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Recertification of the NIST Standard Reference Material® 2372, human DNA quantitation standard
Forensic Science International: Genetics Supplement Series 4 (2013) e256–e257
Contents lists available at ScienceDirect
Forensic Science International: Genetics Supplement Series journal homepage: www.elsevier.com/locate/FSIGSS
Recertification of the NIST Standard Reference Material1 2372, human DNA quantitation standard Peter M. Vallone *, Erica L.R. Butts, David L. Duewer, Margaret C. Kline U.S. National Institute of Standards and Technology, United States
A R T I C L E I N F O
A B S T R A C T
Article history: Received 13 August 2013 Accepted 2 October 2013
During the process of recertification of SRM 2372 (human DNA quantitation standard) a significant (14–26%) increase in decadic attenuance (absorbance) was detected. SRM 2372 consists of extracted human genomic DNA solutions assumed to be in the double stranded state. After further investigation by UV spectroscopy revealed that the materials were slowly dissociating to a single-stranded state. This did not affect the use of the materials in qPCR assays, but was in fact out of the limit of the NIST certified absorbance value. A method was developed to fully convert the materials to the single stranded state and absorbance was the re-measured. The recertified absorbance values were found to be within the error of common qPCR assays and allowed the materials to be returned to market. Published by Elsevier Ireland Ltd.
Keywords: Reference materials Absorbance qPCR
1. Introduction
2. Methods, results and discussion
The NIST Certified Reference Material SRM 2372 was designed for use in the value assignment of human genomic DNA forensic quantitation materials. SRM 2372 consists of three human genomic DNA extracts in TE 4 buffer, each originally certified to have spectrophotometric absorbance of 1.0 at 260 nm. However, by five years after production the absorbance in all materials had increased to the point that the certified values were no longer valid. Investigation revealed that the absorbance increases resulted from the slow conversion of double-stranded DNA (dsDNA) to single-stranded (ssDNA). The conventional conversion factor for dsDNA is 50 ng/mL per absorbance unit while that for ssDNA is 37 ng/mL. There was no evidence of any decrease in fragment size or change in behavior of the materials in numerous qPCR assays. Since the materials remain fit for their designed use, the remaining SRM 2372 units have been recertified for the spectroscopic properties of ssDNA. Users interested in the spectroscopic properties of these materials are instructed to force complete conversion to ssDNA with sodium hydroxide (NaOH). Users interested in using the materials to benchmark qPCR assays should use the materials as supplied but note that use of the ssDNA measurement procedure resulted in the ‘‘Information values’’ for conventional mass concentration supplied in the SRM 2372 Certificate changing from 50 ng/mL to 60 ng/mL.
2.1. Conversion of dsDNA to ssDNA with NaOH
* Corresponding author. Tel.: +1 301 975 4872; fax: +1 301 975 8505. E-mail address: [email protected] (P.M. Vallone). 1875-1768/$ – see front matter . Published by Elsevier Ireland Ltd. http://dx.doi.org/10.1016/j.fsigss.2013.10.131
The feasibility of using the NaOH treatment to entirely convert the SRM 2372 materials to ssDNA was demonstrated using an in house control DNA solution. The control DNA solution was diluted with TE 4 buffer to have a A260–320 of 1.000 (the difference in Absorbance at 260 nm 320 nm). Two aliquots of this solution were prepared, one diluted with an equal volume of TE 4 buffer and the second diluted with an equal volume of 0.40 mol/L freshly prepared NaOH in distilled water. The A260–320 of the TE 4-diluted material is about one-half of that from the undiluted parent. The ideal conventional ratio of the A260–320 values for complete conversion of ‘‘pure’’ dsDNA to ‘‘pure’’ ssDNA as specified in literature ranges from 50/40 to 50/37 [1–3]. The A260–320 of the NaOH-treated control was 50/40 larger than that of the TE 4diluted material. This is consistent with complete conversion of dsDNA–ssDNA. This validated NaOH dilution method was repeated on 18 unique units of the SRM for each of the three components. 2.2. UV spectroscopy The spectra were acquired over the wavelength range 220– 345 nm using the BioCary 100 spectrophotometer (Agilent, SantaClara, CA, USA). Before and after acquiring the spectra for the DNA solutions, SRM 2031 Metal-on-Fused-Silica Filters for Spectrophotometry and SRM 2034 Holmium Oxide Solution Wavelength from 240 nm to 650 nm were evaluated to enable verifying the wavelength and absorbance calibration of the instrument. The absorbance was measured twice over a period of 3 weeks.
