Seeing genetic and epigenetic information without DNA denaturation using sequence-enabled reassembly (SEER)

Methods Mol Biol. 2010;649:365-82. doi: 10.1007/978-1-60761-753-2_23.

Abstract

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.

MeSH terms

  • DNA Methylation / genetics
  • Epigenesis, Genetic / genetics*
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • Models, Biological
  • Nucleic Acid Denaturation / genetics
  • Protein Engineering / methods
  • Zinc Fingers / genetics*
  • beta-Lactamases / genetics
  • beta-Lactamases / metabolism

Substances

  • Green Fluorescent Proteins
  • beta-Lactamases