In Vitro Isolation and Molecular Characterization of Genomic DNA from Ancient Human Long and Hip Bones

Dinesh Kumar; Lalit Mohan Jeena; Devendra Shekhawat; Ira Verma; Sidd Sana

doi:10.4103/mamcjms.mamcjms_57_22

ORIGINAL ARTICLE

Year : 2023 | Volume : 9 | Issue : 1 | Page : 44-49

In Vitro Isolation and Molecular Characterization of Genomic DNA from Ancient Human Long and Hip Bones

Dinesh Kumar¹, Lalit Mohan Jeena², Devendra Shekhawat¹, Ira Verma³, Sidd Sana³
¹ Department of Anatomy, Maulana Azad Medical College, New Delhi, India
² IVF & Reproductive Biology Centre, Maulana Azad Medical College, New Delhi, India
³ Surajmal Agarwal Private Kanya Mahavidyalaya, Kichha, Uttarakhand, India

Date of Submission	11-Sep-2022
Date of Acceptance	31-Dec-2022
Date of Web Publication	28-Apr-2023

Correspondence Address:
Lalit Mohan Jeena
IVF & Reproductive Biology Centre, Maulana Azad Medical College, New Delhi-110002
India

Source of Support: None, Conflict of Interest: None

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DOI: 10.4103/mamcjms.mamcjms_57_22

Abstract

Background: A simple and effective modified ethanol precipitation-based protocol was described for the extraction of genomic DNA from ancient human bones. The qualitative and quantitative evaluation of genomic DNA was done based on DNA purity (260/280) and the PCR method. Method and Materials: In this study, a total of 50 embalmed ancient bones, including 20 long and 30 hip bone samples, were taken for genomic DNA extraction. The efficiency of the genomic DNA extraction was demonstrated on >50-year-old ancient human HIP and long bone samples. In vitro quantitative and qualitative analysis of extracted genomic DNA was performed by 0.8% agarose gel electrophoresis and PCR amplification. To assess the quality of extracted genomic DNA, a mitochondrial-specific ATPase6 gene primer was used to obtain sequence information of 675 bp. Result: Our data show that a concentration of genomic DNA between 1.6 and 2.0 at 260/280 resulted in successful PCR amplification. Our results demonstrated that the extraction of DNA from ancient bone samples with a manual approach will increase the amplification efficiency of the polymerase chain reaction (PCR). Conclusion: In the present study, a simple, time-saving, and cost-effective protocol is described for the extraction of genomic DNA from ancient human bones. Further, we believe the extraction of genomic DNA from ancient bone samples with this approach will increase the success rate of PCR amplification.

Keywords: ATPase6 gene, ancient bones, genomic DNA, in vitro, polymerase chain reaction

How to cite this article:
Kumar D, Jeena LM, Shekhawat D, Verma I, Sana S. In Vitro Isolation and Molecular Characterization of Genomic DNA from Ancient Human Long and Hip Bones. MAMC J Med Sci 2023;9:44-9

How to cite this URL:
Kumar D, Jeena LM, Shekhawat D, Verma I, Sana S. In Vitro Isolation and Molecular Characterization of Genomic DNA from Ancient Human Long and Hip Bones. MAMC J Med Sci [serial online] 2023 [cited 2023 Jun 6];9:44-9. Available from: https://www.mamcjms.in/text.asp?2023/9/1/44/375335

Introduction

The introduction of new DNA analysis and profiling techniques for human identification is a recent development in molecular and forensic investigations. The application of DNA-based identification method can be used, when recognition cannot be based only on available physical parameters. In the last few decades, serious efforts have been continuously made to identify human degraded bones, teeth, and skeletons found in archaeological sites, natural and man-made disasters or wars in order to identify people responsible for crimes and solve paternity issues.^[1],[21] Extraction of genomic DNA and successful polymerase chain reaction (PCR) amplification from ancient and stored human bones and teeth have a key role in archaeological and forensic investigations. It is noted that it is not easy to extract DNA from such sources because the methods for DNA extraction are not always satisfactory.^[1],[2] This is due to the quality of the postmortem tissue being variably compromised due to multiple factors, such as infections, or disease, or time, cause of death as well as antemortem and postmortem treatment. Ancient DNA is heavily modified, which is mainly attributed to oxidative processes. That is why ancient genomic DNA is mainly responsible for the low recovery rate of high-quality DNA from specimens.^[3],[4],[5]

