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Isolation of rare recombinants without using selectable markers for one-step seamless BAC mutagenesis.
- Source :
-
Nature methods [Nat Methods] 2014 Sep; Vol. 11 (9), pp. 966-970. Date of Electronic Publication: 2014 Jul 13. - Publication Year :
- 2014
-
Abstract
- Current methods to isolate rare (1:10,000-1:100,000) bacterial artificial chromosome (BAC) recombinants require selectable markers. For seamless BAC mutagenesis, selectable markers need to be removed after isolation of recombinants through counterselection. Here we illustrate founder principle-driven enrichment (FPE), a simple method to rapidly isolate rare recombinants without using selectable markers, allowing one-step seamless BAC mutagenesis. As proof of principle, we isolated 1:100,000 seamless fluorescent protein-modified Nodal BACs and confirmed BAC functionality by generating fluorescent reporter mice. We also isolated small indel P1 phage-derived artificial chromosome (PAC) and BAC recombinants. Statistical analysis revealed that 1:100,000 recombinants can be isolated with <40 PCRs, and we developed a web-based calculator to optimize FPE.
Details
- Language :
- English
- ISSN :
- 1548-7105
- Volume :
- 11
- Issue :
- 9
- Database :
- MEDLINE
- Journal :
- Nature methods
- Publication Type :
- Academic Journal
- Accession number :
- 25028895
- Full Text :
- https://doi.org/10.1038/nmeth.3030