**2.3 Reductive approaches**

Nucleic acids are polymers of nucleotide units which consist of base, carbohydrate, and phosphodiester bond (Fig. 2.1). As polymeric nucleic acids are not readily quantifiable even with advanced mass spectrometry due to the multiple charges, the components of nucleic acids could be measurement targets in reductive approaches. Firstly, it is noticeable that each unit of nucleotide bears one phosphorus atom in the phosphodiester backbone. Therefore, the quantity of a DNA sequence of a known length (base pairs: bp) can be accurately determined if the quantity of residual phosphorus is quantified accurately. Isotope dilution-mass spectrometry (IDMS) accepted by the metrology community is not an applicable option for determination of phosphorous as the isotope of phosphorus is radioactive (32P and 33P). Instead, inductively coupled plasma–optical emission spectroscopy (ICP-OES) may be chosen as it is capable of quantification of elemental phosphorus with reasonable precision and accuracy. KRISS scientists first tried this measurement approach where all substantial validation points were carefully examined (Yang et al., 2004). Phosphorus measured by ICP-OES should all come from the target DNA sequence. As a synthetic 20-mer DNA oligomer was quantified as a model DNA, all possible phosphorous contamination in the form of small molecules were removed by applying ultrafiltration of the sample solution. Complete removal was confirmed by the observation of complete disappearance of excessively spiked cytidyl monophosphate (dCMP) after repeated ultrafiltration. In phosphorus measurement, a certified reference material (CRM) of pure phosphorus was used as a calibrant, which provides measurement traceability. With the help of acid and microwave heating, DNA in the sample solution was digested to inorganic phosphates to avoid potential influences from the organic residues of the original molecular structure. An organic compound of a similar structure was excessively spiked into the sample solution to confirm the effect of the spiked organic additive disappears through the digestion process. Analytical performance of ICP-OES in measurement of phosphorus in element solutions has been within 0.2% of accuracy. For the nucleic acids it was estimated within 1% of expanded uncertainty. Sequence impurities of DNA oligomer was minimized by polyacrylamide gel electrophoresis (PAGE) purification, and confirmed using MALDI-TOF MS. As far as such validation points are well confirmed, the ICP-OES method seems to be a fine quantitative method for DNA with measurement traceability and assessable measurement uncertainty. This method has been used to determine the quantity of DNA materials for international comparison studies for DNA quantification. Several publications have been made in an attempt to establish the ICP-OES based phosphorous quantification as a primary analytical method for DNA quantification (English et al., 2006; Holden et al., 2007; Brennan et al., 2009).

One can target different moieties in DNA for the reductive measurement approach. Nucleotide could be the quantification target (O'Connor et al., 2002) whereas nucleoside can be also a measurement target (Donald et al., 2005). Phosphodiesterase produces single nucleotide from a DNA sequence with its activity for hydrolysis of phosphodiester bonds at the 5'-position. If the resultant nucleotides are further subject to phosphatase reactions, then nucleosides are produced. Dependent upon the suitability to available analytical methods, either form can be chosen for quantitation.

Fig. 2.1. Chemical structure of DNA (or RNA)

If the exact base composition of the target DNA is known, then its quantity can be readily deduced from the results of quantitation of nucleotides or nucleosides. O'Connor et al. first reported the use of HPLC-IDMS for accurate quantitation of nucleotides resulted from enzymatic hydrolysis of a DNA sequence (O'Connor et al., 2002). Later, they reported that analysis of the nucleosides instead of nucleotides resulted in better estimation of the quantity of the target DNA (Donald et al., 2005). For both analytical targets, isotope dilutionmass spectrometry (ID-MS) was applied to secure metrological quality of analysis. However, measurement traceability might not be well established due to the lack of high quality reference materials for preparation of nucleotides or nucleosides standard solutions. Some of those chemicals are too hygroscopic to accurately weigh the actual amounts in preparation of standard solutions. Instead, the exact quantity in the standard solutions can be determined by phosphorous measurement using ICP-OES where any phosphorus contamination of the nucleotide standard solutions can be determined using HPLC-ICP-MS in which different phosphorous-bearing species are separated and determined in terms of phosphorous quantity. Using a high resolution separation method, any partially degraded nucleotides can be also determined. Recently, Hong et al. reported a highly reliable capillary electrophoresis method for determination of nucleotides (Hong et al., 2011). The accurately determined nucleotide standard solutions will provide measurement traceability to this reductive approach. Utilizing those analytical techniques, several NMIs are preparing for the certified reference materials (CRMs) of the nucleotide solutions.

