1.28 required reading

Why quantify DNA

• To ensure that DNA recovered from an extraction is human rather than from another source such as bacteria, the FBI’s quality assurance standards require human-specific DNA quantitation

• Only after DNA in a sample has been isolated can its quantity and quality be reliably assessed.

• The primary purpose of DNA quantitation is to determine the appropriate amount of DNA template to include in future tests

• Determination of the amount of DNA in a sample is essential for most polymerase chain reaction (PCR)-based assays because a narrow concentration range works best with multiplex short tandem repeat (STR) typing. Typically 0.5 to 2.0 ng of input human DNA is optimal with current commercial STR kits.

• Too much DNA results in overblown electropherograms that make interpretation of results more challenging and time consuming to review.

• Too little DNA can result in loss of alleles due to stochastic amplification and failure to equally sample the STR alleles present in the sample.

• If the amount of DNA in a sample is outside of the target range for creating a ‘ just right’ DNA profile, then the DNA amount must be adjusted prior to putting it into the PCR reaction.

• The process of achieving a DNA concentration that fits the optimal window for analysis is called normalization. This involves diluting the sample down to the desired range or concentrating it by removing excess fluid.

• Evaluation of human DNA quantity in a sample can be used to screen which samples should be sent forward through the DNA testing process. When having to wade through a large number of samples, a sample screening process, based on the amount of human DNA present, can be very helpful and cost effective. DNA quantitation that is done well can save time and preserve the often-limited DNA extracted from biological evidence.

DNA quantities used

• PCR amplification is dependent on the quantity of template DNA molecules added to the reaction. Based on the amount of DNA determined to be in a sample with a quantitation method, the extracted DNA for each sample is adjusted to a level that will work optimally in the PCR amplification reaction.

• A quantity of 1 ng of human genomic DNA corresponds to approximately 303 copies of each locus that will be amplified

• There are approximately 6 pg (one millionth of one millionth of a gram or 10^-12 grams) of genomic DNA in each cell containing a single diploid copy of the human genome. Thus, a range of typical DNA quantities from 0.1 to 25 ng would involve approximately 30 to 8330 copies of every nuclear DNA sequence to be examined.

• Human-specific DNA quantitation with Applied Biosystems’ Quantifiler kit was then performed to see if samples should be processed further to try to obtain a DNA profile. The human DNA quantitation served as an important screening step in the DNA analysis process.

DNA quantitation methods

• early assays were ‘ home-brew ’ (i.e., prepared by the laboratory performing the test) while most forensic DNA quantitation is now performed using commercial kits from suppliers such as Applied Biosystems or Promega Corporation

UV absorbance and yield gels

• Early methods for DNA quantitation typically involved either measurement of absorbance at a wavelength of 260 nm or fluorescence after staining a yield gel with ethidium bromide. Unfortunately, because these approaches are not very sensitive, they consume valuable forensic specimens that are irreplaceable. In addition, absorbance measurements are not specific for DNA, and contaminating proteins or phenol left over from the extraction procedure can give falsely high signals.

• To overcome these problems, several methods have been developed for DNA quantitation purposes.

Slot blot

• The most commonly used method in forensic labs during the late 1990s and beginning years of the 21st century for genomic DNA quantitation was the so-called slot blot procedure.

• This test was specific for human and other primate DNA due to a 40-bp probe that bound to a region on chromosome 17 called D17Z1.

• The slot blot assay was first described with radioactive probes, but was later modified and commercialized with chemiluminescent or colorimetric detection formats.

• Slot blots involved the capture of genomic DNA on a nylon membrane followed by addition of a human-specific probe. Chemiluminescent or colorimetric signal intensities were then compared between a set of standards and the samples

• As with almost all DNA quantitation methods, the slot blot procedure involved a relative measurement that compared unknown samples to a set of standards. These standard samples were prepared via a serial dilution from a DNA sample of known concentration. The comparison between the standards and unknowns was usually performed visually— and therefore influenced by the subjectivity of the analyst. However, digital capture and quantification of slot blot images was also an option using a charge-coupled device (CCD) camera imaging system.

• Up to 30 samples could be tested on a slot blot membrane with 6 to 8 standard samples run on each side of the membrane for comparison purposes.

• Typically about 5 μL of DNA extract from each sample was consumed in order to perform this DNA quantitation test.

• The slot blot assay took several hours to perform and could detect both single-stranded and double-stranded DNA down to levels of approximately 150 pg.

• Even when no results were seen with this hybridization assay, some forensic scientists still went forward with DNA testing and often obtained a successful STR profile. Thus, the slot blot assay was not as sensitive as would have been preferred.

• In addition, as with most ‘ human-specific ’ tests, primate samples, such as chimpanzees and gorillas, also produced signal due to similarities in human and other primate DNA sequences.

• In 2006, Applied Biosystems (Foster City, CA), the final commercial source for slot blot assay reagents, stopped selling the QuantiBlot Human DNA Quantitation Kit. Thus, this assay is now a thing of the past.

PicoGreen microtiter plate assay

• As higher throughput methods for DNA determination are being developed, more automated procedures are needed for rapid assessment of extracted DNA quantity prior to DNA amplification.

• To this end, in the mid-1990s, the Forensic Science Service (Birmingham, England) developed a PicoGreen assay that is capable of detecting as little as 250 pg of double-stranded DNA in a 96-well microtiter plate format.

