**2.1 Backgrounds**

Nucleic acids consist of deoxyribose nucleic acids (DNA) and ribose nucleic acids (RNA). It is well known that chemically more stable DNA carries genetic information in long terms whereas RNA promptly response to physiological demands by transferring genetic information to protein productions. For DNA, its base sequences, the genetic codes, are of

several decades. We can extend this approach whatever biological materials that have a discrete (countable) nature in principle. Quantification of large DNA particles is described in the following section as well as digital-polymerase chain reaction (digital-PCR) that can be also categorized into count-based quantification. In quantification of microorganisms,

Fig. 1.2. Approaches for establishment of technical basis for metrology for biology suggested by KRISS scientists (from the presentation at 2005 CCQM workshop on "New Challenges for the development of Primary or Higher Order Measurement methods and Procedures").

Several important biological quantities such as activity, viability, efficacy, and toxicity are not the quantities of biological molecules as above. Instead, these quantities are defined by the experts group of the related fields. These quantities are often called "method-dependent quantities" as those are determined by following the analytical procedures agreed among the experts groups. Metrology for such quantities is therefore of complexity and is often thought to be out of the domain of metrology. However, comparability of the measurement of those quantities is of great importance, and a great deal of improvement can be made by applying metrological rigorousness to the physical or chemical conditions involved in each analytical procedure. An example of such aspect is demonstrated in PCR in the following section. Noticeable technical advances have been made in metrology for quantification of nucleic acids and also some for proteins, but not yet for other complex biological materials such as cells and glycans. For these materials, a great deal of research should be done to visualize the practical paths forward realization of metrology. Here, only currently

Nucleic acids consist of deoxyribose nucleic acids (DNA) and ribose nucleic acids (RNA). It is well known that chemically more stable DNA carries genetic information in long terms whereas RNA promptly response to physiological demands by transferring genetic information to protein productions. For DNA, its base sequences, the genetic codes, are of

counting of cultured colonies is of a relatively long history.

conceivable technical issues are introduced.

**2.1 Backgrounds** 

**2. Metrology in quantification of nucleic acids** 

primary concerns. Base sequencing of DNA is a full-blown technique these days. The trend in technical advancement was in increment of throughput of analysis. Multiple channel capillary gel electrophoresis has been the most effective and prevailing technique in this regard. Recently, next-generation sequencing (NGS) technology featuring markedly higher throughput than multiple capillary sequencers have been commercialized for more widespread investigation and application of genetic information (Mardis, 2008). Here, the potential bias in DNA sequencing or accuracy in base-calls could be an important issue (Harismendy et al., 2009). However, this area is not in the main scope of metrology as metrology primarily concerns on the quantity. On the other hand, RNA has to be assessed not only about their identities but also regarding their quantities. The expression levels of RNA responding to physiological demands are of critical importance in understanding life processes. Accurate determination of the levels of RNA is especially important to globally share the obtained information. In this regard, related communities such as the Functional Genomics Data Society (FGDS, www.mged.org) actively work for achieving satisfactory levels of global harmonization in RNA quantification.

However, accurate assessment of the quantity of DNA is often crucial (O'Connor et al., 2002). One of the simplest forms of DNA is oligonucleotide. Synthetic oligonucleotides are widely used as an essential component of PCR (Dieffenbach et al., 1993; Halford, 1999), probes for various detection schemes (Mitsuhashi et al., 1994), even therapeutic agents as anti-sense drug (Pirollo et al., 2003; Tewary and Iversen, 1997) and agents for RNA interference (Doran, 2004). Some of these applications demand accurate quantitation of oligonucleotides. For example, accurate quantitation of oligonucleotide preparations is one of the most important concerns in the field of DNA-chip technology, where quantitative information from the applied oligonucleotide probes is crucial (Peterson et al., 2001). Accuracy in the quantity of DNA may govern the confidence and inter-comparability of the results of various experiments. In regulations of genetically modified organisms (GMOs), the quantity of a modified gene relative to an endogenous gene is the basis of legal judgments, and technical feasibility in determination of the quantity has been a crucial point for the successful implementation of the legal systems. In modern forensics, revolutionary technical advance has been made by short-tandem repeats (STR)-based human identification. This technique itself is not in the category of DNA quantification, but its reliability is strongly dependent upon the quantity as well as the quality of the obtained DNA samples. Therefore, accurate determination of the quantity of DNA in the sample specimen is an important prerequisite to enable its powerful utility in courts (Brettell et al., 2009). Among various biological substances, nucleic acids come first as the target materials for establishment of metrology for biology. Success in establishment of quantitative metrology for nucleic acids would be the litmus paper for the future success on establishment of metrology for biology.

