Mark-recapture cloning: a straightforward and cost-effective cloning method for population genetics of single-copy nuclear DNA sequences in diploids
Mark-recapture cloning: a straightforward and cost-effective cloning method for population genetics of single-copy nuclear DNA sequences in diploids
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DOI：
10.1111/j.1471-8286.2007.01685.x
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发表时间：
2007-07-01
期刊：
影响因子：
--
通讯作者：
David, P.
中科院分区：
文献类型：
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作者：
Bierne, N.;Tanguy, A.;David, P.
我们描述了一个简单的协议，以减少二倍体生物群体遗传学研究中的核DNA序列的克隆反应的数量。克隆是在这些生物体中获得正确单倍型的必要步骤，虽然传统方法在将单个个体的许多基因克隆在一起时是有效的，但群体遗传学家需要在许多个体中克隆相同的基因座。我们的方法包括在聚合酶链反应（PCR）过程中使用带有小多核苷酸标签的5 '尾引物标记单个序列。在克隆反应前将PCR产物混合在一起，并用通用质粒引物对克隆进行测序。序列所来自的个体通过每个初始引物上游的标签序列来识别。我们称我们的协议标记-再捕获（MR）克隆。我们目前的结果，从57个实验中进行的MR克隆在四个不同的实验室使用不同长度的核基因座在不同的无脊椎动物物种。描述了凭经验获得的捕获率（检索到一个或多个序列的个体比例）和多重捕获（检索到两个或多个序列的个体比例）。我们估计，与传统的基于个体的克隆相比，MR克隆可以降低高达70%的成本。然而，我们建议调整标记：再捕获比率，以便从同一个体获得多个序列，并避免PCR、克隆和测序的固有技术缺陷。我们认为，MR克隆是一种有效和可靠的高通量方法，提供的序列数量超过最初扩增的个体数量。
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.