The majority of previously published methods involved powdering bone material and incubating it in various extraction buffers as the first step in DNA extraction.^[2],[3],[4] There is a plethora of published methods for DNA extraction, including those from kits, phenol-chloroform^[8],[9],[10] or dialyzed against EDTA and Tris–HCl buffer solution.^[3],[4],[5] Factors including DNA degradation processes such as cross-linking, deamination, and fragmentation, are mainly responsible for ancient DNA degradation in comparison with contemporary genetic material.^[1],[2],[3] Even in the best preservation conditions, there is an upper boundary of 0.4–1.5 million years for a sample to contain sufficient DNA for sequencing technologies.^[2] Genomic DNA has been recovered from mummified tissues,^[13] archival collections of non-frozen medical specimens,^[14] paleo-archaeological and historical skeletal material, preserved plant tissues,^[15],[16] ice and permafrost cores, marine and lake sediments, and excavation dirt.^{[13],[17],[18],[19],[20]}

In this study, a simple and effective ethanol precipitation-based method was used for genomic DNA extraction from dried and treated bones. This protocol was found to be more efficient, cost-effective, and capable of extracting appreciable amount of quality and quantity of DNA with less contamination. This method does not involve any hazardous or expensive chemicals, special devices/kits, or any kind of specific enzyme. The efficiency of this protocol was demonstrated on 50 damaged human bone samples from the Anatomy Bone Bank. The quantitative analysis of extracted DNA was assessed by electrophoresis (0.8% agarose gel), and the qualitative analysis was performed using a nanodrop spectrophotometer with the DNA concentration set at 260/280. A mitochondrial-specific primer pair was used to obtain sequence amplification of the ATPase6 gene 675bp to assess the amplifiability of extracted genomic DNA.

Materials and Methods

All the chemicals and media were purchased from Sigma Chemicals Co. (St. Louis, MO, USA) and disposable plasticware from Tarsons (Private Limited).

Biological sample collection

The study used 50 dried and damaged human bones, including 30 long lower-limb bones and 20 hip bones. These bones were stored in the bone bank of the Anatomy Department, Maulana Azad Medical College (MAMC), New Delhi, India, for >50 years.

Cleaning and decalcification of bones

For this study, bone samples were used that had been preserved in the bone bank of the anatomy department for >50 years. Bone samples were cleaned by scraping off dirt with scalpels and forceps. Approximately 2 cm³ size of bones were taken for the DNA extraction. Bone samples were washed 3–4 times with molecular grade water and subsequently with a 10% solution of sodium hypochlorite. Further, to remove the sodium hypochlorite, the bone samples were kept in molecular grade water for 30 minutes. The bone samples were air-dried in a sterile environment at room temperature (25°C) for 3–5 hours. Thereafter, the bone samples were placed in a sterile mortar-pestle for pulverization. Liquid nitrogen (−196°C) was poured on the partially crushed bone samples to make a fine powder [Figure 1]. Approximately 300 mg of bone powder was transferred into a sterile 15 mL centrifuge tube containing 12 mL of 0.5 M EDTA (pH 8.0), and stored at 4°C overnight. Genomic DNA was extracted from the bone samples using a standard procedure^[21] with few modifications under sterile and aseptic conditions.

Figure 1 Bone samples (a) were turned into fine powder by crushing manually with liquid nitrogen (b–d).

