Using Standard Length DNA Molecules For Accurate Measurement And Analysis Of DNA Fragment By AFM

Atomic force microscopy (AFM), as a new characterization tool in the natural state ofbiological molecular imaging, is widely used to study DNA molecules and other biologicalsamples. We can analyze the data of the biological samples through the characterization, andsamples structural and physical properties has made significant progress. However, the biologicalsample has a certain flexibility, affected by temperature and environmental factors and the processof sample preparation. Therefore, because the impact of the differences in sample preparationprocess and the probe radius of curvature, AFM are still some systematic errors for measuring thebiological samples. Currently, AFM standardized quantification of sample still only at apreliminary stage of exploration. The standard of surveying is to ensure measurement accuracyand repeatability. The present study was designed to address the core problem of biologicalmolecules accurate measurement, aimed to establish a reference scale to achieve precisemeasurement of biological samples.In order to achieve precise measurement of biological samples, our solution is to add astandard length DNA Marker as a reference scale, use the high precision, with the functions ofimage processing and automated software measurement, and establish a standard curve by thedifference method to realize the standardized measurements of biological sample. Because of thephysical and chemical properties of DNA Marker is close to the tested nucleic acid sample, in thesame field of vision, we can relatively accurately reflect a real size of the tested nucleic acidsample,by measurement and analysis of the standard. In this paper, by characterizing andobserving the single-molecule DNA, we can determine the three-dimensional index of single-molecule DNA at an atomic force microscope.On this basis, imaging a sample of DNA Marker,ranging from the length of the DNA mixture to be dimensional data statistical analysis ofmeasurement and analysis of DNA molecular physics nucleotide length relationship AFMmeasurement number (or theoretical value), establish a standard curve, and then on the DNAmolecule of unknown length for accurate measurement and analysis. On this basis, we image a sample of DNA Marker standard, analyze its three-dimensional data, andanalysis the relationship between the length of AFM measurement and the base pairsnumber (or theoretical value) to establish a standard curve, and thus the length of theunknown DNA molecule was precisely measured and analyzed.The results indicate that we determine the sample preparation conditions of thesingle-molecule DNA for characterization and observation, and obtain the heightrange of the single-molecule DNA in the AFM is1.6-2.3nm, and its width range is22-24nm. The physical length of the DNA molecule and its number of base pairsshowed a significant linear positive correlation (R2=0.9991). In addition, under anatomic force microscope, we can clearly observe different length DNA Markerfragment, and calculates the distance between adjacent bases of DNA molecules inAFM approximately0.355nm. With the above methods, our actual DNA samples(measles virus nucleoprotein DNA fragment, double digestion products of West Nileenvelope protein DNA fragments) can be measured and verified. The results showthat the measurement value of the measles virus nucleoprotein DNA fragment is247.8nm/678nm (theoretical length of660bp), the value of West Nile envelopeprotein DNA fragments is1869.74nm/5107bp and747.81nm/2089bp (theoreticallength of5071bp and2066bp). According to the reference scale, we can preciselymeasure unknown biological samples, the measurement deviation is18-36bp(6.5-14nm). The research improve the measurement accuracy of biological samplesby using DNA marker as a reference scale to provide a new method in the study ofnucleic acids and other biological samples.