Sequencing method of single DNA molecule based on high-resolution transmission electron microscopy
A technology of transmission electron microscopy and DNA sequencing, which is applied in the measurement/inspection of microorganisms, biochemical equipment and methods, etc., can solve the problems that gene sequencing technology cannot enter ordinary people, single-molecule sequencing technology has no breakthrough, and achieve equipment and the effect of simple operation and cost reduction
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Embodiment 1
[0041] A single-molecule DNA sequencing technology based on high-resolution transmission electron microscopy, including the following steps:
[0042] 1. Preparation of single-atom labeling solution
[0043] 101. The single-atom markers should be heavy metal salts with different atomic numbers, and the purity should be above 99.9% when dried. Dissolve metal salts in 0.2M phosphate buffer (pH=7.4), at least three heavy metal salt solutions with different atomic numbers should be prepared, and four are used in this example, namely silver nitrate, gold perchlorate, and uranyl acetate , Chromium chlorate, respectively used to label oligonucleotides with four different bases.
[0044] 102. Modification of DNA oligonucleotides: Dilute the above four kinds of salt solutions with 0.2M phosphate buffer (pH=7.4) to 0.1mmol / L, four kinds of dNTPs (dA, dT, dC, dG) or their single The oligomer solution of the body was diluted to 0.2mmol / L (excess relative to the marker). The four markers...
Embodiment 2
[0054] Method is with reference to embodiment 1, but changes as follows:
[0055] 101. Use three heavy metal salt solutions with different atomic numbers, namely silver nitrate, gold perchlorate, and uranyl acetate to label A / T / G oligonucleotides, and base C is not labeled;
[0056] 102.dC is directly diluted to 0.1mmol / L with phosphate buffer solution with pH=7.4, without further labeling—elution process;
[0057] 301. Change to low-temperature baking method below 50 degrees Celsius for TEM sample preparation;
[0058] 401. Use the TEM imaging mode to first form a bright field image (BF-TEM), and then use the characteristic X-spectrum (EDS-Mapping) method to perform three characteristic element spectrum imaging of the area of interest; for the illumination field image, fusion In the obtained 3 spectra, the positions of the labeled atoms are sequentially recorded, and the vacancies represent the unlabeled C bases in the sequence, and the sequence information of the DNA frag...
Embodiment 3
[0060] Method is with reference to embodiment 1, but changes as follows:
[0061] 401. Use the TEM imaging mode to form a bright field image first, and then use the electron energy loss spectrum (EELS-Mapping) method to perform 4 characteristic element spectrum imaging of the area of interest; for the illumination field image, the fusion obtained 4 Create a spectrogram and sequentially record the positions of the labeled atoms to obtain the sequence information of the DNA fragment to be tested.
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