- Email: [email protected]
DNA extraction with silica membrane column from teeth left in soil for more than 60 years
Forensic Science International: Genetics Supplement Series 5 (2015) e133–e134
Contents lists available at ScienceDirect
Forensic Science International: Genetics Supplement Series journal homepage: www.elsevier.com/locate/FSIGSS
DNA extraction with silica membrane column from teeth left in soil for more than 60 years K. Tsukada* , Y. Harayama Department of Forensic Medicine, Criminal Investigation Laboratory, Nagano Pref. Police H.Q., Japan
A R T I C L E I N F O
A B S T R A C T
Article history: Received 25 August 2015 Accepted 14 September 2015 Available online 16 September 2015
We performed DNA extraction from four teeth left in soil for more than 60 years by three methods (AP method, EP method and AEP method). DNA extraction was performed with QIAamp DNA Investigator Kit, DNA typing with AmpFlSTR Identifiler Plus PCR amplification Kit, and electrophoresis with the Applied Biosystems 3130xl Genetic Analyzer. The AEP method is highly efficient in extracting DNA from teeth left in soil for more than 60 years. ã 2015 Elsevier Ireland Ltd. All rights reserved.
Keywords: Teeth Bone Ancient STR Identifiler QIAamp
1. Introduction DNA typing is relatively easy to perform with blood, saliva, semen, and so forth. The same is generally true of DNA typing of teeth or bone. However, it is often difficult to perform DNA typing of teeth or bone discovered in the soil typically encountered in Japan, due to the acidity and moisture content of the soil. In this study, we performed DNA extraction and DNA typing from four teeth exposed to the soil environment for more than 60 years. 2. Materials and methods The teeth in question were discovered along with four bones left in the soil for more than 60 years. The teeth were dried in an oven after washing and brushing of their surface. The teeth were pulverized using a Multi-Beads Shocker MB701(S) (Yasui Kikai, Osaka, Japan). After transferring 200 mg each of the powdered teeth into a 2.0 ml tube, we performed DNA extraction by the AP method (600 ml of Buffer ATL +30 ml of Proteinase K), EP method (600 ml of 0.5 M EDTA (pH 8.0) +30 ml of Proteinase K), and the AEP method (300 ml of Buffer ATL + 300 ml of 0.5 M EDTA (pH 8.0) +30 ml of Proteinase K). Overnight incubation was performed at 37 C in a Thermomixer Comfort (shaken at 900 rpm for 10 s at 1 min intervals) (Eppendorf AG, Hamburg, Germany). Following
* Corresponding author. E-mail address: [email protected] (K. Tsukada). http://dx.doi.org/10.1016/j.fsigss.2015.09.054 1875-1768/ ã 2015 Elsevier Ireland Ltd. All rights reserved.
incubation, the tubes were centrifuged at 15,000 rpm for 5 min, after which the supernatant was transferred to a new 2.0 ml tube. We added 600 ml of Buffer AL to each tube, then continued with the protocol for the QIAamp DNA Investigator kit (QIAGEN GmbH, Hilden, Germany). DNA quantification was performed using Applied Biosystems 7500 RealTime PCR System (Life Technologies/Thermo Fisher Scientific, South San Francisco, CA, USA) [1]; PCR amplification was performed using AmpFlSTR Identifiler Plus PCR Amplification Kit. Electrophoresis was performed using the Applied Biosystems 3130xl Genetic Analyzer. Signal intensities exceeding 150 RFU were regarded as valid. 3. Results We performed DNA extraction from four teeth left in soil for more than 60 years by three methods. The efficiency of DNA extraction by the EP method was about 4 times greater than by the AP method. The efficiency of DNA extraction by the AEP method was more than 5 times greater than by the AP method (Table 1). When PCR amplification was performed using DNA extracted by the three methods, the AEP method proved most effective in detecting signals. When PCR amplification was performed using DNA extracted by the AP method, detectable signals exceeded those detected by the EP method, although the DNA yield with the AP method was less than that with the EP method. When PCR amplification was performed using DNA extracted by the EP method, few signals were detected, with the exception of Sample 3 (Table 2).
e134
K. Tsukada, Y. Harayama / Forensic Science International: Genetics Supplement Series 5 (2015) e133–e134
Table 1 Results of DNA extraction. Method
AP method EP method AEP method
adsorption into silica membrane and pH by adding sodium acetate (3 M, pH 5.0) [3]. The results showed that DNA concentrations extracted by the AEP method did not differ with or without sodium acetate. When we performed PCR amplification using DNA extracted by the EP method, we detected fewer loci than the AP method, except for Sample 3, although the total amount of DNA extracted by the EP method exceeded the amount extracted by the AP method. We performed DNA quantification using 1 ml of DNA extracted in 12 ml
DNA concentration (ng/ml) Sample 1
Sample 2
Sample 3
Sample 4
0.0005 0.0019 0.0023
0.0006 0.0004 0.0009
0.1537 0.156 0.1659
0.057 0.1928 0.292
Table 2 Results of DNA typing. Method
Sample 1 a
AP method EP method AEP method
Sample 2 b
a
Sample 3 b
Partial
Complete
Partial
Complete
2
4
3
2
7
3 2 3
a
Partial
Sample 4 b
Complete 16 16 16
6
Partialb
Completeb
1
11 4 12
Abcent: not detected. a Amount of loci of not full DNA profile. bAmount of loci of full DNA profile.
