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Assessing DNA recovery from highly degraded skeletal remains by using silica-based extraction methods
Journal Pre-proof ASSESSING DNA RECOVERY FROM HIGHLY DEGRADED SKELETAL REMAINS BY USING SILICA-BASED EXTRACTION METHODS Diana C. Vinueza-Espinosa, Cristina Santos, Cristina Mart´ınez-Labarga, Assumpcio´ Malgosa
PII:
S1875-1768(19)30457-3
DOI:
https://doi.org/10.1016/j.fsigss.2019.10.204
Reference:
FSIGSS 1837
To appear in:
Forensic Science International: Genetics Supplement Series
Received Date:
16 September 2019
Accepted Date:
16 October 2019
Please cite this article as: Vinueza-Espinosa DC, Santos C, Mart´ınez-Labarga C, Malgosa A, ASSESSING DNA RECOVERY FROM HIGHLY DEGRADED SKELETAL REMAINS BY USING SILICA-BASED EXTRACTION METHODS, Forensic Science International: Genetics Supplement Series (2019), doi: https://doi.org/10.1016/j.fsigss.2019.10.204
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier.
ASSESSING DNA RECOVERY FROM HIGHLY DEGRADED SKELETAL REMAINS BY USING SILICA-BASED EXTRACTION METHODS Diana C. Vinueza-Espinosaa*, Cristina Santosa, Cristina Martínez-Labargab, Assumpció Malgosaa. a
Biology Anthropology Research Group, Department of Animal Biology, Vegetal Biology and Ecology, Autonomous University of Barcelona, Spain.
b
Centre of Molecular Anthropology for Ancient DNA Studies. Department of Biology, University of Rome Tor Vergata, Italy Corresponding Author Assumpció Malgosa: [email protected]
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ABSTRACT
re
Keyword: silica extraction; degraded human bones; PCR.
-p
Through this study, we extracted human DNA as for five different type of skeletal remains: petrous bone, tooth pulp cavity, tooth cementum, rib and at least two different upper limbs bones (radial, ulna, metacarpal or phalange) from five individuals (5-12th centuries AD) by using four extraction methods; three based on DNA adsorption to silicon dioxide (silica) particles, Non-Columns Silica (NCSi), Silica-HE Spin Columns (SiHEC), Silica-XS Spin Columns (SiXSC) and the traditional organic extraction method (P-Chl) (N:100 DNA extracts). The results showed that the NCSi and SiHEC extraction methods, petrous bones and pulp cavity allowed recovering the highest amount of PCR-amplifiable human mtDNA. The autosomal STRs profiles from petrous DNA presented >70% reportable alleles and peak heights between 50-498 RFUs using both extraction methods. The lower amount of DNA was showed from rib and P-Chl extraction method.
Introduction
lP
During the last decade, cases of missing persons, mass disasters, wars and socio-political disturbances have been solved through genetic identification. However, one of the most important challenges is the extraction of genetic material from highly-degraded bones and teeth since DNA is present in small amounts and fragmented in small pieces, which makes extraction and purification difficult.
na
Many methodological strategies for DNA extraction from skeletal remains have been published, some coming from the ancient DNA research [1–3] and others established in forensic laboratories [4,5]. In forensics, the most common extraction method has been phenol/chloroform; nonetheless, recent works showed that silica-based (silica in suspension, with columns or beads) methodology has better DNA recovery and more efficient removal of inhibitors. However, the variation in DNA yield depends not only on the extraction method but also on the type of skeletal remains used.
ur
The main goal of this study is to determine the best extraction method (the traditional organic extraction method and three silicabased extraction methods - in suspension and with membrane-columns) and the type of skeletal remain that could maximize the recovery of PCR-amplifiable DNA relevant for forensic genetic identification.
Jo
Material and Methods
Sample Preparation and DNA extraction methods Five individuals from the Casseres medieval archaeological site located in Catalonia-Spain dated between 5-11th century AD were selected. Petrous bone, two areas of the teeth: pulp cavity and cementum, rib and at least two different upper limbs bones (radial, ulna, metacarpal or phalange) were collected from each individual. The samples’ surfaces were cleaned using a dental instrument machine; then we collected roughly 120-800 mg total sample powder per each type of skeletal remain and 30-200 mg of powder was used to perform each kind of DNA extraction. Four extraction methods were tested. A negative extraction control without powder per each set of five samples was also included, representing a total of 100 DNA extracts and 25 controls.
