) or DNA methylation (epigenetic) information.
Porter
Jason R
JR
Department of Chemistry, University of Arizona, Tucson, AZ, USA.
Lockwood
Sarah H
SH
Segal
David J
DJ
Ghosh
Indraneel
I
D019175:DNA Methylation / Q000235:genetics; D044127:Epigenesis, Genetic / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008954:Models, Biological; D009691:Nucleic Acid Denaturation / Q000235:genetics; D015202:Protein Engineering / Q000379:methods; D016335:Zinc Fingers / Q000235:genetics*; D001618:beta-Lactamases / Q000235:genetics / Q000378:metabolism
D049452:Green Fluorescent Proteins; D001618:beta-Lactamases