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-construct target genomic DNA with really strong promoter with retrovirus recognition sequence (R U5 PBS etc to ensure that only this is reverse transcribed)<br> | -construct target genomic DNA with really strong promoter with retrovirus recognition sequence (R U5 PBS etc to ensure that only this is reverse transcribed)<br> | ||
-transcribe into RNA using a crap RNA pol that introduces mutations<br> | -transcribe into RNA using a crap RNA pol that introduces mutations<br> | ||
| - | -reverse transcribe into DNA using an overexpressed reverse transcriptase (overexpressed to ensure that this happens before DNA is degraded)<br> | + | -reverse transcribe into DNA using an overexpressed reverse transcriptase (overexpressed to ensure that this happens before DNA is degraded). The RT should be coupled to an inducible promoter to ensure that mutagenesis only takes place in bursts.<br> |
-insert into genome via homologous recombination using an equivalent of infusion enzyme (maybe RecA?)<br> | -insert into genome via homologous recombination using an equivalent of infusion enzyme (maybe RecA?)<br> | ||
</html> | </html> | ||
Revision as of 16:20, 7 July 2011
Rebekka Bauer
novel in vivo mutagenesis mechanism:
-construct target genomic DNA with really strong promoter with retrovirus recognition sequence (R U5 PBS etc to ensure that only this is reverse transcribed)
-transcribe into RNA using a crap RNA pol that introduces mutations
-reverse transcribe into DNA using an overexpressed reverse transcriptase (overexpressed to ensure that this happens before DNA is degraded). The RT should be coupled to an inducible promoter to ensure that mutagenesis only takes place in bursts.
-insert into genome via homologous recombination using an equivalent of infusion enzyme (maybe RecA?)
