Mark-recapture cloning: a straightforward and cost-effective cloning method for population genetics of single-copy nuclear DNA sequences in diploids
|Author(s)||Bierne Nicolas1, Tanguy A2, Faure Michel1, Faure B2, 3, David E4, Boutet I4, Boon E1, Quere N2, Plouviez S1, 2, Kemppainen P5, Jollivet D2, Moraga D4, Boudry Pierre3, David P6|
|Affiliation(s)||1 : Univ Montpellier 2, CNRS, IFREMER, UMR 5171,Stn Mediterraneenne Environm Littoral, F-34200 Sete, France.
2 : CNRS, UPMC, Equipe Evolut & Genet Populat Marines, UMR 7144,Stn Biol Roscoff, F-29682 Roscoff, France.
3 : IFREMER, Lab Genet & Pathol, F-17390 La Tremblade, France.
4 : Univ Bretagne Occidentale, Inst Univ Europeen Mer, CNRS, UMR 6539,Lab Sci Environm Marin, F-29280 Plouzane, France.
5 : Tjarno Marine Biol Lab, Dept Marine Ecol, S-45296 Stromstad, Sweden.
6 : CNRS, Ctr Ecol Fonct & Evolut, F-34293 Montpellier 5, France.
|Source||Molecular Ecology Notes (1471-8278) (Blackwell science), 2007-07 , Vol. 7 , N. 4 , P. 562-566|
|WOS© Times Cited||14|
|Keyword(s)||Sequence, Population genetics, High throughput allele recognition, DNA polymorphism|
|Abstract||We describe a simple protocol to reduce the number of cloning reactions of nuclear DNA sequences in population genetic studies of diploid organisms. Cloning is a necessary step to obtain correct haplotypes in such organisms, and, while traditional methods are efficient at cloning together many genes of a single individual, population geneticists rather need to clone the same locus in many individuals. Our method consists of marking individual sequences during the polymerase chain reaction (PCR) using 5'-tailed primers with small polynucleotide tags. PCR products are mixed together before the cloning reaction and clones are sequenced with universal plasmid primers. The individual from which a sequence comes from is identified by the tag sequences upstream of each initial primer. We called our protocol mark-recapture (MR) cloning. We present results from 57 experiments of MR cloning conducted in four distinct laboratories using nuclear loci of various lengths in different invertebrate species. Rate of capture (proportion of individuals for which one or more sequences were retrieved) and multiple capture (proportion of individuals for which two or more sequences were retrieved) empirically obtained are described. We estimated that MR cloning allowed reducing costs by up to 70% when compared to conventional individual-based cloning. However, we recommend to adjust the mark:recapture ratio in order to obtain multiple sequences from the same individual and circumvent inherent technical artefacts of PCR, cloning and sequencing. We argue that MR cloning is a valid and reliable high-throughput method, providing the number of sequences exceeds the number of individuals initially amplified.|