Biology Reference
In-Depth Information
a single band while degraded DNA or DNA that has been sheared during extraction
appears as a smear. This makes comparison to the standards difficult as the DNA is
spread out over a range a random sizes.
The advantages of agarose gel electrophoresis are that it is quick and easy to carry
out and also gives an indication of the size of the DNA molecules that have been
extracted. The disadvantages are that quantifications are subjective, based on relative
band intensities; it is difficult to gauge the amounts of degraded DNA as there is no
suitable reference standard; total DNA is detected that can be a mixture of human
and microbial DNA, and this can lead to overestimates of the DNA concentration;
the sensitivity of the dye under UV light is poor, so low level DNA will not be
visible; it cannot be used to quantify samples extracted using the Chelex
method,
as this produces single-stranded DNA and the fluorescent dyes that intercalate with
the double-stranded DNA do not bind to the single-stranded DNA.
Ultraviolet spectrophotometry
DNA absorbs light maximally at 260 nm. This feature can be used to estimate
the amount of DNA in an extract and by measuring a range of wavelengths from
220 nm to 300 nm it is also possible to assess the amount of carbohydrate (maximum
absorbance 230 nm) and protein (maximum absorbance 280 nm) that may have co-
extracted with the sample. The DNA is placed in a quartz cuvette and light is shone
through and the absorbance is measured against a standard. A clean DNA extract
will produce a curve as shown in Figure 4.8; if the DNA extract is clean, the ratio
of the absorbance at 260 nm and 280 nm should be between 1.8 and 2.0 [12].
Spectrophotometry is commonly used for quantification in molecular biology lab-
oratories but has not been widely adopted by the forensic community. The major
disadvantage is that it is difficult to quantify small amounts of DNA accurately
using spectrophotometry; also, it is not human specific and other chemicals, for
Figure 4.8
UV absorbance by a solution containing DNA is maximal at 260 nm. The 260:280 ratio
of 1.91 indicates that the extract is not contaminated with proteins