Non-Columns Silica-Based (NCSi): according to Allentoft et al. [6] Silica-HE Spin Columns-Based (SiHEC): according to Rohland et al. 2018 [1]
Silica-XS Spin Columns -Based (SiXSC): the protocol was performed based on manufacturer’s guidelines (NucleoSpin® Plasma XS kit). Phenol-Chloroform (P-Chl): according to Hagelberg & Clegg (1991) [7] Additionally, a final DNA phase concentration by using Amicon ® Ultra 0.5 ml Filters (Merck, Germany).
PCR amplification and sequencing of mitochondrial DNA For all the DNA extracts, a mitochondrial DNA fragment (mtDNA) of 203-bp located in HVS-I between the position 16030 and 16230 was amplified with primers developed by Simon et al. 2011 [8]. The PCR was carried out using the Rotor-Gene®-Q with the 2X Type-it HRM PCR Kit (Qiagen, Germany). Two µl of the extract were used in the PCR reaction. The conditions were: hold at 95ºC for 10 min, denaturation at 95ºC for 10 seconds, annealing at 58ºC for 45 seconds and extension at 72ºC for 10 seconds. The product amplified was quantified by Qubit™ dsDNA HS Assay Kits. MtDNA sequences were analyzed with the sequence Scanner v1.0 (Thermo Fisher Scientific ®, UK) and the alignment with the BioEdit Sequence software version 7.0.9.0. Analysis of nuclear DNA
ro of
For the DNA samples extracted with the two methods that reveal best performance in mtDNA analysis (NCSi and SiHEC), nuclear DNA was also analyzed. First, a 85-bp region that encompasses the -13910C/T polymorphism strongly associate with Lactase Persistent in Europeans (LCT) was amplified with primers developed by Burger et al. 2007 [9]. The PCR conditions were performed as described for mtDNA but using an annealing temperature of 53ºC. The product amplified through PCR was quantified by Qubit™ dsDNA HS Assay Kits. Second, the extracts were amplified using the GlobalFiler® PCR amplification Kit (Thermo Fisher Scientific), 4 µl of the extract were used in the PCR protocol was selected according to manufacturer’s recommendation. Results
-p
Amplification and sequencing of mitochondrial DNA
re
The best results evaluated by the amplified fragment concentration of mtDNA were attributed to the presence of amplification using the NCSi (15.72 ng/µl) and SiHEC (14.32ng/µl) extraction methods; regarding the type of skeletal remains were petrous DNA (18.12 ng/µl) and DNA pulp cavity (12.80 ng/µl). Rib (2.29ng/µl), and Ph-Cl extraction method (1.40ng/µl) showed the lowest results attributed to the absence of amplification. Samples extracted with NCSi and SiHEC extraction method successfully amplified were sequenced and a good sequence quality was obtained in all the cases. Moreover, the concordance between mtDNA profiles of the same individual obtained with different methods and from different bone pieces was verified.
lP
Amplification of nuclear DNA
na
The quantification of the LCT gene amplified indicated that the highest mean values were of 5.37ng/µl from petrous DNA and 4.85ng/µl from pulp cavity DNA using NCSi extraction method (Fig 1A). However, the differences obtained were not statistically significant (p=0.069-0.89) paired with all the trials except for petrous DNA by NCSi extraction protocol paired with the pulp cavity DNA by SiHEC extraction technique (p=0.015). The pulp cavity DNA using SiHEC extraction method did not show amplification of any STR. Regarding peaks height (RFUs), 498.60 RFUs and 228.50 RFUs were the highest values obtained from the petrous DNA using NCSi and SiHEC extraction method respectively. There were significant differences between this assays (p=0.016). (Figure 1B)
Jo
ur
The Peaks Height Ratio (PHR) showed the highest percentage of balance between heterozygous alleles of 53.08 % and 62.89% from the petrous extracts using both extraction methods. However, the differences observed were not statistically significant (p=0.090.47) (Figure 1C). The highest percentages of reportable alleles were of 79.09 % and 71.81% from the petrous DNA using both extraction methods, being the differences with the pulp cavity DNA statistically significant (p=0.035-0.049) (Figure 1D)
Discussion
The DNA extraction from petrous using the NCSi and SiHEC extraction methods showed the best results in the analyses of mtDNA and nuclear DNA, which demonstrates that the success of the DNA recovery does not only depend on the extraction methods used but also on the amount of DNA preserved inside of sample. This is related to that the endogenous genetic material from petrous bones and teeth are more protected against extreme environmental factors as humidity, sunlight and presence of microorganism, avoiding its complete degradation. The rib DNA using the four extraction methods showed the lowest results. However, the NCSi and SiHEC protocol recovered some genetic material, conversely when Ph-Cl extraction method was used almost no DNA was recovered. This could be related with the fact that based-silica methods are more efficient removing, avoiding and minimizing the co-extraction of inhibitors.
