Fast and Non-Toxic In Situ Hybridization without Blocking of Repetitive Sequences
Fast and Non-Toxic In Situ Hybridization without Blocking of Repetitive Sequences
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DOI：
10.1371/journal.pone.0040675
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发表时间：
2012-07-24
期刊：
影响因子：
3.7
通讯作者：
Hansen, Charles M.
中科院分区：
文献类型：
--
作者：
Matthiesen, Steen H.;Hansen, Charles M.
甲酰胺是原位杂交（ISH）中降低核酸链的熔点和退火温度的优选溶剂。甲酰胺的一个主要优点是由于较低的孵育温度而更好地保持形态。然而，在荧光原位杂交（FISH）中，针对组织切片中的独特DNA靶标，需要过夜杂交以获得足够的信号强度。在这里，我们确定了替代溶剂，并开发了一种新的杂交缓冲液，将所需的杂交时间缩短至1小时（IQFISH方法）。值得注意的是，不需要针对重复DNA序列进行变性和阻断以防止非特异性结合。此外，新的杂交缓冲液比含甲酰胺的缓冲液危害更小。结果表明，在较低的变性和杂交温度下，DNA和PNA（肽核酸）探针的杂交率显着增加。我们预计，这些甲酰胺替代溶剂将成为改变核酸变性和杂交的理解和性能的基础。例如，用于癌症诊断中基因畸变测试的基于组织的ISH的处理时间可以从几天减少到几个小时。此外，理解核酸链的相互作用和双链体形成可以受益于这些溶剂的性质。
Formamide is the preferred solvent to lower the melting point and annealing temperature of nucleic acid strands in in situ hybridization (ISH). A key benefit of formamide is better preservation of morphology due to a lower incubation temperature. However, in fluorescence in situ hybridization (FISH), against unique DNA targets in tissue sections, an overnight hybridization is required to obtain sufficient signal intensity. Here, we identified alternative solvents and developed a new hybridization buffer that reduces the required hybridization time to one hour (IQFISH method). Remarkably, denaturation and blocking against repetitive DNA sequences to prevent non-specific binding is not required. Furthermore, the new hybridization buffer is less hazardous than formamide containing buffers. The results demonstrate a significant increased hybridization rate at a lowered denaturation and hybridization temperature for both DNA and PNA (peptide nucleic acid) probes. We anticipate that these formamide substituting solvents will become the foundation for changes in the understanding and performance of denaturation and hybridization of nucleic acids. For example, the process time for tissue-based ISH for gene aberration tests in cancer diagnostics can be reduced from days to a few hours. Furthermore, the understanding of the interactions and duplex formation of nucleic acid strands may benefit from the properties of these solvents.