Appeal No. 1997-4145
Application No. 08/361,328
BACKGROUND
The claimed invention is directed to methods for stretching DNA (claims 1-10)
and to intact stretched or "(super)extended" DNA (claims 26-29 and 33-40), which can
be combined with fluorescent hybridization procedures as a means for physically
mapping DNA sequences or gene clusters (specification, pp. 6 and 7).
The method provides extended and super-extended forms of DNA, the
latter stretched to a length beyond the calculated "unwound" length of
native DNA. By comparison, the DNA from typical "spreads" (Lehninger,
...) is two-dimensional in the sense of exhibiting two directions in a single
plane (including curves and winding). The form of DNA disclosed in the
present invention is virtually straight (linear), has no contour and little, if
any, randomness. It is thus visualized as extending in only one direction
in a single plane. The invention therefore provides a procedure to stretch
DNA into novel, linear, one-dimensional forms up to and beyond 0.34 :m
per kilobase pair. The DNA can be used as a target for hybridization of
labelled probes to visually observe a high resolution map of probes along
a single strand of DNA. Mapping resolutions as high as 1 kb are possible
with the extended DNA. Super-extended DNA allows even further
increases in resolution to at least 0.4 kb, although the extended form is
practical for many applications.
Extended DNA is DNA stretched to a substantially one-dimensional
form up to about 0.34 :m per kilobase pair. Super-extended DNA is an
essentially one-dimensional form stretched beyond what is commonly
held to be the maximal length of fully extended DNA (0.34 :m per
kilobase pair). [Specification, p. 6, ll. 12-35.]
The specification (p. 23, ll. 4-17) exemplifies stretching duplex DNA
by placing individual cells (100-5000) in two :l of PBS on one end of a
glass slide and letting the drop dry. Immediately after drying, 5 :l of 0.5%
SDS\50 mM EDTA\200 mM Tris, pH 7.4 solution was placed on the dried
spot to dissolve the cells and release the DNA. After 5 minutes of
dissolving, the slide was tilted to allow the drop of SDS and DNA to run
down the slide. This resulted in a DNA stream extending down the slide
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