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Genomic DNA extraction

After overnight incubation at 4°C, the tube was centrifuged at 4000 rpm for 5 minutes. The supernatant was discarded, and the pellet was dissolved in 300 µL of cell lysis buffer (TKM-1) supplemented with Tris–HCl (1 M), NaCl (1 M), MgCl₂ (1 M), EDTA (0.5 M, pH 8.0), SDS (10%, pH 8.0), and 40 µL (20 mg/mL) of proteinases-K. After adding lysis buffer, the samples were incubated at 56°C for 3 hours. Thereafter, 100 µL of TKM-3 (lysis buffer) supplemented with Tris–HCl (1 M), 1 M KCl (1 M), MgCl₂ (1 M), EDTA (0.5 M, pH 8.0), NaCl (4 M, pH 8.0) was added and incubated again for 30 minutes at 70°C. The solution was then centrifuged for 5 minutes at 4000 rpm, and the supernatant was discarded. Add 100 µL of 5M NaCl into the pellet and vortex. An equal volume of chloroform (100 µL) was added along with 70 µL of 5M sodium acetate (0.1 volumes), and 1 mL of 100% ethanol. This was kept overnight at −20°C. On the following day, it was centrifuged at 10,000 rpm for 10 minutes. The supernatant was discarded, and the pellet was washed twice with 1 mL of 70% ethanol to hydrate the DNA. Thereafter, the pellet was air-dried and resuspended by adding 50 µL of TE buffer. The quantitative analysis of genomic DNA samples was checked by a Nanodrop spectrophotometer (IMPLEN, BioRed) at wavelength 260/280 nm and the integrity was checked by using 0.8% agarose gel electrophoresis.

Primer selection details

A positive control, mitochondrial ATPase6 gene^[22] was taken [Table 1] for the PCR amplification. The specificity of primer sequence was tested by an online available database from BLAST, NCBI. Amplified PCR products were subjected to electrophoresis in 1.8% agarose gel electrophoresis. The specific amplicon was visualized with a gel documentation system.

Table 1 Primer sequences and information of the ATPase6 gene

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Amplification of ATPase6 gene

PCR was performed with a total volume of 25 μL containing 2× Master mixture (Thermo-scientific), 1 µL of forward and reverse primer (Eurofins, 10 pmol), and 1 µL genomic DNA (50 ng/µL). The following PCR amplification conditions were used to optimize the reaction accuracy performed in the Thermal cycler (ABI, USA) at 95°C for 5 minutes, 95°C for 30 seconds, annealing temperature 56°C [Table 1] for 1 minute, 72°C for 30 seconds, and a final extension at 72°C for 10 minutes for 35 cycles. The specificity of the amplification product was confirmed by the product size of 675bp of ATPase6 gene on 1.8% agarose gel electrophoresis [Figure 5]. The yield of the gel analysis amplified product of DNA samples demonstrated that the size and quantity of the DNA achieved were 675bp in length.

Results

A total of 50 dried human ancient bone samples were collected from the bone bank of the Anatomy Department, MAMC, New Delhi. The concentration of genomic DNA was checked by a Nanodrop spectrophotometer (IMPLEN, BioRad) at a wavelength of 260/280nm. [Figure 3] show the graphical presentation of genomic DNA concentrations and their optical densities (OD). The integrity of the genomic DNA was checked by using 0.8% agarose gel electrophoresis [Figure 4].

We could successfully extracted genomic DNA from 13 of 20 long bone samples with a concentration ranging from 50–250 ng/L at 260–280 and an OD range between 1.6 and 2.0. All samples were successfully amplified in PCR for gene ATPase6 [Figure 2]. The PCR amplification rate was 65% (13/20). The rest of the seven samples were not amplified by PCR. Those samples which were not amplified with OD ranged between 1.2 and 1.5. Whereas with hip bone samples, we could successfully extract genomic DNA from 17 out of 30 samples, with an OD ranging between 1.6 and 2.0 and a concentration between 50 and 250 ng/μL. The rest of the 13 samples were not amplified in PCR [Figure 5].

Figure 2 Graphical presentation of concentration and OD of 20 genomic DNA samples extracted from ancient long bones.

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Figure 3 Graphical presentation of concentration and OD of 30 genomic DNA samples extracted from ancient Hip bones.

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Figure 4 Integrity of the genomic DNA on 0.8% agarose gel electrophoresis.

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Figure 5 Amplified PCR product of ATPase6 gene. Lane 1: 100bp ladder; lane 2: amplified PCR product with 100–150 ng/μL concentration with 260/280 at 1.7–1.9; Lane 3: amplified PCR product with 150–200 ng/μL concentration with 260/280 at 1.6–1.9; Lane 4: amplified PCR product with 10–50 ng/μL concentration with 260/280 at 1.6–2.0. Lane 5: PCR product with 260/280 at 1.2–1.5; Lane 6: Negative control.