One can target different moieties in DNA for the reductive measurement approach. Nucleotide could be the quantification target (O'Connor et al., 2002) whereas nucleoside can be also a measurement target (Donald et al., 2005). Phosphodiesterase produces single nucleotide from a DNA sequence with its activity for hydrolysis of phosphodiester bonds at the 5'-position. If the resultant nucleotides are further subject to phosphatase reactions, then nucleosides are produced. Dependent upon the suitability to available analytical methods,

If the exact base composition of the target DNA is known, then its quantity can be readily deduced from the results of quantitation of nucleotides or nucleosides. O'Connor et al. first reported the use of HPLC-IDMS for accurate quantitation of nucleotides resulted from enzymatic hydrolysis of a DNA sequence (O'Connor et al., 2002). Later, they reported that analysis of the nucleosides instead of nucleotides resulted in better estimation of the quantity of the target DNA (Donald et al., 2005). For both analytical targets, isotope dilutionmass spectrometry (ID-MS) was applied to secure metrological quality of analysis. However, measurement traceability might not be well established due to the lack of high quality reference materials for preparation of nucleotides or nucleosides standard solutions. Some of those chemicals are too hygroscopic to accurately weigh the actual amounts in preparation of standard solutions. Instead, the exact quantity in the standard solutions can be determined by phosphorous measurement using ICP-OES where any phosphorus contamination of the nucleotide standard solutions can be determined using HPLC-ICP-MS in which different phosphorous-bearing species are separated and determined in terms of phosphorous quantity. Using a high resolution separation method, any partially degraded nucleotides can be also determined. Recently, Hong et al. reported a highly reliable capillary electrophoresis method for determination of nucleotides (Hong et al., 2011). The accurately determined nucleotide standard solutions will provide measurement traceability to this reductive approach. Utilizing those analytical techniques, several NMIs are preparing for

either form can be chosen for quantitation.

Fig. 2.1. Chemical structure of DNA (or RNA)

the certified reference materials (CRMs) of the nucleotide solutions.

Regardless of the analytical methods applied for analysis of nucleotides or nucleosides, any imperfection of enzymatic hydrolysis will lead to bias in the final results. Therefore, the completeness of the enzymatic hydrolysis has to be carefully assured. Either an insufficient or excessive amount of hydrolysis enzymes may lead to abnormality in the final results. Fortunately, however, any substantial imperfection in hydrolysis can be unambiguously found by comparison of the quantitation results of four nucleotides or nucleosides. This aspect is a great advantage of the methods simultaneously determining four nucleotides or nucleosides. Compared to the ICP-OES method above, this group of analytical procedures requires substantially less amounts of samples, which dramatically expands the applicability. Therefore, the best approach in metrological quantification of nucleic acids will be as follows: 1) measurement traceability is established by the ICP-OES procedure; then 2) quantification of nucleic acids is performed by measuring nucleotides or nucleosides after enzyme hydrolysis where CRMs determined by the ICP-OES procedure is applied.

## **2.4 Count-based quantification**

Another interesting approach in metrological quantification of DNA is a count-based quantitation of a trace level DNA. Two methods have been reported in this category. Firstly, several research groups have explored the potential of digital PCR (d-PCR) that aims amplify a single copy of a target gene in a microplate well (Sykes et al., 1992; Vogelstein and Kinzler, 1999). With appropriate dilution, the target gene is to be at the concentration level that only a single copy goes into a microwell or not (Fig. 2.2). Based on Poisson approximation of binomial distribution, at this concentration range, the population of wells that contain a single copy of the target gene among the given number wells will reveal the original concentration of the target gene if the dilution factor is properly considered. Occupation of each well can be determined by the fluorescence signal from the PCR amplicons stained with a fluorescent dye. The concept of digital PCR was first successfully transformed to a commercial instrumentation by Fluidigm (www.fluidigm.com), and commercialization was followed by several other companies. Interesting applications of digital PCR can be readily found (Zimmermann et al., 2008).