Conflict of interest statement
None
Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Acknowledgments The work was supported by Generalitat of Cataluña (AGAUR. Ref 2017SGR1630)
References N. Rohland, I. Glocke, A. Aximu-petri, et al. Extraction of highly degraded DNA from ancient bones , teeth and sediments for high-throughput sequencing, 13 (2018) 2447–2461.
[2]
C. Gamba, K. Hanghøj, C. Gaunitz, et al. Comparing the performance of three ancient DNA extraction methods for highthroughput sequencing, (2016) 459–469. doi:10.1111/1755-0998.12470.
[3]
N. Rohland and M. Hofreiter, Comparison and optimization of ancient DNA extraction, Biotechniques. 42 (2007) 343– 352. doi:10.2144/000112383.
[4]
P.L. Marshall, M. Stoljarova, S.E. Schmedes, et al. A high volume extraction and purification method for recovering DNA from human bone, Forensic Sci. Int. Genet. 12 (2014) 155–160. doi:10.1016/J.FSIGEN.2014.06.011.
[5]
R. Huel, S. Amory, A. Bili, et al. DNA Electrophoresis Protocols for Forensic Genetics, 830 (2012) 185–198. doi:10.1007/978-1-61779-461-2.
[6]
M.E. Allentoft, M. Sikora, K.-G. Sjögren, et al. Population genomics of Bronze Age Eurasia, Nature. 522 (2015) 167. https://doi.org/10.1038/nature14507.
[7]
E. Hagelberg and J.B. Clegg, Isolation and characterization of DNA from archaeological bone, Proc. R. Soc. B Biol. Sci. 244 (1991) 45–50. doi:10.1098/rspb.1991.0049.
[8]
M. Simón, X. Jordana, N. Armentano, et al., The presence of nuclear families in prehistoric collective burials revisited: The bronze age burial of montanissell cave (Spain) in the light of aDNA, Am. J. Phys. Anthropol. 146 (2011) 406–413. doi:10.1002/ajpa.21590.
[9]
J. Burger, M. Kirchner, B. Bramanti, et al. Absence of the lactase-persistence-associated allele in early Neolithic Europeans, Proc. Natl. Acad. Sci. 104 (2007) 3736–3741. doi:10.1073/pnas.0607187104.
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ur
na
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[1]
Figures
Petrous-NCSi
Petrous-SiHEC
Pulp cavity-NCSi
Pulp cavity-SiHEC
5.37
A
Petrous-NCSi
Petrous-SiHEC
Pulp cavity-NCSi
Pulp cavity-SiHEC
B
498.6
4.85 4.1
228.5 0.85
57.82
Nuclear DNA amplified concentration (ng/ul) Petrous-SiHEC
Pulp cavity-NCSi
Pulp cavity-SiHEC
Peak Height (RFUs)
C
Petrous-NCSi
Petrous-SiHEC
Pulp cavity-NCSi
Pulp cavity-SiHEC
79.09
62.89 53.08 23.46
D
ro of
Petrous-NCSi
0
71.81
24.54
0
0
%PHR
% Reportable Alelles
Jo
ur
na
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re
-p
Figure 1: Means of the values of (A) the concentration of nuclear DNA amplified (ng/µl) and comparison of the quality parameters of short tandem repeat (STRs) profile between the petrous and pulp cavity using NCSi and SiHEC extraction methods.(B) Peak height (RFUs) (C) Peaks height ratio (%), (D) Alleles reported (%)
PII:
S1875-1768(19)30457-3
DOI:
https://doi.org/10.1016/j.fsigss.2019.10.204
Reference:
FSIGSS 1837
To appear in:
Forensic Science International: Genetics Supplement Series
Received Date:
16 September 2019
Accepted Date:
16 October 2019
Please cite this article as: Vinueza-Espinosa DC, Santos C, Mart´ınez-Labarga C, Malgosa A, ASSESSING DNA RECOVERY FROM HIGHLY DEGRADED SKELETAL REMAINS BY USING SILICA-BASED EXTRACTION METHODS, Forensic Science International: Genetics Supplement Series (2019), doi: https://doi.org/10.1016/j.fsigss.2019.10.204
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier.
