Appeal No. 94-0120
Application 07/208,512
1. A method of isolating duplex DNA fragments which are present in a mixture of
different-sequence duplex DNA fragments derived from a positive source, but absent from
a mixture of different-sequence duplex DNA fragments derived from a negative source,
said method comprising:
attaching a double-strand linker to the positive-source fragments, and separately, to
the negative-source fragments, by ligating the linker to both strands of said positive-source
and negative-source fragments, at both ends of said positive-source and negative-source
fragments,
amplifying the number of each linker-carrying fragment in each fragment mixture by
successively repeating the steps of (I) denaturing the fragments to produce single fragment
strands with linker regions at each strand end, (ii) hybridizing the single strands with a
single-strand primer whose sequence is complementary to the linker region at one end of
each strand, to form a strand/primer complex, and (iii) converting the strand/primer
complexes to double-strand fragments in the presence of polymerase and
deoxynucleotides,
denaturing the amplified fragments in the two amplified fragment mixtures and
hybridizing the denatured fragments in the two mixtures under conditions in which the linker
regions associated with the positive-source strands do not hybridize with the linker regions
associated with the negative-source strands, and
selectively isolating DNA species which are not hybridized with DNA fragment
strands from the negative source.
12. A method of amplifying a mixture of different sequence duplex DNA fragments,
comprising
attaching a double-strand linker to the fragments, by ligating the linkers to both
strands of the fragments, at both fragment ends,
denaturing the fragments to produce single fragment strands with linker regions at
both strand ends,
hybridizing the single strands with a primer whose sequence is complementary to a
linker region on each fragment strand, to form strand/primer complexes,
2
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Last modified: November 3, 2007