As discussed in the following section, conventional analytical methods such as UVabsorption and fluorescence measurements as well as recent PCR-based measurements lack measurement traceability to the SI unit of mole. Even the most advanced mass spectrometry (MS) is not suitable to accurate quantify nucleic acids. Numerous reports on applications of MS techniques, mostly matrix assisted laser desorption ionization-time of flight (MALDI-TOF) MS (Bruenner et al., 1996; Zhang and Gross, 2000) are found but are not of adequate accuracy. The complex nature of the ionization of poly-ionic DNA material causes substantial uncertainty in quantitative analysis. Rigorous evaluations on quantitative performances of mass spectrometry in analysis of polymeric DNA molecules should be further carried out. Meanwhile, other forms of primary analytical methods for establishment of metrology in DNA quantification are demanded. In this regard, notable new approaches are discussed in the following sections.

### **2.2 Conventional methods**

Measurement of UV absorbance is most widely used for quantification of DNA. UV absorbance is commonly described in the unit of optical density (OD) of which definition is absorbance of UV light through 1 cm absorption path-length. For DNA, OD for 260 nm (OD260) is preferentially used since a local absorption maximum of DNA is at 260 nm. It is known that 1.0 OD260 corresponds to the absorption by a single strand oligonucleotide of 30-38 µg/mL, where the most commonly accepted value is 33 µg/mL. Although measurement of UV absorbance is quick and easy, the quantity of DNA estimated from OD is a crude approximation. Bases of DNA consist of four different kinds such as adenine (A), guanine (G), thymine (T) and cytosine (C), and the absorbance of a DNA string varies dependent upon the base composition. The purines of A and G featuring double hetero cyclic rings obviously show stronger molar absorption than the pyrimidine bases of T and C having one ring. The effect of base composition could be reflected in conversion of a UV OD value to a DNA quantity. However, such reflection is not simple due to hypochromicity effects caused by stacking among neighboring bases. A simple summing up method ignoring hypochromicity could lead to as highly as 24% overestimation of the extinction coefficient of a tested oligonucleotide (Cavaluzzi and Borer, 2004). Refinement in consideration of the sequence dependency of the extinction coefficient has been made to 'nearest-neighbor estimates' of extinction coefficients based on mono- and dinucleotide additivity rules (Kallansrud and Ward, 1996). Although quick and convenient, calculation of the concentrations of nucleic acids from the extinction coefficients and UV OD values can't be used as a definitive quantitation method for DNA analysis.

Measurement of fluorescence from dyes intercalated into DNA is known to be a highly sensitive quantitation method of DNA. The dyes are intercalated into DNA proportionally to the number of base units (Yan et al., 1999). Therefore, a linear relationship exists between the intensity of fluorescence and the quantity of the bases. However, the intensity of fluorescence depends on other parameters such as dye concentration, the degree of bleaching, intensity of the excitation light, the optics, and the geometry of a measurement cell as well as the quantity of DNA (Heid et al., 1996). For this reason, calibration using accurate measurement standards is indispensable to obtain accurate quantitative results in fluorometric determination of DNA.

