Mark-recapture cloning: a straightforward and cost-effective cloning method for population genetics of single-copy nuclear DNA sequences in diploids
2007
Bierne, Nicolas | Tanguy, Arnaud | Faure, Matthieu | Faure, Jean-Baptiste | David, Elise | Boutet, Isabelle | Boon, E. | Quéré, Nolwenn | Plouviez, Sophie | Kemppainen, P. | Jollivet, Didier | Moraga, Dario | Boudry, Pierre | David, Patrice | Génome, populations, interactions, adaptation (GPIA) ; Université Montpellier 2 - Sciences et Techniques (UM2)-Institut Français de Recherche pour l'Exploitation de la Mer (IFREMER)-Centre National de la Recherche Scientifique (CNRS) | Adaptation et diversité en milieu marin (AD2M) ; Station biologique de Roscoff [Roscoff] (SBR) ; Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS) | Unité Amélioration génétique, Santé animale et Environnement (AGSAE) ; Institut Français de Recherche pour l'Exploitation de la Mer (IFREMER) | Laboratoire des Sciences de l'Environnement Marin (LEMAR) (LEMAR) ; Institut de Recherche pour le Développement (IRD)-Institut Français de Recherche pour l'Exploitation de la Mer (IFREMER)-Université de Brest (UBO)-Centre National de la Recherche Scientifique (CNRS) | Department of Marine Ecology ; Tjarno Marine Biological Laboratory | Centre d’Ecologie Fonctionnelle et Evolutive (CEFE) ; Université Paul-Valéry - Montpellier 3 (UPVM)-Institut National de la Recherche Agronomique (INRA)-Centre international d'études supérieures en sciences agronomiques (Montpellier SupAgro)-École Pratique des Hautes Études (EPHE) ; Université Paris Sciences et Lettres (PSL)-Université Paris Sciences et Lettres (PSL)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche pour le Développement (IRD [Occitanie])-Institut national d’études supérieures agronomiques de Montpellier (Montpellier SupAgro)
International audience
اظهر المزيد [+] اقل [-]إنجليزي. 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.
اظهر المزيد [+] اقل [-]الكلمات المفتاحية الخاصة بالمكنز الزراعي (أجروفوك)
المعلومات البيبليوغرافية
تم تزويد هذا السجل من قبل Institut national de la recherche agronomique