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Discussion

Bones are one of the best sources of genomic DNA from persevered specimens as they undergo the least decomposition, especially for abandoned dead bodies.^[3],[4] But still, just like any ancient biological material, contamination and degradation of DNA material are a major problem, especially for ancient specimens, where the desired quality and quantity are not achieved.

In the present study, it has been seen that genomic DNA integrity was positive in all of the samples on 0.8% agarose gel electrophoresis. In a similar study by Bender et al. in 2000,^[23] they successfully extracted the DNA from the ancient bone samples, and the success of amplification of genomic DNA was done with the amplification size of human mitochondrial DNA of 400bp size using the phenol/chloroform extraction method and glass of milk to enhance it.^[23],[24] Nadin Rohland and Michael Hofreiter also demonstrated extracting DNA from bones using powdered bone and buffers containing EDTA and Proteinase-K and purifying with silica binding in the presence of high concentrations of guanidinium thiocyanate.^[25] The extraction of high-quality DNA is a crucial step for studying the genetic cause of disease and for the development of diagnostic tools. It also plays an important role in various fields of research, Forensic science, animal domestication, genome sequencing, paternity determination, and the identification of ancestor relationships for generations.^{[26],[27],[28]}

Many studies have reported different methods for DNA extraction from healthy bones, but extracting DNA from severely damaged bones is still challenging.^[29],[30] Here, we tried to isolate DNA from damaged bones that were more than 40 years old and managed to successfully extract sufficient genomic DNA from all samples in a sufficient quantity. The time since death, environmental effects (humidity, temperature, pressure, etc.), chemical factors that permeate inside bone tissue, physical destruction of skeletal remains, and type of bone available for sampling are all important factors that affect DNA quality. The optical density (OD) of the extracted DNA ranged from 1.2 to 2.0 (including both types of bones). However, a DNA concentration ranging between 1.6 and 2.0 (this might be due to the contamination by RNA and protein in the genomic DNA) gives the best amplification in PCR for the ATPase6 gene, as shown in [Figure 5].

Conclusion

Our study demonstrates that manual protocol to quickly DNA extracts from ancient bone specimens with minimal hands-on time. Although DNA yields were relatively low, but the DNA amplifies well and informative DNA profiles suitable for profile comparison could be obtained. This protocol can be easily practised by laboratory personnel and produce DNA concentrations that are sufficient for molecular studies, and the DNA profiling.

Financial support and sponsorship

Nil.

Conflicts of interest

The authors report no conflicts of interest.

