**2.5 Summary**

Metrology for biology has been pursued first in quantification of nucleic acids. Due to the polymeric nature of nucleic acids, conventional methods are not as accurate as a metrological standard should be. In addition, measurement traceability was hardly established. Efforts of many scientists, mostly scientists at various NMIs, have been poured for establishment of metrology for DNA quantification, and substantial achievements have been made. Reductive approaches have been well developed to establish measurement traceability, and count-based quantification methods for trace level DNA copies were also successfully progressed. Although there are some technical challenges still remained, the overall technical structure for metrology in this area is visible now (Fig. 2.4).

Soon, the products of metrology such as certified reference materials in DNA quantification will be available and their widespread uses are expected. Consequently, the credibility in data of DNA quantification will be dramatically improved in various areas such as R&D, industrial, medical, and regulation sectors. More important, the success in the effort to establish metrology for nucleic acid quantification will encourage the scientists who are gazing over the technical challenges in establishment of technical infrastructures for modern biology.

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

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

Metrology for biology has been pursued first in quantification of nucleic acids. Due to the polymeric nature of nucleic acids, conventional methods are not as accurate as a metrological standard should be. In addition, measurement traceability was hardly established. Efforts of many scientists, mostly scientists at various NMIs, have been poured for establishment of metrology for DNA quantification, and substantial achievements have been made. Reductive approaches have been well developed to establish measurement traceability, and count-based quantification methods for trace level DNA copies were also successfully progressed. Although there are some technical challenges still remained, the overall technical structure for metrology in this area is

Soon, the products of metrology such as certified reference materials in DNA quantification will be available and their widespread uses are expected. Consequently, the credibility in data of DNA quantification will be dramatically improved in various areas such as R&D, industrial, medical, and regulation sectors. More important, the success in the effort to establish metrology for nucleic acid quantification will encourage the scientists who are gazing over the technical challenges in establishment of technical infrastructures for modern

accurately determined by other reliable methods like the direct counting method.

of such methods.

**2.5 Summary** 

visible now (Fig. 2.4).

biology.

Fig. 2.4. Core analytical techniques developed for establishment of metrology in DNA quantification, and their links to validations or applications
