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 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, it is not human specific and other chemicals, for exam- ple, dyes from clothing and humic acids from bone samples, can interfere with the analysis.
Figure 4.5 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
QUANTIFICATION OF DNA
Ethidium bromide or DAPI (4′,6-diamidino-2-phenylindole) can be used to visualize DNA in agarose gels – these are both examples of chemicals that fluoresce at much higher levels when they intercalate with DNA. In addition to staining agarose gels, fluorescent dyes can also be used as an alternative to UV spectrophotometry for DNA quantitation. A range of dyes has been developed that can be used with fluorescent mi- croplate readers and these are very sensitive. The PicoGreen® dye is specific for double stranded DNA and can detect as little as 25 pg/ml of DNA . When PicoGreen® binds to DNA the fluorescence of the dye increases over 1000-fold – ethidium bromide in comparison increases in fluorescence 50-100-fold when it intercalates with dou- ble stranded DNA . PicoGreen® is very sensitive and is a powerful technique for quantifying total DNA – it does however have the drawback that it is not human specific.
Hybridization based quantification methods have been widely used in forensic genetics since the early 1990s, in particular a commercially available kit Quantiblot® (Applied Biosystems). Extracted DNA is applied to a positively charged nylon membrane using a slot or dot blot process; the DNA is then challenged with a probe that is specific to human DNA. A commonly used target is the D17S1 alpha satellite repeat that is on human chromosome 17 in 500-1000 copies. The probe can be labelled in a number of ways including colorimetric and chemiluminescent. A series of standards is applied to the membrane, and comparison of the signal from the extracted DNA with the standards allows quantification. The advantage of hybridization-based methods is that the quantification is human specific – agarose gel electrophoresis and spectrophotometry detect total DNA and forensic samples that have been exposed to the environment for any length of time are prone to colonization by bacteria and fungi. The hybridization systems do suffer from a lack of sensitivity. For samples producing a negative result in many cases it is still possible to generate a profile after PCR. The analysis of the results is also subjective, leading different operators to come to different conclusions. In addition to the limited sensitivity, the process is labour intensive, taking approximately 2 hours to produce the blot. Hybridization-based methods are being widely replaced by real-time PCR systems.
When generating a DNA profile, the PCR products are normally analysed at the end point after 28-34 cycles. It is, however, possible to monitor the generation of PCR products as they are generated – real time. This was first developed using ethidium bro- mide: as PCR products are generated in each cycle, more ethidium bromide intercalates with the double-stranded DNA molecule and fluoresces under UV light. The increase in fluorescence can be detected using a suitable ‘camera’ . Increasingly sensitive
Figure 4.6 (a) The TaqMan® quantification system consists of two PCR primers and an internal probe that hybridizes within the region that is the amplified region; (b) as the primer extends it encounters the probe, the 5′ exonuclease activity of the Taq polymerase degrades the probe: (c) the reporter molecule is no longer in proximity to the quencher and fluoresces
assays have been developed, such as SYBR® Green and the TaqMan® system. Using SYBR® Green, as PCR products are generated, the dye binds to the double-stranded productandthefluorescenceincreases.TheTaqMan® systemusesadifferentapproach, with two primers and a probe; the probe is within the region defined by the primers and is labelled on the 5′ end with a fluorescent molecule and on the 3′ end with a molecule that quenches the fluorescence. As the primers are extended by the Taq polymerase, one of them meets the probe, which is degraded by the polymerase, releasing the probe and the quencher into solution – efficient quenching of the fluorescent molecules only occurs when they are in close proximity on the probe molecule (Figure 4.6). AsmorePCRproductsaregenerated,morefluorescentmoleculesarereleasedandthe fluorescence from the sample increases. Real-time assays are highly sensitive, human specific and are not labour intensive.