P.M. Vallone et al. / Forensic Science International: Genetics Supplement Series 4 (2013) e256–e257 Table 1 Recertified Values, A260–320, for SRM 2372 components diluted with equal volumes of 0.4 mol/L NaOH. A
B
C
0.777 0.060
0.821 0.095
0.804 0.068
Parameter Value U95
Table 2 Information values for untreated solutions. Parameter A260–320 Conventional concentration (ng ssDNA/mL solution)
e257
assays and the rather wide tolerances of commercial STR multiplex assays, the approximately 10% (5 ng/mL) increase in this conventional concentration estimate will have minimal practical impact. Nor will certification of the A260–320 values using an approximate 95% level of confidence prediction interval rather than the much narrower spectrophotometric confidence interval of the originally certified D10 values. Role of funding
A
B
C
1.2 57
1.3 61
1.3 59
Forensic DNA research conducted at NIST is supported by an interagency agreement between the National Institute of Justice and the NIST Law Enforcement Standards Office. Conflict of interest
2.3. Recertified values Table 1 lists the recertified values: the apparent absorbance, A260–320, of the three components calculated as the difference in D10 at 260 nm D10 at 320 nm. Each value is the mean of 18 replicate measurements per SRM component on two separate days. These A260–320 BioCary 100 results are traceable through the SRM 2031 and SRM 2034 measurements to NIST’s high-accuracy reference spectrophotometer and through it to the International System of Units’ ratio unit ‘‘1’’. 2.4. Informational values We are also providing the conventional DNA concentration in ng/mL, based upon the proposed certified A260–320 values, the dilution factor of 2 for equal-volume dilution, and the conventional conversion factor of 37 specified in [2] (Table 2). 3. Conclusions Forcing the nominally dsDNA to all ssDNA prior to measurement enables certification of an apparently stable property of the solutions, the A260–320 of ssDNA. Given the variability among qPCR
None. Acknowledgements Points of view in this document are those of the authors and do not necessarily represent the official position or policies of the U.S. Department of Commerce. Certain commercial equipment, instruments, and materials are identified in order to specify experimental procedures as completely as possible. In no case does such identification imply a recommendation or endorsement by NIST, nor does it imply that any of the materials, instruments, or equipment identified are necessarily the best available for the purpose. References [1] EU-JRC, Protocol NK603 – Community Reference Laboratory, ‘‘Event-specific method for the quantification of maize line NK603 using real-time PCR’’, Section 3.3 Spectrophotometric Measurement of DNA Concentration, http://gmo-crl.jrc.ec.europa.eu/summaries/NK603-WEB-ProtocolValidation.pdf. [2] ISO 21571:2005(E), Foodstuffs – Methods of analysis for the detection of genetically modified organisms and derived products – Nucleic acid extraction. Annex B Methods for quantitation of extracted DNA, pp. 34–36. [3] J. Sambrook, D.W. Russell, Molecular Cloning a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001.
Contents lists available at ScienceDirect
Forensic Science International: Genetics Supplement Series journal homepage: www.elsevier.com/locate/FSIGSS
Recertification of the NIST Standard Reference Material1 2372, human DNA quantitation standard Peter M. Vallone *, Erica L.R. Butts, David L. Duewer, Margaret C. Kline U.S. National Institute of Standards and Technology, United States
A R T I C L E I N F O
A B S T R A C T
Article history: Received 13 August 2013 Accepted 2 October 2013
During the process of recertification of SRM 2372 (human DNA quantitation standard) a significant (14–26%) increase in decadic attenuance (absorbance) was detected. SRM 2372 consists of extracted human genomic DNA solutions assumed to be in the double stranded state. After further investigation by UV spectroscopy revealed that the materials were slowly dissociating to a single-stranded state. This did not affect the use of the materials in qPCR assays, but was in fact out of the limit of the NIST certified absorbance value. A method was developed to fully convert the materials to the single stranded state and absorbance was the re-measured. The recertified absorbance values were found to be within the error of common qPCR assays and allowed the materials to be returned to market. Published by Elsevier Ireland Ltd.
Keywords: Reference materials Absorbance qPCR
1. Introduction
2. Methods, results and discussion
The NIST Certified Reference Material SRM 2372 was designed for use in the value assignment of human genomic DNA forensic quantitation materials. SRM 2372 consists of three human genomic DNA extracts in TE 4 buffer, each originally certified to have spectrophotometric absorbance of 1.0 at 260 nm. However, by five years after production the absorbance in all materials had increased to the point that the certified values were no longer valid. Investigation revealed that the absorbance increases resulted from the slow conversion of double-stranded DNA (dsDNA) to single-stranded (ssDNA). The conventional conversion factor for dsDNA is 50 ng/mL per absorbance unit while that for ssDNA is 37 ng/mL. There was no evidence of any decrease in fragment size or change in behavior of the materials in numerous qPCR assays. Since the materials remain fit for their designed use, the remaining SRM 2372 units have been recertified for the spectroscopic properties of ssDNA. Users interested in the spectroscopic properties of these materials are instructed to force complete conversion to ssDNA with sodium hydroxide (NaOH). Users interested in using the materials to benchmark qPCR assays should use the materials as supplied but note that use of the ssDNA measurement procedure resulted in the ‘‘Information values’’ for conventional mass concentration supplied in the SRM 2372 Certificate changing from 50 ng/mL to 60 ng/mL.
