Work
at Opgen
Research Aims: To determine whether E. Coli K-12 high molecular weight (HMW) DNA from commercially available kits was suitable for optical mapping.To determine whether long term storage at room temperature results in the formation of a DNA gradient in storage tube. To determine if E.Coli K12 plug DNA can be mixed by inverting or gently “flicking”the solution in a microfuge tube to produce a homogeneous concentration of DNA within the tube and determine whether the inverted DNA can be used to produce an optical map similar in all parameters to uninverted or “unflicked “ DNA. To compare measurements of microbial cell growth between a turbidimeter (1) and a spectrophotometer (2) and to determine the range of use with regard to number of cells.
Opgen Inc.(3) performs optical mapping (4)services] for research customers and fedral organizations . Optical mapping can be used to compare genomes of different microbial strains Comparative genomics), for sequencing whole genomes or for strain typing for epidemiological studies. The overall process depends on the isolation of high molecular weight DNA (HMW) and digestion with appropriate restriction enzymes (5) HMW is generally t obtained by imbedding the cells of interest into low-melting point agarose (6). The cells of interest in the agarose plug are lysed in situ and the plugs containing the DNA are washed thoroughly. The agarose plug is melted at 70oC and the agarose is digested using β-agarase (7) to release the HMW DNA.HMW DNA spiked with a DNA size standard is loaded into channels of an microfluidic (8) optical chip device resulting in long parallel arrays of single strand DNA molecules on a treated glass coverslip. The electrical charge of the coverslip interacts with the electrical charge of the DNA molecule which “holds” the DNA molecule onto the coverslip. The DNA is digested with a restriction enzyme. Restriction enzyme digestion results in small gaps in the parallel arrays of DNA on the coverslip. The restriction digested DNA chip is stained with a fluorescent dye, JOJO /2-ME], and the size, compared to DNA standards, and order of the DNA fragments are determined through the use of an automated fluorescence microscope an and image analysis software. The restriction enzyme maps of each single molecule of HMW are assembled to produce a consensus optical map (9).
During my tenure as a scientist II at Opgen, few if any, commercially available kits for DNA isolation were appropriate for optical mapping. Most of the kits produced DNA molecules which were not long enough for the mapping process. Many of the kits used methodologies that resulted in shearing of the DNA. The low melting point agarose plug method, while tedious and time consuming, produced the highest quality HMW DNA.
At Opgen, HMW DNA was stored at room temperature for long periods of time in either H2O or in TBE buffer (10) .We were concerned that the DNA might settle in the tube creating a gradient of DNA concentration. If long term storage did result in a gradient could we produce a more homologous sample by gentle inversion, flicking or rocking of the DNA sample? Long term storage did produce a DNA concentration gradient in tubes stored at room temperature for long periods of time. The highest DNA concentration of HMW DNA , measured by the Qubit dsDNA High Sensitivity Assay, was found at the bottom of the storage tubes. Rocking or gentle flicking did not produce a significantly homologous sample and did not produce optical maps. Gentle Inversion of the DNA in the storage tube produced a homologous DNA concentration and that DNA was suitable for mapping purposes.
Ideally, we would like to determine optical maps of microbes
in real life situations with a minimal number of cells. This would obviate the
need for growing the microbe of interest in the laboratory. In an effort to
determine the minimal number of cells that could be measured, we compared the accuracy
and range of a Turbimeter vs
a spectrophotometer for determining the number of cells. The accuracy of
measurement was determined using McFarland standards 0.5 (11). A standard curve
was created with using dilutions of McFarland standards for both instruments.
The Turbidimeter had a much wider range of
measurement, 104 – 1.5 x 10 8 cells, compared
to the spectrophotometer, 1.5 x 108 – 2.5 X 109 cells.
The narrow range with the spectrophotometer is due to the fact that the
spectrophotometer measures optical density which is dependent on absorptance of light passed through the sample. The turbidimeter, also called nephelometer, measures the scattering of light with
higher measurements of light as more particles in the sample scatter light to
the detector.
1. http://en.wiktionary.org/wiki/turbidimeter
2. http://en.wikipedia.org/wiki/Spectrophotometry]
4. http://www.opgen.com/Technology/What_Is_Optical_Mapping.php
5. http://en.wikipedia.org/wiki/Restriction_enzyme.
6. http://en.wikipedia.org/wiki/Agar
7. http://en.wikipedia.org/wiki/Agarase
8. http://en.wikipedia.org/wiki/Microfluidics
9. http://en.wikipedia.org/wiki/Optical_mapping
10. http://en.wikipedia.org/wiki/TBE_buffer
11. http://en.wikipedia.org/wiki/McFarland_standards
Page last updated: July 26, 2011 16:43