DNA quantification

Commercially produced DNA profiling kits, such as the AmpFlSTR SGM Plus PCR are optimised to produce DNA profiles from a narrow range of template DNAconcentration, usually between 0.5ng and 2.0ng DNA. The addition of too little template DNA can result in stochastic amplification which causes unbalanced amplification of heterozygote loci and can result in allelic drop out, making heterozygotes appear as homozygotes at the affected loci. The addition of too much template DNA can lead to the production of large stutter peaks that complicate DNA profile interpretation. By quantifying all DNA samples before performing DNA profiling PCR, the production of such artefacts can be reduced or completely avoided.

Spectrophotometry

There are a number of methods available to quantify DNA. The traditional method of DNA quantitation involves measuring the absorbance of the sample at 260nm on a spectrophotometer. This method is simple to perform and shows little sample-to-sample variation, making it a desirable technique. But this technique is not species-specific; meaning any bacterial or fungal DNA that has co-purified with the DNA of interest cannot be distinguished from the human target DNA. Additionally, singlestranded DNA and nucleotides cannot be distinguished from double-stranded DNA by this method which can also lead to falsely high quantitation readings.

Slot blot

A different method of quantification is known as the slot blot technique. This method is based on the specific hybridisation of a 40bp probe that binds to the human alpha satellite locus, D17Z1. Using this technique,DNA is quantified by visual comparison of sample band intensities with the band intensities of standards, produced from known amounts of DNA. This comparison can be performed manually but this allows for the subjectivity of individual interpretation which can lead to between-operator differences in quantitation, or it can be performed electronically by computationally converting the band intensities into numerical values.

This technique shows a high degree of species specificity (human and primate) and can detect as little as 150pg DNA in a standard assay. Similarly to the development of commercial DNA profiling kits, the development of commercial DNA quantitation kits has also progressed. Applied Biosystems produced the Quantiblot Human DNA.

PicoGreen

Another method involves the use of a fluorescent dye, PicoGreen dsDNAquantitation reagent. This method was developed to allow high-throughput quantitation of multiple samples concurrently whilst increasing the sensitivity DNA quantitation methods. The method utilises the increased fluorescent intensity that is observed when PicoGreen binds to dsDNA. The fluorescent intensity of the PicoGreen dye is measured with a spectrofluorometer capable of producing the excitation wavelength of ~480 nm and recording at the emission wavelength of ~520 nm. The DNA is then quantified by comparison of the sample fluorescence to the fluorescence of a set of standards that are included in every sample run. The biggest disadvantage to this method over the hybridisation methods discussed previously is that this method is not specific for human DNA. Any animal, bacterial or fungal DNA co-purified with the human DNA of interest will contribute to the final reading and could give a falsely high DNA quantification. This method is, however, much more sensitive than previously described methods, with a reported lower detection limit of 25pg/ml.

Real time PCR

The latest development in DNA quantitation is based on the technique of real time PCR. The methodology was first proposed 15 years ago by Higuchi and co-workers who, by including ethidium bromide in the PCR reaction, were able to continuously monitor the production of doublestranded DNA in 'real time'. By capturing the change in fluorescent intensity on video camera, the potential of this new development for specific DNAquantitation was realised. There are several different approaches to real time quantitation of DNA; they are all, however, based around the principle of fluorescent dye binding double-strandedDNA as it accumulates during the PCR process. As the technique is based on the polymerase chain reaction, DNA quantitation can be

undertaken by targeting any specific region of template DNA, with many systems targeting the multicopy Alu sequence, which appears 500,0001,000,000 times throughout the human genome. This not only ensures that the technique is species-specific, but has allowed numerous variations, many designed to perform additional functions, such as independent quantitation of nuclear and mitochondrial DNA in a single reaction and assessment of DNA quality. There are now several real time PCR kits commercially available, such as the Quantifiler systems from Applied Biosystems and the HY Plexor system from Promega, that allow for the accurate quantification of human specific DNA, and even the separate quantification of human and human-male DNA in the same reaction.

1 picogram = 0.001 nanogram 1 pg = 0.001 ng TT?1 picogram = 1.0 10-6 microgram 1 pg = 1.0 10-6 g TT?1 picogram = 1.0 10-9 milligram 1 pg = 1.0 10-9 mg TT?1 picogram = 1.0 10-12 gram 1 pg = 1.0 10-12 g TT?1 picogram = 1.0 10-15 kilogram 1 pg = 1.0 10-15 kg TT?1 picogram = 1.0 10-17 centner 1 pg = 1.0 10-17 centner TT?1 picogram = 1.0 10-18 tonne 1 pg = 1.0 10-18 t TT?1 picogram = 5.0 10-12 carat 1 pg = 5.0 10-12 ct TT?1 picogram = 602 214 151 134 atomic mass units 1 pg = 602 214 151 134 u TT?1 picogram = 3.527396198 10-14 ounce 1 pg = 3.527396198 10-14 oz TT?1 picogram = 2.204622622 10-15 pound 1 pg = 2.204622622 10-15 lb TT?1 picogram = 1.54323583529 10-11 grain 1 pg = 1.54323583529 10-11 gr TT?1 picogram = 6.85217796477 10-17 slug 1 pg = 6.85217796477 10-17 sl TT?1 picogram = 1.102311311 10-18 short ton 1 pg = 1.102311311 10-18 short tn TT?1 picogram = 9.84206527679 10-19 long ton 1 pg = 9.84206527679 10-19 long tn TT?1 picogram = 1.57473044429 10-16 stone 1 pg = 1.57473044429 10-16 st TT?1 picogram = 2.67922888303 10-15 troy pound 1 pg = 2.67922888303 10-15 troy

TT?1 picogram = 3.21507465964 10-14 troy ounce 1 pg = 3.21507465964 10-14 oz t TT?1 picogram = 6.43014931371 10-13 pennyweight 1 pg = 6.43014931371 10-13 dwt