2.1. Conversion of dsDNA to ssDNA with NaOH
* Corresponding author. Tel.: +1 301 975 4872; fax: +1 301 975 8505. E-mail address: [email protected] (P.M. Vallone). 1875-1768/$ – see front matter . Published by Elsevier Ireland Ltd. http://dx.doi.org/10.1016/j.fsigss.2013.10.131
The feasibility of using the NaOH treatment to entirely convert the SRM 2372 materials to ssDNA was demonstrated using an in house control DNA solution. The control DNA solution was diluted with TE 4 buffer to have a A260–320 of 1.000 (the difference in Absorbance at 260 nm 320 nm). Two aliquots of this solution were prepared, one diluted with an equal volume of TE 4 buffer and the second diluted with an equal volume of 0.40 mol/L freshly prepared NaOH in distilled water. The A260–320 of the TE 4-diluted material is about one-half of that from the undiluted parent. The ideal conventional ratio of the A260–320 values for complete conversion of ‘‘pure’’ dsDNA to ‘‘pure’’ ssDNA as specified in literature ranges from 50/40 to 50/37 [1–3]. The A260–320 of the NaOH-treated control was 50/40 larger than that of the TE 4diluted material. This is consistent with complete conversion of dsDNA–ssDNA. This validated NaOH dilution method was repeated on 18 unique units of the SRM for each of the three components. 2.2. UV spectroscopy The spectra were acquired over the wavelength range 220– 345 nm using the BioCary 100 spectrophotometer (Agilent, SantaClara, CA, USA). Before and after acquiring the spectra for the DNA solutions, SRM 2031 Metal-on-Fused-Silica Filters for Spectrophotometry and SRM 2034 Holmium Oxide Solution Wavelength from 240 nm to 650 nm were evaluated to enable verifying the wavelength and absorbance calibration of the instrument. The absorbance was measured twice over a period of 3 weeks.
P.M. Vallone et al. / Forensic Science International: Genetics Supplement Series 4 (2013) e256–e257 Table 1 Recertified Values, A260–320, for SRM 2372 components diluted with equal volumes of 0.4 mol/L NaOH. A
B
C
0.777 0.060
0.821 0.095
0.804 0.068
Parameter Value U95
Table 2 Information values for untreated solutions. Parameter A260–320 Conventional concentration (ng ssDNA/mL solution)
e257
assays and the rather wide tolerances of commercial STR multiplex assays, the approximately 10% (5 ng/mL) increase in this conventional concentration estimate will have minimal practical impact. Nor will certification of the A260–320 values using an approximate 95% level of confidence prediction interval rather than the much narrower spectrophotometric confidence interval of the originally certified D10 values. Role of funding
A
B
C
1.2 57
1.3 61
1.3 59
Forensic DNA research conducted at NIST is supported by an interagency agreement between the National Institute of Justice and the NIST Law Enforcement Standards Office. Conflict of interest
2.3. Recertified values Table 1 lists the recertified values: the apparent absorbance, A260–320, of the three components calculated as the difference in D10 at 260 nm D10 at 320 nm. Each value is the mean of 18 replicate measurements per SRM component on two separate days. These A260–320 BioCary 100 results are traceable through the SRM 2031 and SRM 2034 measurements to NIST’s high-accuracy reference spectrophotometer and through it to the International System of Units’ ratio unit ‘‘1’’. 2.4. Informational values We are also providing the conventional DNA concentration in ng/mL, based upon the proposed certified A260–320 values, the dilution factor of 2 for equal-volume dilution, and the conventional conversion factor of 37 specified in [2] (Table 2). 3. Conclusions Forcing the nominally dsDNA to all ssDNA prior to measurement enables certification of an apparently stable property of the solutions, the A260–320 of ssDNA. Given the variability among qPCR
None. Acknowledgements Points of view in this document are those of the authors and do not necessarily represent the official position or policies of the U.S. Department of Commerce. Certain commercial equipment, instruments, and materials are identified in order to specify experimental procedures as completely as possible. In no case does such identification imply a recommendation or endorsement by NIST, nor does it imply that any of the materials, instruments, or equipment identified are necessarily the best available for the purpose. References [1] EU-JRC, Protocol NK603 – Community Reference Laboratory, ‘‘Event-specific method for the quantification of maize line NK603 using real-time PCR’’, Section 3.3 Spectrophotometric Measurement of DNA Concentration, http://gmo-crl.jrc.ec.europa.eu/summaries/NK603-WEB-ProtocolValidation.pdf. [2] ISO 21571:2005(E), Foodstuffs – Methods of analysis for the detection of genetically modified organisms and derived products – Nucleic acid extraction. Annex B Methods for quantitation of extracted DNA, pp. 34–36. [3] J. Sambrook, D.W. Russell, Molecular Cloning a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001.