| 摘要 |
Methods and compositions for producing single-stranded cDNA (ss-cDNA) in eukaryotic cells, specifically, a DNA cassette that produces ss-cDNA in vivo. The cassette contains the Moloney murine leukemia virus reverse transcriptase/RNAse H coding gene, a bacterial restriction endonuclease gene, and a sequence of interest which produces an RNA template from which the reverse transcriptase synthesizes ss-cDNA of a specified sequence. The ss-cDNA is then modified to remove all flanking vector sequences by taking advantage of the "stem-loop" structure of the ss-cDNA, which forms as a result of the inclusion of an inverted tandem repeat that allows the ss-cDNA to fold back on itself, forming a double stranded DNA stem, in the sequence of interest. The double-stranded stem contains one or more functional genetic elements such as a restriction endonuclease recognition site and the loop, which remains as ss-DNA, is comprised of any desired nucleotide sequence. This design allows the double-stranded stem of the stem-loop intermediate to be cleaved by the desired corresponding restriction endonuclease(s) specific for the site in the stem and the loop portion, or sequence of interest, is then relased as a linearized, single-stranded piece of DNA. This released (or cleaved) ss-DNA piece contains minimal, if any, sequence information either upstream 5' or downstream 3' from the previous double stranded stem portion which contains the restriction endonuclease cut site. In vivo tranfections using the DNA vector system described herein demonstrate the use of this system to produce ss-DNA in host cells.
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