Polymerase chain reaction (PCR), especially in real-time fluorescence detection mode (rt-PCR) or quantitative PCR (q-PCR), is widely used for quantification of specific genes in a DNA mixture for various purposes (Heid et al., 1996; Ponchel et al., 2003). The exponential amplification of PCR renders unprecedented sensitivity to the detection of DNA. If the degree of amplification is monitored at the early stage of the exponential amplification, a linear relationship between the logarithm of the quantity of DNA and the PCR cycle for a given amplification level is obtained (Heid et al., 1996). In PCR amplification, high

substantial uncertainty in quantitative analysis. Rigorous evaluations on quantitative performances of mass spectrometry in analysis of polymeric DNA molecules should be further carried out. Meanwhile, other forms of primary analytical methods for establishment of metrology in DNA quantification are demanded. In this regard, notable new approaches

Measurement of UV absorbance is most widely used for quantification of DNA. UV absorbance is commonly described in the unit of optical density (OD) of which definition is absorbance of UV light through 1 cm absorption path-length. For DNA, OD for 260 nm (OD260) is preferentially used since a local absorption maximum of DNA is at 260 nm. It is known that 1.0 OD260 corresponds to the absorption by a single strand oligonucleotide of 30-38 µg/mL, where the most commonly accepted value is 33 µg/mL. Although measurement of UV absorbance is quick and easy, the quantity of DNA estimated from OD is a crude approximation. Bases of DNA consist of four different kinds such as adenine (A), guanine (G), thymine (T) and cytosine (C), and the absorbance of a DNA string varies dependent upon the base composition. The purines of A and G featuring double hetero cyclic rings obviously show stronger molar absorption than the pyrimidine bases of T and C having one ring. The effect of base composition could be reflected in conversion of a UV OD value to a DNA quantity. However, such reflection is not simple due to hypochromicity effects caused by stacking among neighboring bases. A simple summing up method ignoring hypochromicity could lead to as highly as 24% overestimation of the extinction coefficient of a tested oligonucleotide (Cavaluzzi and Borer, 2004). Refinement in consideration of the sequence dependency of the extinction coefficient has been made to 'nearest-neighbor estimates' of extinction coefficients based on mono- and dinucleotide additivity rules (Kallansrud and Ward, 1996). Although quick and convenient, calculation of the concentrations of nucleic acids from the extinction coefficients and UV OD values can't be used as a definitive quantitation method for DNA

Measurement of fluorescence from dyes intercalated into DNA is known to be a highly sensitive quantitation method of DNA. The dyes are intercalated into DNA proportionally to the number of base units (Yan et al., 1999). Therefore, a linear relationship exists between the intensity of fluorescence and the quantity of the bases. However, the intensity of fluorescence depends on other parameters such as dye concentration, the degree of bleaching, intensity of the excitation light, the optics, and the geometry of a measurement cell as well as the quantity of DNA (Heid et al., 1996). For this reason, calibration using accurate measurement standards is indispensable to obtain accurate quantitative results in

Polymerase chain reaction (PCR), especially in real-time fluorescence detection mode (rt-PCR) or quantitative PCR (q-PCR), is widely used for quantification of specific genes in a DNA mixture for various purposes (Heid et al., 1996; Ponchel et al., 2003). The exponential amplification of PCR renders unprecedented sensitivity to the detection of DNA. If the degree of amplification is monitored at the early stage of the exponential amplification, a linear relationship between the logarithm of the quantity of DNA and the PCR cycle for a given amplification level is obtained (Heid et al., 1996). In PCR amplification, high

are discussed in the following sections.

**2.2 Conventional methods** 

analysis.

fluorometric determination of DNA.

selectivity to a specific gene sequence is achieved by using the unique sequences of a primer pair. Therefore, the unknown amount of a target gene sequence can be determined from a calibration curve. However, q-PCR requires calibration standards as the amplification yield substantially depends upon various experimental conditions. Calibration materials of accurately determined quantity are not available yet, and the calibration curves are usually on the scale of the relative quantity of DNA. Although less precise in quantification (~ 10% RSD) and needs to be carefully performed (Halford, 1999), q-PCR is a prevailing quantification method for specific genes and requires urgent development of suitable calibration standards to have measurement traceability to the SI unit of mole.
