此前科學(xué)家們?cè)岢觯陔娮语@微鏡的第三代DNA測(cè)序技術(shù)將能夠得到超常的讀長(zhǎng),。今年有兩項(xiàng)研究分別展示了這一理論的實(shí)用性，使電鏡測(cè)序成為來(lái)年備受期待的新測(cè)序方法,。
近年來(lái),，第二代測(cè)序技術(shù)得到了長(zhǎng)足發(fā)展,，而第三代測(cè)序技術(shù)也逐步商業(yè)化走入尋常百姓家，例如Pacific BioSciences和Helicos的單分子合成平臺(tái),。不過,，盡管這些技術(shù)都在測(cè)序通量和測(cè)序成本上實(shí)現(xiàn)了實(shí)質(zhì)性突破，但對(duì)于高等真核生物（尤其是植物）DNA串聯(lián)重復(fù)區(qū)域中的一些長(zhǎng)重復(fù)序列而言,，目前的測(cè)序讀長(zhǎng)還是不夠,，使人們難以對(duì)這些基因組區(qū)域進(jìn)行可靠測(cè)序。
研究顯示,，以納米孔為基礎(chǔ)的第三代測(cè)序技術(shù)可以使測(cè)序讀長(zhǎng)達(dá)到megabases（Mb）級(jí)甚至更長(zhǎng),，該技術(shù)被認(rèn)為會(huì)在不久的將來(lái)進(jìn)入市場(chǎng)?；陔娮语@微鏡的第三代DNA測(cè)序方法也能夠達(dá)到類似的讀長(zhǎng),，近期發(fā)表的兩篇文章就展示了這一技術(shù)的巨大應(yīng)用潛力，電鏡測(cè)序絕對(duì)是2013年最值得關(guān)注的新測(cè)序技術(shù)之一,。
利用電子顯微鏡EM直接讀取DNA序列,，這一概念并不新鮮但人們卻一直沒有實(shí)現(xiàn)這一技術(shù)。這是因?yàn)镈NA四種堿基之間只有幾個(gè)輕原子的差異,，使人們很難通過電鏡進(jìn)行區(qū)分,。用于增加透射電鏡樣本對(duì)比度的標(biāo)準(zhǔn)技術(shù)，被證明無(wú)法提供足夠的對(duì)比度來(lái)區(qū)分DNA序列的差異,。
用重原子對(duì)DNA 進(jìn)行化學(xué)標(biāo)記以提供區(qū)分堿基所需的對(duì)比度,，這被認(rèn)為是最值得一試的方法，不過人們此前的種種嘗試都未能成功,。Bell等人在研究中利用DNA聚合酶在DNA合成時(shí)編入了重金屬標(biāo)記的堿基（汞標(biāo)記的dUTP）,。在標(biāo)記后，DNA被固定在一個(gè)薄薄的支持物上,，通過DNA分子梳平行地獲得分開且拉直了的單個(gè)DNA分子,。隨后研究人員使用環(huán)形暗場(chǎng)掃描透射電子顯微鏡對(duì)標(biāo)記了的DNA進(jìn)行成像，成功讀取了3.2 kb DNA合成片段和7.2 kb病毒基因組中被標(biāo)記的堿基,，展現(xiàn)了電鏡測(cè)序理論的實(shí)用性,。研究者們正在進(jìn)一步改進(jìn)方法以識(shí)別更多的堿基類型，減少標(biāo)記損失并讀取更長(zhǎng)的DNA片段,。希望2013年這一新型測(cè)序技術(shù),，能夠幫助人們大大超越現(xiàn)有測(cè)序讀長(zhǎng)。
在Bell的文章發(fā)表之后,，Mankos及其同事也公布了自己的研究,，他們通過另一種電鏡進(jìn)行測(cè)序，研究顯示這一技術(shù)比Bell的方法更具優(yōu)勢(shì)。Mankos等人并未使用透射電鏡TEM,，而是采用了低能電子顯微鏡LEEM,，這是一種高靈敏度的表面成像技術(shù)，能夠得到單個(gè)原子層面的高對(duì)比度圖像,。理論上,，使用改良版的LEEM可以直接在天然DNA中獲得足夠的對(duì)比度來(lái)區(qū)分不同堿基。這將是一大重要進(jìn)步,，因?yàn)槿藗儗⒉辉傩枰g盡腦汁地對(duì)DNA樣本進(jìn)行標(biāo)記,，可以直接測(cè)序天然DNA。此外這一技術(shù)還有一個(gè)好處,，與高分辨率TEM所需的高能電子相比,，LEEM的低能電子不會(huì)對(duì)DNA樣本產(chǎn)生可能引起錯(cuò)讀的放射性損傷。
Mankos等研究人員提出的方法是,，使DNA在分子梳上拉長(zhǎng)，然后在對(duì)比度足以區(qū)分堿基的情況下進(jìn)行LEEM成像,。他們?cè)诔醪綄?shí)驗(yàn)中對(duì)大量DNA聚合物樣本進(jìn)行了研究,，研究中的對(duì)比度足以區(qū)分DNA樣本與背景，而且研究顯示電子能量的微小變化會(huì)對(duì)DNA對(duì)比度產(chǎn)生重要影響,。研究人員希望通過新設(shè)計(jì)的LEEM（單色,、相差校正、雙光束LEEM）來(lái)拓展他們的初步成果,，獲得能夠區(qū)分DNA鏈中不同堿基的對(duì)比度,。如果這一成果能夠在2013年實(shí)現(xiàn)，將成為該領(lǐng)域中的重要里程碑,。(生物谷Bioon.com)
doi: 10.1017/S1431927612012615
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DNA base identification by electron microscopy.
