Patent Number: 6,165,726

Title: Non-radioactive methods for chemical cleavage sequencing and footprinting of nucleic acids

Abstract: A method of sequencing a nucleic acid is provided, comprising: a) labeling the nucleic acid with biotin; b) subjecting the nucleic acid of step a) to base specific chemical modifications; c) cleaving the nucleic acid of step b) at the modified bases with hot piperidine; d) contacting the nucleic acid of step c) with immobilized streptavidin, whereby biotin-containing fragments are bound to immobilized streptavidin and unbound fragments are washed off; e) extracting the streptavidin bound fragments of step d); f) resolving the fragments of step e) on a denaturing polyacrylamide gel; and g) staining the fragments in the gel of step f) by silver staining. A further method of sequencing a nucleic acid is provided, comprising a) labeling the nucleic acid with DMT (dimethyl trityl protective group); b) subjecting the nucleic acid of step a) to base specific chemical modifications; c) cleaving the nucleic acid of step b) at the modified bases with hot piperidine; d) contacting the nucleic acid of step c) with an OPC (Oligo Purification Column) whereby DMT-containing fragments are bound to the OPC and unbound fragments are washed off; e) eluting the fragments that bound to the OPC in step d); f) resolving the fragments of step e) on a denaturing polyacrylamide gel; and g) staining the fragments in the gel of step f) by silver staining. Methods for DNAse I footprinting, footprinting with Exonuclease III, hydroxyl radical footprinting, methylation protection and methylation interference studies and ethylation protection studies are provided. Kits containing some or all of the components needed to practice one or more steps of the present method are provided.

Inventors: Iyyalasomayazula; Narayana Rao (Oak Ridge, TN)

Assignee: UT-Battelle, LLC

International Classification: C12Q 1/68 (20060101); C12Q 001/68 ()

Expiration Date: 12/26/2017