4. Discussion The phenol-chloroform method is a popular DNA extraction method for bone and teeth. However, in this study, DNA extraction was not performed by organic-based method, because the aqueous phase was colored. PCR reactions are often inhibited when color is observed in the aqueous phase. In general, when DNA is extracted from bone or teeth, a decalcification step is performed for a few days with a change in the EDTA solution, followed by a digestion step after the EDTA solution is discarded. However, our results showed high DNA concentrations in the EDTA solution after decalcification (Table 1). This indicates the EDTA solution after decalcification is critical as a crude DNA extraction solution. The efficiency of DNA extraction by the AP method was equivalent to that by the EP method or lower, whereas that of AEP method was highest. These results show that decalcification may be required for DNA extraction from bone or teeth, but is not necessarily a requirement before Proteinase K treatment. The results support the findings from a previous study [2]. Based on reports that DNA is not adsorbed by silica membrane under alkaline conditions, we examined the relationship between DNA
of reaction mixture but performed PCR amplification using 10 ml of DNA extracted in 25 ml of reaction mixture (in the case of Sample 4, 5 ml of extracted DNA usage as template). The proportion of DNA extract in the PCR mix was higher than in the quantification mix, suggesting that something in the extract inhibits the PCR reaction. Our results underscore the importance of the decalcification solution following decalcification. Additionally, our results suggest that the AEP method is the most efficient method for extracting DNA from bone or teeth. 5. Conclusion We demonstrated the presence of high concentrations of DNA in decalcification solutions. The AEP method is highly efficient in extracting DNA from teeth left in soil for more than 60 years. References [1] K. Tsukada, et al., Influence of presumptive reagents on DNA typing, Forensic Sci. Int.: Genet. Suppl. Ser. 3 (2011) e375–e376. [2] D.N.A. Bone, extraction and purification using silica-coated paramagnetic beads, Am. J. Phys. Anthropol. 148 (2012) 473–482. [3] MinElute Handbook, QIAGEN GmbH.
Contents lists available at ScienceDirect
Forensic Science International: Genetics Supplement Series journal homepage: www.elsevier.com/locate/FSIGSS
DNA extraction with silica membrane column from teeth left in soil for more than 60 years K. Tsukada* , Y. Harayama Department of Forensic Medicine, Criminal Investigation Laboratory, Nagano Pref. Police H.Q., Japan
A R T I C L E I N F O
A B S T R A C T
Article history: Received 25 August 2015 Accepted 14 September 2015 Available online 16 September 2015
We performed DNA extraction from four teeth left in soil for more than 60 years by three methods (AP method, EP method and AEP method). DNA extraction was performed with QIAamp DNA Investigator Kit, DNA typing with AmpFlSTR Identifiler Plus PCR amplification Kit, and electrophoresis with the Applied Biosystems 3130xl Genetic Analyzer. The AEP method is highly efficient in extracting DNA from teeth left in soil for more than 60 years. ã 2015 Elsevier Ireland Ltd. All rights reserved.
Keywords: Teeth Bone Ancient STR Identifiler QIAamp
1. Introduction DNA typing is relatively easy to perform with blood, saliva, semen, and so forth. The same is generally true of DNA typing of teeth or bone. However, it is often difficult to perform DNA typing of teeth or bone discovered in the soil typically encountered in Japan, due to the acidity and moisture content of the soil. In this study, we performed DNA extraction and DNA typing from four teeth exposed to the soil environment for more than 60 years. 2. Materials and methods The teeth in question were discovered along with four bones left in the soil for more than 60 years. The teeth were dried in an oven after washing and brushing of their surface. The teeth were pulverized using a Multi-Beads Shocker MB701(S) (Yasui Kikai, Osaka, Japan). After transferring 200 mg each of the powdered teeth into a 2.0 ml tube, we performed DNA extraction by the AP method (600 ml of Buffer ATL +30 ml of Proteinase K), EP method (600 ml of 0.5 M EDTA (pH 8.0) +30 ml of Proteinase K), and the AEP method (300 ml of Buffer ATL + 300 ml of 0.5 M EDTA (pH 8.0) +30 ml of Proteinase K). Overnight incubation was performed at 37 C in a Thermomixer Comfort (shaken at 900 rpm for 10 s at 1 min intervals) (Eppendorf AG, Hamburg, Germany). Following
* Corresponding author. E-mail address: [email protected] (K. Tsukada). http://dx.doi.org/10.1016/j.fsigss.2015.09.054 1875-1768/ ã 2015 Elsevier Ireland Ltd. All rights reserved.