ASSESSING DNA RECOVERY FROM HIGHLY DEGRADED SKELETAL REMAINS BY USING SILICA-BASED EXTRACTION METHODS Diana C. Vinueza-Espinosaa*, Cristina Santosa, Cristina Martínez-Labargab, Assumpció Malgosaa. a
Biology Anthropology Research Group, Department of Animal Biology, Vegetal Biology and Ecology, Autonomous University of Barcelona, Spain.
b
Centre of Molecular Anthropology for Ancient DNA Studies. Department of Biology, University of Rome Tor Vergata, Italy Corresponding Author Assumpció Malgosa: [email protected]
ro of
ABSTRACT
re
Keyword: silica extraction; degraded human bones; PCR.
-p
Through this study, we extracted human DNA as for five different type of skeletal remains: petrous bone, tooth pulp cavity, tooth cementum, rib and at least two different upper limbs bones (radial, ulna, metacarpal or phalange) from five individuals (5-12th centuries AD) by using four extraction methods; three based on DNA adsorption to silicon dioxide (silica) particles, Non-Columns Silica (NCSi), Silica-HE Spin Columns (SiHEC), Silica-XS Spin Columns (SiXSC) and the traditional organic extraction method (P-Chl) (N:100 DNA extracts). The results showed that the NCSi and SiHEC extraction methods, petrous bones and pulp cavity allowed recovering the highest amount of PCR-amplifiable human mtDNA. The autosomal STRs profiles from petrous DNA presented >70% reportable alleles and peak heights between 50-498 RFUs using both extraction methods. The lower amount of DNA was showed from rib and P-Chl extraction method.
Introduction
lP
During the last decade, cases of missing persons, mass disasters, wars and socio-political disturbances have been solved through genetic identification. However, one of the most important challenges is the extraction of genetic material from highly-degraded bones and teeth since DNA is present in small amounts and fragmented in small pieces, which makes extraction and purification difficult.
na
Many methodological strategies for DNA extraction from skeletal remains have been published, some coming from the ancient DNA research [1–3] and others established in forensic laboratories [4,5]. In forensics, the most common extraction method has been phenol/chloroform; nonetheless, recent works showed that silica-based (silica in suspension, with columns or beads) methodology has better DNA recovery and more efficient removal of inhibitors. However, the variation in DNA yield depends not only on the extraction method but also on the type of skeletal remains used.
ur
The main goal of this study is to determine the best extraction method (the traditional organic extraction method and three silicabased extraction methods - in suspension and with membrane-columns) and the type of skeletal remain that could maximize the recovery of PCR-amplifiable DNA relevant for forensic genetic identification.
Jo
Material and Methods
Sample Preparation and DNA extraction methods Five individuals from the Casseres medieval archaeological site located in Catalonia-Spain dated between 5-11th century AD were selected. Petrous bone, two areas of the teeth: pulp cavity and cementum, rib and at least two different upper limbs bones (radial, ulna, metacarpal or phalange) were collected from each individual. The samples’ surfaces were cleaned using a dental instrument machine; then we collected roughly 120-800 mg total sample powder per each type of skeletal remain and 30-200 mg of powder was used to perform each kind of DNA extraction. Four extraction methods were tested. A negative extraction control without powder per each set of five samples was also included, representing a total of 100 DNA extracts and 25 controls.
