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; the absorbance is measured against a standard. A clean DNA extract will produce a curve as shown in Figure 4.5; 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 .
Spectrophotometry is commonly used for quantification in molecular biology laboratories 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, it is not human specific and other chemicals, for example, dyes from clothing and humic acids from bone samples, can interfere with the analysis.
Was this article helpful?