SINGLE MOLECULAR DNA/RNA SEQUENCING WITH MICROSCOPY
SINGLE MOLECULAR DNA/RNA SEQUENCING WITH MICROSCOPY
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使用显微镜进行单分子 DNA/RNA 测序
DOI：
10.1016/j.ajps.2015.11.113
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发表时间：
2016
影响因子：
10.2
通讯作者：
Hidehiro Oana
中科院分区：
文献类型：
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作者：
Masanori Kataokaa;Kuniaki Nagayama;Hidehiro Oana
超高通量DNA测序已经成为基因组学研究领域之外的一个非常热门的话题。已经有各种方法来解决这个问题，从直接观察个体DNA合成[1]，使用纳米孔[2，3]的碱基安培/光学检测和使用高分辨率探针显微镜[4]的直接测序。最近，我们开发了一种技术，可以化学修饰DNA中的所有核碱基[5]，因此可以通过使用高分辨率电子显微镜（EM）区分碱基A，T，G，C。对这种依赖于EM能力的测序的要求是：（i）单链DNA的拉伸，（ii）在DNA中没有损伤的高产率结构修饰，以及（iii）保护或减少对碱基的电子剂量损伤的方式。最关键的一步，特别是对于在不依赖标记重元素的情况下观察完整的有机分子来说，是最后一个要求，我们仍在寻找如何做到这一点。在本文中，我们将报告我们针对前两个要求所做的工作。单链DNA分子的拉伸和修饰有两种方法，即“修饰前”拉伸和“修饰后”拉伸，但我们发现DNA链变得脆弱，容易断裂，因此本文采用“修饰前”拉伸方案。首先将DNA拉伸并通过分子梳理固定在无定形碳薄层或无定形碳膜（微网格）上。然后将固定的DNA链暴露于乙酸盐缓冲液中的1.75 M氯乙醛中以修饰腺苷。为了可视化乙烯并加合物的程度，加入抗乙烯并腺苷抗体，其被携带Qdot的二抗荧光化。然后，用PBS缓冲液冲洗固体表面，并使用AFM和EM进行观察。拉伸和修饰的DNA的AFM成像结果示于图1A中。从尺寸分析，照片中的点被识别为量子点，其覆盖延伸约20 mm的DNA的整个长度。如在最上面（最放大）的照片中所见，DNA被量子点相当均匀地标记，具有几个10 nm的间距。考虑到抗体-Qdot复合物的尺寸为直径约20 nm，它们沿DNA沿着密集堆积。对同一样品进行的TEM观察结果如图1B所示。高分辨率TEM照片显示，量子点复合物排列在DNA骨架上，并显示了量子点的详细形状。这些结果证明了TEM测序仪的可能性。
Ultra high-throughput DNA sequencing has been a very hot topic beyond the field of genomic researches. There have been various approaches to this issue ranging from direct observation of individual DNA synthesis [1], amperometric/optical detection of bases using a nanopore [2, 3] and direct sequencing with a high-resolution probe microscope [4]. Recently, we have developed a technique to chemically modify all nucleobases in DNA [5], so that the bases A, T, G, C can be differentiated in the sequence by using high-resolution electron microscopy (EM). The requirements toward this sequencing, which relies on the EM capability are;(i) stretching of singlestranded DNA,(ii) high yield structural modification without damage in DNA and (iii) a way to protect or reduce the electron dose damage to bases. The most crucial step particularly for the case of observing intact organic molecules without relying on labeled heavy elements is the last requirement and we are still underway to find how to do it. In this paper we will report what we have done for the first two requirements. There are two ways to have stretched and modified single-stranded DNA molecules; stretch “before” modification or “after,” but we found that DNA strands become fragile and break easily, so that “stretch before” scheme is employed in this paper.DNA (48.5 kb, 16.5 mm) was employed as the sample. DNA was first stretched out and immobilized onto anamorphous carbon thin layer or anamorphous carbon film (micro grid) by molecular combing. Then the fixed DNA strands were exposed to 1.75 M chloroacetaldehyde in the acetate buffer for modification of adenosine. To visualize the degree of etheno adduct, anti-ethenoadenosine antibody was added, which was fluoresceinated by a secondary antibody carrying Qdot. Then, the solid surface was rinsed with PBS buffer and observation was carried out using AFM and EM. The result of AFM imaging for the stretched and modified DNA is shown in Fig. 1A. From the size analysis, the dots in the photo are identified as Qdots, which cover the entire length of DNA that extends about 20 mm. As seen in the uppermost (most enlarged) photo, DNA is labeled by Qdots rather uniformly, with the spacing of several 10 nm. Considering that the size of an antibodies-Qdotcomplex is about 20 nm in diameter, they are packed densely along the DNA. The observation of TEM carried out for the same sample is shown in Fig. 1B. The high-resolution TEM picture shows that the Qdotcomplex is lined on a DNA backbone and a detailed shape of Qdots. These results demonstrate a possibility of a TEM sequencer.