Methods to Quantify DNA
UV absorbance and yield gels: Quantification of absorbance at a wavelength of 260nm or fluorescence after staining a yield gel with ethidium bromide were common early techniques for DNA quantitation. Furthermore, because absorbance measurements are not particular for DNA, contaminants such as proteins or phenol leftover from the extraction process can result in falsely high signals.
Slot blot: The slot blot assay was originally characterized using radioactive probes, but it was later altered and commercialized using chemiluminescent or colorimetric detection methods. The capture of genomic DNA on a nylon membrane was accompanied by the addition of a human-specific probe in slot blots. The intensity of chemiluminescent or colorimetric signals was then evaluated between the specimens and a series of standards.
PicoGreen microtiter plate assay: In a 96-well microtiter plate template, a PicoGreen assay can identify as little as 250pg of double-stranded DNA. PicoGreen is a fluorescent intercalating dye that emits more light when bound to double-stranded DNA.
End-point PCR: An end-point PCR test is a less elegant (but less expensive) method of determining the 'amplifiability' of a DNA specimen. In this method, DNA specimens of recognized concentrations are used to amplify a single STR locus or other areas of the human genome, such as an Alu repeat. A standard curve can be created by comparing specimens with known concentrations to specimens with unknown concentrations.
Real-time quantitative PCR (qPCR): The main goal of a DNA quantification test is to figure out how much 'amplifiable' DNA there is. Coextracted inhibitors, highly degraded DNA, insufficient DNA quantity, or a combination of these factors may cause a PCR amplification reaction to fail. As a result, an experiment that can accurately represent both the quality and quantity of the DNA framework existing in an extracted specimen is helpful in determining how to proceed. PCR assays that are 'real-time' allow for evaluation.
Controls, Standards, Reference Standards, and Calibrators: To Avoid Variation in DNA Quantitation
Positive control or standard: Good positive control will have a value that is at or near the detection threshold. The assay should always be positive (qualitative) or capable of detecting that amount (quantitative). Standards or reference materials with known numbers can be difficult to come by for quantitative assays. Physical or biochemical methods are commonly used to quantify the standard material. Synthetic controls have the benefit of being simple to use and inexpensive, but they are extremely likely to contaminate specimens.
Extraction control: This control can be used in conjunction with the first if the appropriate starting material is available in sufficient quantities. As an example, as a viral extraction control, plasma spiked with a known number of viral particles could be used. However, if an assay fails, this control will be unable to distinguish between a problem with the assay and a problem with the extraction.
Nucleic Acid control: This control will determine whether the specimen contains enough DNA/RNA. Quantitating a housekeeping gene can help with this.
Inhibition controls: Molecular biology enzymes, particularly polymerases, are inhibited by many substances found in normal specimens. Heme, hemoglobin, bile, glycoproteins, particularly lactoferrin, urine crystals, heparin, and even EDTA anticoagulants, and phenol are just a few illustrations (manual extraction protocols).
Contamination control: Amplification procedures are susceptible to contamination by controls, standards, other specimens, and even airborne organisms due to the same characteristics that make them powerful. Contamination can be reduced to a large extent by regulating the environment but controls should be included in the assay run as well.
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References
- Butler JM. Fundamentals of forensic DNA typing/John M. Butler.
- White HE, Matejtschuk P, Rigsby P, et al. Establishment of the first World Health Organization international genetic reference panel for quantitation of BCR-ABL mRNA. Blood. 2010, 116(22).
