Fragmentation and labeling of probe DNA for whole-mount FISH in Drosophila.
Fragmentation and labeling of probe DNA for whole-mount FISH in Drosophila.
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DOI：
10.1101/pdb.prot066886
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发表时间：
2011-12-01
影响因子：
--
通讯作者：
Dernburg, Abby F
中科院分区：
文献类型：
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作者：
Dernburg, Abby F
良好的荧光原位杂交（FISH）探针必须满足两个标准：DNA片段必须非常小，并且必须高度标记。本文描述了一种有效的标记方案，包括片段化探针DNA，然后使用末端脱氧核苷酸转移酶（TdT）将标记和未标记核苷酸的混合物添加到3'末端。该方法可用于标记各种DNA探针，而不管它们的初始大小（例如，质粒、粘粒或P1克隆、聚合酶链式反应[PCR]产物或总基因组DNA）。短的寡核苷酸也可以以这种方式标记而无需消化，因为它们的小尺寸允许它们扩散通过厚组织。末端标记的潜在优点是修饰的核苷酸不掺入互补探针序列本身中，因此可以较少地干扰杂交。游离的3'尾也可以使半抗原更容易接近检测试剂。
Good probes for whole-mount fluorescent in situ hybridization (FISH) must meet two criteria: The DNA fragments must be very small and they must be highly labeled. This article describes an effective labeling scheme that involves fragmenting the probe DNA and then adding a mixture of labeled and unlabeled nucleotides to the 3' ends using the enzyme terminal deoxynucleotidyl transferase (TdT). This method can be used to label a variety of DNA probes, regardless of their initial size (e.g., plasmid, cosmid, or P1 clones, polymerase chain reaction [PCR] products, or total genomic DNA). Short oligonucleotides may also be labeled in this way without digestion, because their small size allows them to diffuse through thick tissues. A potential advantage of end-labeling is that the modified nucleotides are not incorporated into the complementary probe sequence itself and may thus interfere less with hybridization. The free 3' tail may also make haptens more accessible to detection reagents.