Detection of Homologous Recombination Intermediates <em>via</em> Proximity Ligation and Quantitative PCR in <em>Saccharomyces cerevisiae</em>

Diedre Reitz; J&#233;r&#244;me Savocco; Aur&#232;le Piazza; Wolf-Dietrich Heyer

doi:10.3791/64240

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Genetics

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JoVE Journal Genetics

Detection of Homologous Recombination Intermediates via Proximity Ligation and Quantitative PCR in Saccharomyces cerevisiae

通过邻近连接和定量PCR检测酿酒酵母同源重组中间体

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07:55 min

September 11, 2022

DOI: 10.3791/64240-v

Diedre Reitz¹, Jérôme Savocco², Aurèle Piazza², Wolf-Dietrich Heyer^1,3

¹Department of Microbiology & Molecular Genetics,University of California, Davis, ²Laboratory of Biology & Modeling of the Cell,École Normale Supérieure de Lyon, ³Department of Molecular & Cellular Biology,University of California, Davis

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D环捕获（DLC）和D环延伸（DLE）测定利用邻近连接原理与定量PCR一起量化酿酒酵母中诱导双链断裂部位的D环形成，D环延伸和产物形成。

D环捕获和延伸测定是第一个允许在有丝分裂生长的酵母细胞同源重组期间对D环形成和延伸步骤进行无偏和直接定量的测定。这种与生存能力无关的技术允许研究D环代谢中的蛋白质，否则不可能从以后的角色中解脱出来，只需查看BIR产品。在未来，我们设想将这些检测转移到人类细胞中，以更好地了解关键蛋白质的作用，包括BRCA2以及bloom和Warner kilo病例在同源重组中的作用。

首先将样品离心五分钟。将沉淀重悬于2.5毫升1x补骨脂素溶液中，并将其转移到60×15毫米的培养皿中。对于无交联对照，将沉淀重悬于2.5毫升TE1溶液中。

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