Chemistry: molecular biology and microbiology – Treatment of micro-organisms or enzymes with electrical or... – Modification of viruses
Patent
1996-01-13
1999-11-02
Jones, W. Gary
Chemistry: molecular biology and microbiology
Treatment of micro-organisms or enzymes with electrical or...
Modification of viruses
435 6, 435 912, C12N 1500
Patent
active
059768460
ABSTRACT:
The subject invention relates to a method referred to as multifragment in vivo cloning (MFIVC). In the method, the polymerase chain reaction or the cleavage by restriction enzyme(s) are used to generate a series of double-stranded DNA fragments. Each fragment contains a region homologous to a portion of the fragment to which it is to be joined. These homologous regions undergo recombination in vivo following transformation into a host with efficient and precise homologous recombination (such as the yeast S. cerevisiae). A series is designed so that the last fragment in the series contains a region homologous to a portion of the first fragment in the series, thus forming a circular DNA molecule after recombination in vivo. A circular DNA molecule can be selected in vivo if the circular DNA molecule created contains both a suitable DNA replication origin and a suitable marker for genetic selection. A series may be designed so that the first and last fragment in the series contain telomeric sequence elements, forming a linear DNA molecule with telomeric sequence elements at its ends, after recombination in vivo. One preferred embodiment of this method includes a means for mapping a phenotypically expressed mutation within a gene. A second embodiment of this method includes a means for constructing plasmids using DNA cassettes. A third embodiment of this method includes a means of reasserting mutations in a double-stranded DNA molecule. The invention also includes kits containing reagents for conducting the method.
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Marykwas Donna L.
Passmore Steven E.
Jones W. Gary
Whisenant Ethan
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