References

1.	Paabo S. Ancient DNA: extraction, characterization, molecular cloning, and enzymatic amplification. Proc Natl Acad Sci U S A 1989;86:1939-43.
2.	Balayan A, Kapoor A, Chaudhary G, Raina AX. Evaluation of techniques for human bone decalcification and amplification using sixteen STR markers. Egypt J Forensic Sci 2015;5:30-5.
3.	Booncharoen P, Khacha-ananda S, Kanchai C, Ruengdit S. Factors influencing DNA extraction from human skeletal remains: bone characteristic and total demineralization process. Egypt J Forensic Sci 2021;11-12.
4.	Jeffreys AJ, Allen MJ, Hagelberg E, Sonnberg A. Identification of DNA the skeletal remains of Josef Mengele by DNA analysis. Forensic Sci Int 1992;56:65-6.
5.	Latham KE, Miller JJ. DNA recovery and analysis from skeletal material in modern forensic contexts. Forensic Sci Res 2019;4:51-9.
6.	Hagelherg E, Gray IC, Jeffreys AJ. Identification of the skeletal remains of a murder victim by DNA analysis. Nature 1991;352:427-29.
7.	Honda K, Harihara S, Nakamura T, Hirai M, Misawa S. Sex identification by analysis of DNA extracted from hard tissues. Jpn J Legal Med 1990;44:293-301.
8.	Amory S, Huel R, Bilic A, Loreille O, Parsons TP. Automatable full demineralization DNA extraction procedure from degraded skeletal remains. Forensic Sci. Int. Genet 2012;6:398-406.
9.	Loreille OM, Diegoli TM, Irwin JA, Coble MD, Parsons TJ. High efficiency DNA extraction from bone by total demineralization. Forensic Sci Int Genet 2007;1:191-5.
10.	Jakubowska J, Maciejewska A, Pawłowski R. Comparison of three methods of DNA extraction from human bones with different degrees of degradation. Int J Legal Med 2012;126:173-8.
11.	Boer HH, Maat JRG, Kadarmo DA, Widodo TP, Kloosterman DA, Kal JA. DNA identification of human remains in Disaster Victim Identification (DVI): an efficient sampling method for muscle, bone, bone marrow and teeth. Forensic Sci Int 2018;289:253-9.
12.	Hagelberg E, Bell LS, Allen T, Boyde A, Jones SJ, Clegg JB. Analysis of ancient bone DNA: techniques and applications. Philos Trans R Soc Lond B Biol Sci 1991;333:399-407.
13.	Duijs FE, Sijen T. A rapid and efficient method for DNA extraction from bone powder. Forensic Sci Int Rep 2020;2:100099.
14.	Brown TA, Brown KA. Ancient DNA and the archaeologist. Antiquity 1992;66:10-23.
15.	Waltenberger L, Pany-Kucera D, Rebay-Salisbury K, Mitteroecker P. The association of parturition scars and pelvic shape: a geometric morphometric study. Am J Phys Anthrop 2021;174:19-31.
16.	Abitbol MM. Obstetrics and posture in pelvic anatomy. J Hum Evol 1987;16:243-55.
17.	Auerbach BM, King KA, Campbell RM, Campbell ML, Sylvester AD. Variation in obstetric dimensions of the human bony pelvis in relation to age-at-death and latitude. Am J Phys Anthrop 2018;167:628-43.
18.	Yang DY, Eng B, Waye JS, Dudar JC, Saunders SR. Improved DNA extraction from ancient bones using silica-based spin columns. Am J Biol Anthrop 1998;105:539-43.
19.	Pusch CM, Giddings I, Scholz M. A polymerase chain reaction inhibitor of ancient hard and soft tissue DNA extracts is determined as human collagen type I. Nucleic Acids Res 1998;259:283-86.
20.	Pusch CM, Scholz M. An efficient isolation method for high-quality DNA from ancient bones. Tech Tips Online 1997;2:T01217.
21.	Mohammadi A, Ghorbani AA, Khafaei M et al. A new and efficient method for DNA extraction from human skeletal remains usable in DNA typing. J Appl Biotechnol Rep 2017;4:609-14.
22.	Elrahman Abd MM, El-Makawy AI, Hassanane MS, Alam SS, Hassan NHA, Amer MK. Assessment of correlation between asthenozoospermia and mitochondrial DNA mutations in Egyptian infertile men. J Genet Eng Biotechnol 2021;19:2-15.
23.	Bender K, Schneider PM, Rittner CC. Application of mtDNA sequence analysis in forensic casework for the identification of human remains. Forensic Sci Int Genet 2000;113:103-07.
24.	Ayala FJ, Escalante A, O’hUigin C, Klein J. Molecular genetics of speciation and human origins. Proc Natl Acad Sci U S A 1994;91:6787-94.
25.	Nadin R, Michael H. Ancient DNA extraction from bones and teeth. Nat Protoc 2007;2:1756-62.
26.	Salamon M, Tuross N, Arensburg B, Weiner S. Relatively well-preserved DNA is present in the crystal aggregates of fossil bones. Proc Natl Acad Sci U S A 2005;102:13783-8.
27.	Tibor K, Csanad ZB, Antonia M, Istvan R. A simple and efficient method for PCR amplifiable DNA extraction from ancient bones. Nucleic Acids Res 2000;28:e67.
28.	Thuesen I, Engberg J. Recovery and analysis of human genetic material from mummified tissue and bone. J Archaeol Sci 1990; 17: 679–89.
29.	Meijer H, Perizonius WK, Geraedts JPM. Recovery and identification of DNA sequences harboured in preserved ancient human bone. Biochem Biophys Res Commun 1992;183:367-74.
30.	Sidstedt M, Steffen CR, Kiesler KM, Vallone PM, Radstrom P, Hedman J. The impact of common PCR inhibitors on forensic MPS analysis. Forensic Sci Int Genet 2019;40:182-91.