Fluorescence imaging of single-copy DNA sequences within the human genome using PNA-directed padlock probe assembly.
Fluorescence imaging of single-copy DNA sequences within the human genome using PNA-directed padlock probe assembly.
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DOI：
10.1016/j.chembiol.2013.02.012
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发表时间：
2013-03-21
影响因子：
--
通讯作者：
Smolina IV
中科院分区：
文献类型：
--
作者：
Yaroslavsky AI;Smolina IV
我们提出了一种新的方法，用于荧光原位检测人类细胞基因组DNA中的短的单拷贝序列。我们的方法的单拷贝灵敏度和单碱基特异性是由于三种组分的组合而实现的。首先，肽核酸（PNA）探针局部打开选定的靶位点，这允许挂锁DNA探针进入该位点并连接。其次，滚环扩增（RCA）产生数千个靶序列的单链拷贝。最后，荧光原位杂交（FISH）用于显示扩增的DNA。我们通过成功检测人类线粒体和常染色体DNA上的六个独特靶位点来验证这种新技术。我们还通过检测人类性染色体上的X和Y特异性序列以及同时检测三个独特的靶位点来证明该方法的高特异性。最后，我们区分两个靶位点，不同的两个核苷酸。PNA-RCA-FISH方法是一种独特的原位杂交方法，能够在人类染色体和细胞核内进行多靶点可视化，不需要DNA变性，并且具有极强的序列特异性。
We present a novel approach for fluorescent in situ detection of short, single-copy sequences within genomic DNA in human cells. The single copy sensitivity and single base specificity of our method is achieved due to the combination of three components. First, a peptide nucleic acid (PNA) probe locally opens a chosen target site, which allows a padlock DNA probe to access the site and become ligated. Second, rolling circle amplification (RCA) generates thousands of single-stranded copies of the target sequence. Finally, fluorescent in situ hybridization (FISH) is used to visualize the amplified DNA. We validate this new technique by successfully detecting six unique target sites on human mitochondrial and autosomal DNA. We also demonstrate the high specificity of this method by detecting X- and Y- specific sequences on human sex chromosomes and by simultaneously detecting three unique target sites. Finally, we discriminate two target sites that differ by two nucleotides. The PNA-RCA-FISH approach is a unique in situ hybridization method capable of multi-target visualization within human chromosomes and nuclei that does not require DNA denaturation and is extremely sequence specific.
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影响因子：
4.8
作者：
Demidov, VV;Kuhn, H;Frank-Kameneteskii, MD
通讯作者：
Frank-Kameneteskii, MD
影响因子：
48
作者：
Larsson, C;Koch, J;Nilsson, M
通讯作者：
Nilsson, M
DOI：
10.1073/pnas.092127999
发表时间：
2002-04-30
影响因子：
11.1
作者：
Demidov, VV;Protozanova, E;Frank-Kamenetskii, MD
通讯作者：
Frank-Kamenetskii, MD
影响因子：
30.8
作者：
Lizardi, PM;Huang, XH;Ward, DC
通讯作者：
Ward, DC
影响因子：
3.5
作者：
Smolina, Irina V.;Kuhn, Heiko;Frank-Kamenetskii, Maxim D.
通讯作者：
Frank-Kamenetskii, Maxim D.