US 6,982,145 B1
Isolation and identification of control sequences and genes modulated by transcription factors
Daniel Mercola, P.O. Box 3752, Rancho Santa Fe, Calif. 92067 (US); Eileen Adamson, P.O. Box 3752, Rancho Santa Fe, Calif. 92067 (US); and Ian de Belle, 3158 Via Alicante, Apt. D, La Jolla, Calif. 92037 (US)
Filed on Dec. 20, 2001, as Appl. No. 10/32,260.
Application 10/032260 is a continuation in part of application No. 09/270391, filed on Mar. 16, 1999, granted, now 6,410,233.
This patent is subject to a terminal disclaimer.
Int. Cl. C12Q 1/68 (2006.01)
U.S. Cl. 435—6 10 Claims
 
1. A method for identifying one or more cDNA molecules that correspond to one or more genes regulated by a transcription factor, comprising:
a) cross-linking at least one transcription factor to at least one nucleic acid molecule in at least one cell or at least one nucleus, forming one or more transcription factor-nucleic acid molecule complexes;
b) fragmenting said at least one nucleic acid molecule to form one or more transcription factor-nucleic acid molecule fragment complexes;
c) isolating one or more nucleic acid molecule fragments from said one or more transcription factor-nucleic acid molecule fragment complexes to form one or more isolated nucleic acid molecule fragments;
d) combining said one or more isolated nucleic acid molecule fragments with either:
1) a cDNA library, or
2) cDNA obtained by reverse transcription of a population of RNA molecules, to form a mixture comprising isolated nucleic acid molecule fragment/cDNA complexes; and
e) amplifying one or more cDNAs that binds with said one or more isolated nucleic acid molecule fragments using said one or more nucleic acid molecule fragments as primers to obtain one or more isolated cDNA molecules, said one or more isolated cDNA molecules comprising at least a portion of a gene operably linked to or in close proximity to a nucleic acid sequence that binds with at least one transcription factor; and
f) identifying said one or more cDNAs by either:
1) sequencing said one or more cDNAs and comparing said sequence to the sequences of DNA molecules of known sequence, or
2) hybridizing said one or more cDNAs to one or more nucleic acid molecules corresponding to known genes or nucleic acid sequences.