Fig. 2.2. Schematic illustration of the measurement principle of digital PCR for absolute quantification of DNA

Digital PCR, in principle, does not require calibration as the results of quantification comes from the fundamental distribution (Poission binomial distribution). Therefore, scientists at NMIs pay great attention to this particular measurement technique as it may accurately quantify the amount of DNA without calibration when no appropriate calibration materials are available. A number of publications exploring such possibility can be found (Bhat et al., 2010; Bhat et al., 2009; Corbisier et al., 2010; Sanders et al., 2011). However, there is an important validation point for the use of digital PCR as a metrological DNA quantification method. Calculation for the results of a digital PCR method assumes a 100% success rate for single-copy PCR amplification. Any deviation from 100% success rate will lead to underestimation. In practice, however, 100% success rate for a single-copy PCR is not readily achievable. As reported by Bhat et al., applied PCR conditions such as the status of template DNA or priming sites could result in variations in measurement results (Bhat et al., 2009). For this reason, digital PCR should be carefully validated to draw metrological determination of DNA quantity. Therefore, one would do his or her best to accomplish the 100% success rate for PCR amplification. The judgment for reaching the condition for 100% success rate can be surely made only if a certified reference material (CRM) with actual quantity of the target gene sequence is given.

As described above, digital PCR is a count-based DNA quantitation method for which commercial instruments can be conveniently utilized. However, its validity can be easily checked or demonstrated only if a suitable CRM is available. The other approach of the count-based quantitation of DNA may function in this purpose. KRISS scientists have worked on developing a method and instrumentation for counting individual DNA single molecules on a flow stream. DNA is stained with an intercalating dye and detected by laser induced fluorescence (LIF) detection. For securing enough fluorescence intensity, however, DNA particles need to be large enough. They succeeded in counting lambda phage DNA (45.8 kbp) single molecules (Fig. 2.3) (Lim et al., 2009) and now is capable of counting plasmid DNAs as small as 2 kbp. The LIF detection in this approach does not involve PCR but directly measure fluorescence intensity from the target DNA. Therefore, failure in PCR is not an issue. Direct measurement of fluorescence intensity is rather straight forward and less likely involves errors. In addition, the fluorescence intensity is proportional to the size of the DNA, which gives information on the size of each counted DNA particle. In this counting method, the molar concentration of target DNA is determined by the following simple equation:

#### *C* = (*Nc*/*NA*)/*V*

Where *C*: molar concentration; *Nc*: countednumber; *NA*: Avogadro's number; *V*: sample volume.

As indicated by the equation above, measurement uncertainty may rise from uncertainty in counting of DNA particles (Nc) and estimation of sample volume (V). There are several check points for assuring accuracy of Nc as described in detail in the previous publication (Lim et al., 2009). All DNA particles should pass the detection points of a flow channel. Concentration of DNA should be limited under a certain level that simultaneous passing of a number of DNA particles is avoided. Occasional simultaneous passage of two DNA particles should be detectable and counted accordingly by the help of an algorithm for analysis of superimposed peaks. Signal to noise ratio should be large enough to reject background noise in counting. Validation of count-based determination of lambda phage DNA was attempted by comparison with the result of CE-dNMP analysis (Hong et al., 2011), for which the sample concentration was about 100 thousand-fold thicker than the sample for counting. The counting method resulted in about 30% less quantity for lambda phage DNA.

Digital PCR, in principle, does not require calibration as the results of quantification comes from the fundamental distribution (Poission binomial distribution). Therefore, scientists at NMIs pay great attention to this particular measurement technique as it may accurately quantify the amount of DNA without calibration when no appropriate calibration materials are available. A number of publications exploring such possibility can be found (Bhat et al., 2010; Bhat et al., 2009; Corbisier et al., 2010; Sanders et al., 2011). However, there is an important validation point for the use of digital PCR as a metrological DNA quantification method. Calculation for the results of a digital PCR method assumes a 100% success rate for single-copy PCR amplification. Any deviation from 100% success rate will lead to underestimation. In practice, however, 100% success rate for a single-copy PCR is not readily achievable. As reported by Bhat et al., applied PCR conditions such as the status of template DNA or priming sites could result in variations in measurement results (Bhat et al., 2009). For this reason, digital PCR should be carefully validated to draw metrological determination of DNA quantity. Therefore, one would do his or her best to accomplish the 100% success rate for PCR amplification. The judgment for reaching the condition for 100% success rate can be surely made only if a certified reference material (CRM) with actual