incubation, the tubes were centrifuged at 15,000 rpm for 5 min, after which the supernatant was transferred to a new 2.0 ml tube. We added 600 ml of Buffer AL to each tube, then continued with the protocol for the QIAamp DNA Investigator kit (QIAGEN GmbH, Hilden, Germany). DNA quantification was performed using Applied Biosystems 7500 RealTime PCR System (Life Technologies/Thermo Fisher Scientific, South San Francisco, CA, USA) [1]; PCR amplification was performed using AmpFlSTR Identifiler Plus PCR Amplification Kit. Electrophoresis was performed using the Applied Biosystems 3130xl Genetic Analyzer. Signal intensities exceeding 150 RFU were regarded as valid. 3. Results We performed DNA extraction from four teeth left in soil for more than 60 years by three methods. The efficiency of DNA extraction by the EP method was about 4 times greater than by the AP method. The efficiency of DNA extraction by the AEP method was more than 5 times greater than by the AP method (Table 1). When PCR amplification was performed using DNA extracted by the three methods, the AEP method proved most effective in detecting signals. When PCR amplification was performed using DNA extracted by the AP method, detectable signals exceeded those detected by the EP method, although the DNA yield with the AP method was less than that with the EP method. When PCR amplification was performed using DNA extracted by the EP method, few signals were detected, with the exception of Sample 3 (Table 2).
e134
K. Tsukada, Y. Harayama / Forensic Science International: Genetics Supplement Series 5 (2015) e133–e134
Table 1 Results of DNA extraction. Method
AP method EP method AEP method
adsorption into silica membrane and pH by adding sodium acetate (3 M, pH 5.0) [3]. The results showed that DNA concentrations extracted by the AEP method did not differ with or without sodium acetate. When we performed PCR amplification using DNA extracted by the EP method, we detected fewer loci than the AP method, except for Sample 3, although the total amount of DNA extracted by the EP method exceeded the amount extracted by the AP method. We performed DNA quantification using 1 ml of DNA extracted in 12 ml
DNA concentration (ng/ml) Sample 1
Sample 2
Sample 3
Sample 4
0.0005 0.0019 0.0023
0.0006 0.0004 0.0009
0.1537 0.156 0.1659
0.057 0.1928 0.292
Table 2 Results of DNA typing. Method
Sample 1 a
AP method EP method AEP method
Sample 2 b
a
Sample 3 b
Partial
Complete
Partial
Complete
2
4
3
2
7
3 2 3
a
Partial
Sample 4 b
Complete 16 16 16
6
Partialb
Completeb
1
11 4 12
Abcent: not detected. a Amount of loci of not full DNA profile. bAmount of loci of full DNA profile.
4. Discussion The phenol-chloroform method is a popular DNA extraction method for bone and teeth. However, in this study, DNA extraction was not performed by organic-based method, because the aqueous phase was colored. PCR reactions are often inhibited when color is observed in the aqueous phase. In general, when DNA is extracted from bone or teeth, a decalcification step is performed for a few days with a change in the EDTA solution, followed by a digestion step after the EDTA solution is discarded. However, our results showed high DNA concentrations in the EDTA solution after decalcification (Table 1). This indicates the EDTA solution after decalcification is critical as a crude DNA extraction solution. The efficiency of DNA extraction by the AP method was equivalent to that by the EP method or lower, whereas that of AEP method was highest. These results show that decalcification may be required for DNA extraction from bone or teeth, but is not necessarily a requirement before Proteinase K treatment. The results support the findings from a previous study [2]. Based on reports that DNA is not adsorbed by silica membrane under alkaline conditions, we examined the relationship between DNA
of reaction mixture but performed PCR amplification using 10 ml of DNA extracted in 25 ml of reaction mixture (in the case of Sample 4, 5 ml of extracted DNA usage as template). The proportion of DNA extract in the PCR mix was higher than in the quantification mix, suggesting that something in the extract inhibits the PCR reaction. Our results underscore the importance of the decalcification solution following decalcification. Additionally, our results suggest that the AEP method is the most efficient method for extracting DNA from bone or teeth. 5. Conclusion We demonstrated the presence of high concentrations of DNA in decalcification solutions. The AEP method is highly efficient in extracting DNA from teeth left in soil for more than 60 years. References [1] K. Tsukada, et al., Influence of presumptive reagents on DNA typing, Forensic Sci. Int.: Genet. Suppl. Ser. 3 (2011) e375–e376. [2] D.N.A. Bone, extraction and purification using silica-coated paramagnetic beads, Am. J. Phys. Anthropol. 148 (2012) 473–482. [3] MinElute Handbook, QIAGEN GmbH.