Non-Columns Silica-Based (NCSi): according to Allentoft et al. [6] Silica-HE Spin Columns-Based (SiHEC): according to Rohland et al. 2018 [1]
Silica-XS Spin Columns -Based (SiXSC): the protocol was performed based on manufacturer’s guidelines (NucleoSpin® Plasma XS kit). Phenol-Chloroform (P-Chl): according to Hagelberg & Clegg (1991) [7] Additionally, a final DNA phase concentration by using Amicon ® Ultra 0.5 ml Filters (Merck, Germany).
PCR amplification and sequencing of mitochondrial DNA For all the DNA extracts, a mitochondrial DNA fragment (mtDNA) of 203-bp located in HVS-I between the position 16030 and 16230 was amplified with primers developed by Simon et al. 2011 [8]. The PCR was carried out using the Rotor-Gene®-Q with the 2X Type-it HRM PCR Kit (Qiagen, Germany). Two µl of the extract were used in the PCR reaction. The conditions were: hold at 95ºC for 10 min, denaturation at 95ºC for 10 seconds, annealing at 58ºC for 45 seconds and extension at 72ºC for 10 seconds. The product amplified was quantified by Qubit™ dsDNA HS Assay Kits. MtDNA sequences were analyzed with the sequence Scanner v1.0 (Thermo Fisher Scientific ®, UK) and the alignment with the BioEdit Sequence software version 7.0.9.0. Analysis of nuclear DNA
ro of
For the DNA samples extracted with the two methods that reveal best performance in mtDNA analysis (NCSi and SiHEC), nuclear DNA was also analyzed. First, a 85-bp region that encompasses the -13910C/T polymorphism strongly associate with Lactase Persistent in Europeans (LCT) was amplified with primers developed by Burger et al. 2007 [9]. The PCR conditions were performed as described for mtDNA but using an annealing temperature of 53ºC. The product amplified through PCR was quantified by Qubit™ dsDNA HS Assay Kits. Second, the extracts were amplified using the GlobalFiler® PCR amplification Kit (Thermo Fisher Scientific), 4 µl of the extract were used in the PCR protocol was selected according to manufacturer’s recommendation. Results
-p
Amplification and sequencing of mitochondrial DNA
re
The best results evaluated by the amplified fragment concentration of mtDNA were attributed to the presence of amplification using the NCSi (15.72 ng/µl) and SiHEC (14.32ng/µl) extraction methods; regarding the type of skeletal remains were petrous DNA (18.12 ng/µl) and DNA pulp cavity (12.80 ng/µl). Rib (2.29ng/µl), and Ph-Cl extraction method (1.40ng/µl) showed the lowest results attributed to the absence of amplification. Samples extracted with NCSi and SiHEC extraction method successfully amplified were sequenced and a good sequence quality was obtained in all the cases. Moreover, the concordance between mtDNA profiles of the same individual obtained with different methods and from different bone pieces was verified.
lP
Amplification of nuclear DNA
na
The quantification of the LCT gene amplified indicated that the highest mean values were of 5.37ng/µl from petrous DNA and 4.85ng/µl from pulp cavity DNA using NCSi extraction method (Fig 1A). However, the differences obtained were not statistically significant (p=0.069-0.89) paired with all the trials except for petrous DNA by NCSi extraction protocol paired with the pulp cavity DNA by SiHEC extraction technique (p=0.015). The pulp cavity DNA using SiHEC extraction method did not show amplification of any STR. Regarding peaks height (RFUs), 498.60 RFUs and 228.50 RFUs were the highest values obtained from the petrous DNA using NCSi and SiHEC extraction method respectively. There were significant differences between this assays (p=0.016). (Figure 1B)
Jo
ur
The Peaks Height Ratio (PHR) showed the highest percentage of balance between heterozygous alleles of 53.08 % and 62.89% from the petrous extracts using both extraction methods. However, the differences observed were not statistically significant (p=0.090.47) (Figure 1C). The highest percentages of reportable alleles were of 79.09 % and 71.81% from the petrous DNA using both extraction methods, being the differences with the pulp cavity DNA statistically significant (p=0.035-0.049) (Figure 1D)
Discussion
The DNA extraction from petrous using the NCSi and SiHEC extraction methods showed the best results in the analyses of mtDNA and nuclear DNA, which demonstrates that the success of the DNA recovery does not only depend on the extraction methods used but also on the amount of DNA preserved inside of sample. This is related to that the endogenous genetic material from petrous bones and teeth are more protected against extreme environmental factors as humidity, sunlight and presence of microorganism, avoiding its complete degradation. The rib DNA using the four extraction methods showed the lowest results. However, the NCSi and SiHEC protocol recovered some genetic material, conversely when Ph-Cl extraction method was used almost no DNA was recovered. This could be related with the fact that based-silica methods are more efficient removing, avoiding and minimizing the co-extraction of inhibitors.
