Müller Stefan, Neusser Michaela, Köhler Daniela, Cremer Marion
Ludwig-Maximilians University Munich, Department Biology II, AG Thomas Cremer (Chair of Anthropology and Human Genetics), 82152 Martinsried-Planegg, Germany.
CSH Protoc. 2007 May 1;2007:pdb.prot4730. doi: 10.1101/pdb.prot4730.
INTRODUCTIONDNA probes for fluorescence in situ hybridization (FISH) can be generated and labeled by various methods. This protocol describes the conjugation of dUTPs with haptens or fluorochromes, as well as the generation and labeling of DNA probes using those modified dUTPs. Sources of probe DNA include genomic DNA, DNA from flow-sorted chromosomes, bacterial artificial chromosomes (BACs), and cosmids. DNA amplification and labeling procedures involving degenerate oligonucleotide-primed PCR (DOP-PCR) and multiple displacement amplification (MDA) are provided. Advice is given for setting up complex probe pools, such as those containing large pools of BAC probes. Also included is a method for probe precipitation and preparation of a hybridization mix ready to be used for 3D fluorescence in situ hybridization (FISH) experiments.
引言
用于荧光原位杂交（FISH）的DNA探针可以通过多种方法生成和标记。本方案描述了dUTP与半抗原或荧光染料的缀合，以及使用这些修饰的dUTP生成和标记DNA探针的过程。探针DNA的来源包括基因组DNA、流式分选染色体的DNA、细菌人工染色体（BAC）和黏粒。提供了涉及简并寡核苷酸引物PCR（DOP-PCR）和多重置换扩增（MDA）的DNA扩增和标记程序。还给出了关于建立复杂探针库的建议，例如那些包含大量BAC探针的库。此外，还包括一种探针沉淀方法以及用于三维荧光原位杂交（FISH）实验的杂交混合物的制备方法。