As described above, digital PCR is a count-based DNA quantitation method for which commercial instruments can be conveniently utilized. However, its validity can be easily checked or demonstrated only if a suitable CRM is available. The other approach of the count-based quantitation of DNA may function in this purpose. KRISS scientists have worked on developing a method and instrumentation for counting individual DNA single molecules on a flow stream. DNA is stained with an intercalating dye and detected by laser induced fluorescence (LIF) detection. For securing enough fluorescence intensity, however, DNA particles need to be large enough. They succeeded in counting lambda phage DNA (45.8 kbp) single molecules (Fig. 2.3) (Lim et al., 2009) and now is capable of counting plasmid DNAs as small as 2 kbp. The LIF detection in this approach does not involve PCR but directly measure fluorescence intensity from the target DNA. Therefore, failure in PCR is not an issue. Direct measurement of fluorescence intensity is rather straight forward and less likely involves errors. In addition, the fluorescence intensity is proportional to the size of the DNA, which gives information on the size of each counted DNA particle. In this counting method, the molar concentration of target DNA is determined by the following simple equation:

*C* = (*Nc*/*NA*)/*V* Where *C*: molar concentration; *Nc*: countednumber; *NA*: Avogadro's number; *V*: sample volume. As indicated by the equation above, measurement uncertainty may rise from uncertainty in counting of DNA particles (Nc) and estimation of sample volume (V). There are several check points for assuring accuracy of Nc as described in detail in the previous publication (Lim et al., 2009). All DNA particles should pass the detection points of a flow channel. Concentration of DNA should be limited under a certain level that simultaneous passing of a number of DNA particles is avoided. Occasional simultaneous passage of two DNA particles should be detectable and counted accordingly by the help of an algorithm for analysis of superimposed peaks. Signal to noise ratio should be large enough to reject background noise in counting. Validation of count-based determination of lambda phage DNA was attempted by comparison with the result of CE-dNMP analysis (Hong et al., 2011), for which the sample concentration was about 100 thousand-fold thicker than the sample for counting. The

counting method resulted in about 30% less quantity for lambda phage DNA.

quantity of the target gene sequence is given.

Fig. 2.3. A single lambda DNA captured at 1 ms intervals (A), signals displayed on an oscilloscope (B), and count result displayed in a histogram (C). In panel B, the upper trace indicates the fluorescence intensity, and the lower trace is the digital pulse to be counted (Lim et al., 2009).

In counting plasmid DNA, however, such underestimation disappeared. Signal intensity from plasmid DNA such as pBR322 (4.8 kbp) is 10-fold less than that from lambda phage DNA. Therefore, the underestimation for lambda phage DNA counting should not be due to the weak fluorescence signal for detection. Instead, the underestimation is highly likely due to the fragmentation of lambda viral DNA in the given sample. Small fragments (< 2 kbp) were not counted in counting of lambda phage DNA as the fluorescence signals were far less than that of lambda phage DNA. However, the CE method measures nucleotides from all DNA fragments regardless of their sizes. Therefore, it is possible that the result of CE analysis is substantially greater than the counting result. In contrast, pBR322 DNA that is smaller than lambda phage DNA is too small to be readily fragmented by shear stress (Yoo et al., 2011). The lack of small DNA fragments in pBR322 sample is concordant to the close agreement between the counting method and the CE method. This feature should be carefully considered in determination of what we want to measure. If we want to measure only the DNA molecules of whole integrity, then the counting method gives the right answer from its resultant histogram (count vs. size). On the other hand, if we are interested in the quantity of all DNA fragments, then what the CE method measures is the right answer. Digital PCR in this regard measures a portion of DNA sequences encompassed by a primer pair, which should not necessarily be the same as the number of integral DNA molecules. The "measurand" (exactly what to measure) of each method is significantly different, and this point should be well considered in comparing the results of such methods.

In conclusion, digital PCR will be widely used in attempts to quantify the copies of DNA sequences as commercial instrumentations have become available. However, such applications may have to be confirmed with certified reference materials (CRMs) that are accurately determined by other reliable methods like the direct counting method.
