In forensics laboratories, one of the most frequently used techniques is DNA analysis. This technique can be performed in several ways, however, precise identification and quantification of small samples has been often complex and time-consuming.
With recent advancements in UV-visible microspectroscopy technology, even trace amounts of DNA evidence can be easily quantified and analyzed for purity and viability. This article outlines the technique of DNA analysis using UV-visible microspectroscopy.
Spectral Characteristics of DNA Components
DNA molecules contain four bases:
These bases form the rungs of the ladder composing the DNA double helix. Hydrogen bonds form between matching bases resulting in a double helix structure.
The four bases have different spectral characteristics and can easily be identified, since these bases have different UV-visible spectra. These spectra are shown in Figure 1.
Figure 1. UV-visible spectra of the bases
Quantification of DNA Samples
It is important to quantify DNA samples, whether collected for research or as evidence. To this end, UV microspectroscopy is used to quantify the DNA samples on microscopic quantities.
For the experiment, the hydrated DNA sample is first vortexed and diluted in the ratio of 1:50 with ultrapure water. The sample is again vortexed and placed into a UV grade microwell plate supplied by CRAIC Technologies.
The above procedure is repeated for all the samples, which are later placed in a separate microwell. Then, a drop of water is placed in one microwell to act as a blank.
The samples are analyzed by taking spectra of each well containing the DNA. Care should be taken that the spectral range remains between 250 and 400nm and the blank solution is auto-optimized as these precautions provide optimum results.
After acquiring the spectra from each microwell, the software can determine the DNA yield or concentration. The software helps in performing the following calculations:
DNA concentration (μg DNA/ml Solution) = (A 260 - A 320 ) x 50 (DNA extinction coefficient) x dilution factor (i.e. 1000/20).
DNA yield in μg = (A 260 - A 320) x 50 (DNA extinction coefficient) x dilution factor (i.e. 1000/20) x final sample volume.
Since the DNA has limited absorbance at 320 nm, this is employed as a base for the calculations. The overall DNA yield in micrograms is calculated using the actual absorbance at 260 nm. Hence, using this method, one can determine both the absolute DNA yield and DNA concentration for that collection method.
Quantification of RNA Samples
It is also important to quantify RNA samples, whether collected for research or as evidence. This can be done by applying the same procedure used for quantifying DNA, but observing a different wavelength.
The software helps in performing the following calculations:
RNA concentration (μg RNA/ml Solution) = (A 260 -A 320 ) x 40 (RNA extinction coefficient) x dilution factor (i.e. 1000/20).
RNA yield in μg = (A 260 -A 320 ) x 40 (RNA extinction coefficient) x dilution factor (i.e. 1000/20) x final sample volume.
Since the RNA has limited absorbance at 320nm, this is employed as a base in the calculations. The overall RNA yield is calculated in micrograms using the actual absorbance at 280nm. Therefore, using this method, one can determine both the absolute RNA yield and the RNA concentration for that collection method.
Purity of DNA Sample
The DNA purity is also determined with the help of software. This is done by measuring the absorbance of the DNA against protein. However, the background must also be considered to obtain precise figures. The calculation is as follows:
DNA Purity = (A 260-A 320) / (A 280-A320).
In case a value of 1.7 to 2.0 is acquired from this calculation, the DNA sample is considered to be pure.
Thus, with the help of modern UV-visible microspectroscopy technology, even trace amounts of DNA evidence can be easily quantified for purity.
Produced from articles authored by Dr. Paul Martin, CRAIC Technologies, Inc.
About CRAIC Technologies
CRAIC Technologies™ specializes in developing superior UV-visible-NIR microanalysis solutions: we build instrument to collect spectra and images of sample features ranging from sub-micron to hundreds of microns. CRAIC Technologies products include UV and NIR microscopes, UV-visible-NIR microspectrophotometers, instruments to measure thin film thickness and colorimetry on the microscopic scale, Raman microspectrometers, automation solutions, traceable standards and more.
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