Bell DC, Thomas WK, Murtagh KM, Dionne CA, Graham AC, Anderson JE, Glover WR.
Advances in DNA sequencing, based on fluorescent microscopy, have transformed many areas of biological research. However, only relatively short molecules can be sequenced by these technologies. Dramatic improvements in genomic research will require accurate sequencing of long (>10,000 base-pairs), intact DNA molecules. Our approach directly visualizes the sequence of DNA molecules using electron microscopy. This report represents the first identification of DNA base pairs within intact DNA molecules by electron microscopy. By enzymatically incorporating modified bases, which contain atoms of increased atomic number, direct visualization and identification of individually labeled bases within a synthetic 3,272 base-pair DNA molecule and a 7,249 base-pair viral genome have been accomplished. This proof of principle is made possible by the use of a dUTP nucleotide, substituted with a single mercury atom attached to the nitrogenous base. One of these contrast-enhanced, heavy-atom-labeled bases is paired with each adenosine base in the template molecule and then built into a double-stranded DNA molecule by a template-directed DNA polymerase enzyme. This modification is small enough to allow very long molecules with labels at each A-U position. Image contrast is further enhanced by using annular dark-field scanning transmission electron microscopy (ADF-STEM). Further refinements to identify additional base types and more precisely determine the location of identified bases would allow full sequencing of long, intact DNA molecules, significantly improving the pace of complex genomic discoveries.
10.1116/1.4764095
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Progress toward an aberration-corrected low energy electron microscope for DNA sequencing and surface analysis
Marian Mankos1, Khashayar Shadman1, Alpha T. N'Diaye2, Andreas K. Schmid2, Henrik H. J. Persson3, and Ronald W. Davis3
Monochromatic, aberration-corrected, dual-beam low energy electron microscopy (MAD-LEEM) is a novel imaging technique aimed at high resolution imaging of macromolecules, nanoparticles, and surfaces. MAD-LEEM combines three innovative electron–optical concepts in a single tool: a monochromator, a mirror aberration corrector, and dual electron beam illumination. The monochromator reduces the energy spread of the illuminating electron beam, which significantly improves spectroscopic and spatial resolution. The aberration corrector is needed to achieve subnanometer resolution at landing energies of a few hundred electronvolts. The dual flood illumination approach eliminates charging effects generated when a conventional, single-beam LEEM is used to image insulating specimens. The low landing energy of electrons in the range of 0 to a few hundred electronvolts is also critical for avoiding radiation damage, as high energy electrons with kilo-electron-volt kinetic energies cause irreversible damage to many specimens, in particular biological molecules. The performance of the key electron–optical components of MAD-LEEM, the aberration corrector combined with the objective lens and a magnetic beam separator, was simulated. Initial results indicate that an electrostatic electron mirror has negative spherical and chromatic aberration coefficients that can be tuned over a large parameter range. The negative aberrations generated by the electron mirror can be used to compensate the aberrations of the LEEM objective lens for a range of electron energies and provide a path to achieving subnanometer spatial resolution. First experimental results on characterizing DNA molecules immobilized on Au substrates in a LEEM are presented. Images obtained in a spin-polarized LEEM demonstrate that high contrast is achievable at low electron energies in the range of 1–10 eV and show that small changes in landing energy have a strong impact on the achievable contrast. The MAD-LEEM approach promises to significantly improve the performance of a LEEM for a wide range of applications in the biosciences, material sciences, and nanotechnology where nanometer scale resolution and analytical capabilities are required. In particular, the microscope has the potential of delivering images of unlabeled DNA strands with nucleotide-specific contrast. This simplifies specimen preparation and significantly eases the computational complexity needed to assemble the DNA sequence from individual reads