• PicoGreen is a fluorescent interchelating dye whose fluorescence is greatly enhanced when bound to double-stranded DNA.

• To perform this microtiter plate assay, 5 μL of sample are added to 195 μL of a solution containing the PicoGreen dye. Each sample is placed into an individual well on a 96-well plate and then examined with a fluorometer. A 96-well plate containing 80 individual samples and 16 calibration samples can be analyzed in under 30 minutes. The DNA samples are quantified through comparison to a standard curve. This assay has been demonstrated to be useful for the adjustment of input DNA into the amplification reaction of STR multiplexes. It has been automated on a robotic workstation as well.

• Unfortunately, this assay quantifies total DNA in a sample and is not specific for human DNA

AluQuant human DNA quantitation system

• Around 2000, the Promega Corporation developed a human DNA quantitation system, known as AluQuant, that enabled fairly sensitive detection of DNA using Alu repeats that are in high abundance in the human genome.

• Probe-target hybridization initiated a series of enzymatic reactions that ended in oxidation of luciferin with production of light. The light intensity was then read by a luminometer with the signal being proportional to the amount of DNA present in the sample. Sample quantities were determined by comparison to a standard curve.

• The AluQuant assay possesses a range of 0.1 to 50 ng for human DNA and can be automated on a robotic liquid-handling workstation. While this assay was used for several years by laboratories such as the Virginia Department of Forensic Sciences, it has been made obsolete with the introduction of real-time quantitative PCR assays.

End point PCR

• A less elegant (and less expensive than qPCR) approach for testing the ‘ amplifiability’ of a DNA sample is to perform an end-point PCR test.

• In this approach a single STR locus or other region of the human genome, such as an Alu repeat, is amplified along with DNA samples of known concentrations. A standard curve can be generated from the samples with known amounts to which samples of unknown concentration are compared.

• A fluorescent intercalating dye such as SYBR Green can be used to detect the generated PCR products.

• Based on the signal intensities resulting from amplification of the single STR marker or Alu repeat region, the level of DNA can be adjusted prior to amplifying the multiplex set of DNA markers in order to obtain the optimal results.

• This method is a functional test because it also monitors the level of PCR inhibitors present in the sample.

Real-time PCR analysis

• Three distinct phases define the PCR process: geometric or exponential amplification, linear amplification, and the plateau region. These regions can be seen in a plot of fluorescence versus PCR cycle number.

• During exponential amplification, there is a high degree of precision surrounding the production of new PCR products.

• When the reaction is performing at close to 100% efficiency, then a doubling of amplicons occurs with each cycle.

• A plot of cycle number versus a log scale of the DNA concentration should result in a linear relationship during the exponential phase of PCR amplification.

• A linear phase of amplification follows the exponential phase as one or more components fall below a critical concentration and amplification efficiency slows down to an arithmetic increase rather than the geometric one in the exponential phase.

• Since components such as deoxynucleotide triphosphates (dNTPs) or primers may be used up at slightly different rates between reactions, the linear phase is not as precise from sample to sample and therefore is not as useful for comparison purposes.

• The final phase of PCR is the plateau region where accumulation of PCR product slows to a halt as multiple components have reached the end of their effectiveness in the assay.

• The fluorescent signal observed in the plateau phase levels out.

• The accumulation of PCR product generally ceases when its concentration reaches approximately 10^-7 mol/L.

• The optimal place to measure fluorescence versus cycle number is in the exponential phase of PCR where the relationship between the amount of product and input DNA is more likely to be consistent.

• Real-time PCR instruments use what is termed the cycle threshold (C_T) for calculations. The C_T value is the point in terms of PCR amplification cycles when the level of fluorescence exceeds some arbitrary threshold, such as 0.2, that is set by the real-time PCR software to be above the baseline noise observed in the early stages of PCR.

• The fewer cycles it takes to get to a detectable level of fluorescence (i.e., to cross the threshold set by the software), the greater the initial number of DNA molecules put into the PCR reaction.

• Thus a plot of the log of DNA concentrations versus the C_T value for each sample results in a linear relationship with a negative slope.

• The cleavage of TaqMan probes or binding of SYBR Green intercalating dye to double-stranded DNA molecules results in an increase in fluorescence signal. This rise in fluorescence can be correlated to the initial DNA template amounts when compared with samples of known DNA concentration.

• Provided that there is good sample-to-sample consistency and precision, a sample with an unknown DNA quantity can be compared to this standard curve to calculate its initial DNA template concentration.

• Commercial kits for detecting human DNA as well as a real-time PCR assay for determining the amount of human Y-chromosome DNA present in a sample are now available. These kits include Quantifiler, Quantifiler Y, and Quantifiler Duo from Applied Biosystems and Plexor HY from Promega Corporation

Comparison of methods

• Several interlaboratory tests to evaluate DNA quantification methods have been conducted by the U.S. National Institute of Standards and Technology (NIST) to better understand the measurement variability seen with various techniques.

• A 10-fold range of reported concentrations was observed in one study.

• Most DNA quantitation measurements are precise to within a factor of two if performed properly.

• While this degree of imprecision may seem excessive, quantitation results are usually sufficiently valid to estimate DNA template amounts that will enable optimal PCR amplification.