Appeal No. 2006-2851
Application No. 09/844,501
follows.
In another embodiment, cellular chromatin is subjected to limited nuclease
action, and fragments having one end defined by nuclease cleavage are
preferentially cloned. For example, isolated chromatin or permeabilized
nuclei are exposed to low concentrations of DNase I, optionally for short
periods of time (e.g., one minute) and/or at reduced temperature (e.g.,
lower than 37°C). DNase-treated chromatin is then deproteinized and the
resulting DNA is digested to completion with a restriction enzyme,
preferably one having a four-nucleotide recognition sequence. …
Preferential cloning of nuclease-generated fragments is accomplished by
a number of methods. For example, prior to restriction enzyme digestion,
nuclease-generated ends can be rendered blunt-ended by appropriate
nuclease and/or polymerase treatment (e.g., T4 DNA polymerase plus the
4 dNTPs). Following restriction digestion, fragments are cloned into a
vector that has been cleaved to generate a blunt end and an end that is
compatible with that produced by the restriction enzyme used to digest the
nuclease treated chromatin. ... Ligation of adapter oligonucleotides, to
nuclease-generated ends and/or restriction enzyme-generated ends, can
also be used to assist in the preferential cloning of fragments containing a
nuclease-generated end. For example, a library of accessible sequences
is obtained by selective cloning of fragments having one blunt end
(corresponding to a site of nuclease action in an accessible region) and
one cohesive end …
In the method of claim 123, it is only after the DNA fragments have been
ligated to a vector molecule that the polynucleotide of interest is selected. See,
Example 15, specification, page 114, lines 6-14, wherein E.coli colonies
harboring insert-containing plasmids were identified and screened.
While both appellants and the examiner rely heavily on argument with respect to
potential limitations within the preamble the claims, we do not find it necessary to reach
this issue to decide the case before us. For the reasons discussed herein, we do not
find the examiner has provided sufficient evidence to support a prima facie case of
obviousness. The rejection of the claims over Grosveld is reversed.
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