In vivo construction of recombinant molecules within the Caenorhabditis elegans germ line using short regions of terminal homology.
Homologous recombination provides a means for the in vivo construction of recombinant DNA molecules that may be problematic to assemble in vitro. We have investigated the efficiency of recombination within the Caenorhabditis elegans germ line as a function of the length of homology between recombining molecules. Our findings indicate that recombination can occur between molecules that share only 10 bp of terminal homology, and that 25 bp is sufficient to mediate relatively high levels of recombination. Recombination occurs with lower efficiency when the location of the homologous segment is subterminal or internal. As in yeast, recombination can also be mediated by either single- or double-stranded bridging oligonucleotides. We find that ligation between cohesive ends is highly efficient and does not require that the ends be phosphorylated; furthermore, precise intermolecular ligation between injected molecules that have blunt ends can also occur within the germ line.
Kemp
Benedict J
BJ
Department of Biological Sciences, Dartmouth College, Hanover, NH 03755, USA.
Hatzold
Julia
J
Sternick
Laura A
LA
Cornman-Homonoff
Joshua
J
Whitaker
Jessica M
JM
Tieu
Pamela J
PJ
Lambie
Eric J
EJ
D000818:Animals; D017173:Caenorhabditis elegans / Q000235:genetics*; D004270:DNA, Circular / Q000737:chemistry; D004274:DNA, Recombinant / Q000737:chemistry*; D004742:Enhancer Elements, Genetic; D005818:Genetic Engineering / Q000379:methods; D005854:Germ Cells; D049452:Green Fluorescent Proteins / Q000235:genetics; D009841:Oligonucleotides / Q000737:chemistry; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011995:Recombination, Genetic; D012689:Sequence Homology, Nucleic Acid
D004270:DNA, Circular; D004274:DNA, Recombinant; D009841:Oligonucleotides; D049452:Green Fluorescent Proteins