Conflict of interest statement
None
Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Acknowledgments The work was supported by Generalitat of Cataluña (AGAUR. Ref 2017SGR1630)
References N. Rohland, I. Glocke, A. Aximu-petri, et al. Extraction of highly degraded DNA from ancient bones , teeth and sediments for high-throughput sequencing, 13 (2018) 2447–2461.
[2]
C. Gamba, K. Hanghøj, C. Gaunitz, et al. Comparing the performance of three ancient DNA extraction methods for highthroughput sequencing, (2016) 459–469. doi:10.1111/1755-0998.12470.
[3]
N. Rohland and M. Hofreiter, Comparison and optimization of ancient DNA extraction, Biotechniques. 42 (2007) 343– 352. doi:10.2144/000112383.
[4]
P.L. Marshall, M. Stoljarova, S.E. Schmedes, et al. A high volume extraction and purification method for recovering DNA from human bone, Forensic Sci. Int. Genet. 12 (2014) 155–160. doi:10.1016/J.FSIGEN.2014.06.011.
[5]
R. Huel, S. Amory, A. Bili, et al. DNA Electrophoresis Protocols for Forensic Genetics, 830 (2012) 185–198. doi:10.1007/978-1-61779-461-2.
[6]
M.E. Allentoft, M. Sikora, K.-G. Sjögren, et al. Population genomics of Bronze Age Eurasia, Nature. 522 (2015) 167. https://doi.org/10.1038/nature14507.
[7]
E. Hagelberg and J.B. Clegg, Isolation and characterization of DNA from archaeological bone, Proc. R. Soc. B Biol. Sci. 244 (1991) 45–50. doi:10.1098/rspb.1991.0049.
[8]
M. Simón, X. Jordana, N. Armentano, et al., The presence of nuclear families in prehistoric collective burials revisited: The bronze age burial of montanissell cave (Spain) in the light of aDNA, Am. J. Phys. Anthropol. 146 (2011) 406–413. doi:10.1002/ajpa.21590.
[9]
J. Burger, M. Kirchner, B. Bramanti, et al. Absence of the lactase-persistence-associated allele in early Neolithic Europeans, Proc. Natl. Acad. Sci. 104 (2007) 3736–3741. doi:10.1073/pnas.0607187104.
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ur
na
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re
-p
ro of
[1]
Figures
Petrous-NCSi
Petrous-SiHEC
Pulp cavity-NCSi
Pulp cavity-SiHEC
5.37
A
Petrous-NCSi
Petrous-SiHEC
Pulp cavity-NCSi
Pulp cavity-SiHEC
B
498.6
4.85 4.1
228.5 0.85
57.82
Nuclear DNA amplified concentration (ng/ul) Petrous-SiHEC
Pulp cavity-NCSi
Pulp cavity-SiHEC
Peak Height (RFUs)
C
Petrous-NCSi
Petrous-SiHEC
Pulp cavity-NCSi
Pulp cavity-SiHEC
79.09
62.89 53.08 23.46
D
ro of
Petrous-NCSi
0
71.81
24.54
0
0
%PHR
% Reportable Alelles
Jo
ur
na
lP
re
-p
Figure 1: Means of the values of (A) the concentration of nuclear DNA amplified (ng/µl) and comparison of the quality parameters of short tandem repeat (STRs) profile between the petrous and pulp cavity using NCSi and SiHEC extraction methods.(B) Peak height (RFUs) (C) Peaks height ratio (%), (D) Alleles reported (%)