Optimization of a DNA Extraction Method for

Technical Note

Optimization of a DNA Extraction Method for Nonhuman and Human Bone

Sulekha Coticone 1 Lyndsey Barna 1 Max Teets 2 Abstract: The present study involves the use of ultrasonic technology to rapidly and effectively extract trace amounts of DNA from bones. This method not only extracts DNA but also preserves its integrity because there is no heating or foaming during the automated extraction process. Additionally, because the system is self contained, there is minimal risk of contamination. This technique allows for the extraction of DNA from bones using acoustic energy which can be manipulated using two different parameters (duty cycle and amplitude). Using pig bones, different acoustic settings were tested to determine which combination produces the highest yield of amplifiable DNA after the entire extraction procedure has been completed. Pig and human DNA were successfully extracted from bones and amplified using this procedure. The results demonstrate the ability to obtain DNA from bone samples using acoustic energy.

Introduction Traditional methods for DNA extraction from bone require cutting and sanding using Dremel tools and pulverizing the bone fragments into a powder using blenders and grinders before extraction [1]. These methods for DNA extraction, though robust, can be time-consuming and labor-intensive. Additionally, the recovery of DNA from bones presents specific difficulties in DNA purif ication and amplif ication using polymerase chain reaction in the presence of inhibitors [2-5]. Existing methods 1 Florida Gulf Coast University, Ft Myers, FL 2 Arthrex Inc, Naples, FL Received September 29, 2009; accepted Janaury 2, 2010 Journal of Forensic Identification 430 / 60 (4), 2010

for DNA extraction in previously published protocols involve incubation of the bone powder in various extraction buffers [6]. In the next step, the proteins are removed with a phenolchloroform mixture [7]. The DNA is then precipitated using ethanol, isoproponol, or a microconcentrator. The DNA can also be separated by using a silica suspension or columns [8]. In the present study, a noninvasive method for extracting DNA f rom bone samples is described. The new technique involves controlled disruption of bone using acoustic power to deliver consistent extraction, with minimal processing steps. The ultrasonic technology is based on adaptive focused acoustics (AFA), which is used primarily for the acoustic disruption of kidney stones and gallstones [9]. Ultrasound energy that is generally safe at low power settings creates “cavitation” at high power settings. Cavitation causes bubbles in a f luid to form and subsequently collapse. Cavitation has been used in many fields such as physical science, engineering, medicine, and dentistry in applications such as cleaning, homogenization, lithotripsy, and cell disruption. In the present instrument, the process involves creating low-level shock waves from a conical shaped transducer that converge on a localized area. This results in a noncontact, controlled isothermal mechanical energy that can be used to disrupt the cells. Compared to the traditional methods of DNA extraction, this method allows for a wide range of variables to control the breakdown process including strength and duration. The testing of these variables is imperative to determine the best setting combination that yields the largest amount of extracted, amplifiable DNA. The system was initially optimized to extract DNA from pig bones. Using parameters obtained from the initial pig bone tests, DNA was successfully extracted from human bones and amplified using standard f luorescent-based STR multiplex methods. Methods Sample Preparation Samples of pig bones were obtained from a grocer. Human bone samples were donated by Arthrex Inc. (Naples, FL). The band saw and the lab surfaces were cleaned using a disinfecJournal of Forensic Identification 60 (4), 2010 \ 431

tant (Cavicide). Each specimen was sectioned transversely into eight 1-cm-thick pieces using a 14" Delta Wood Cutting Band Saw (Jackson, TN). The bone fragments were then placed in the TT1 plastic containers provided by the manufacturer (Covaris Inc, MA). These containers were placed in liquid nitrogen and the bones were pulverized using the Covaris CryoPrep (Covaris Inc, MA). Ultrasonic Treatment Bone samples were placed in tubes containing lysis buffer [10 mM Tris-HCl, 400 mM NaCl, 2.0 % hexadecyltimethylammomium bromide (CTAB), and 0.1 % polyvinylpyrrolidone (PVPP)] and were subjected to ultrasonic waves using the Covaris E200 instrument. Settings were adjusted on the instrument and the samples were loaded, each set of three samples having different settings. The Covaris E200 was initiated, submerging each test tube in water while being treated with ultrasonic waves of energy. DNA Extraction Following ultrasonic treatment, DNA was extracted from each tube by salt extraction [10] followed by ethanol precipitation. Further purification of DNA was performed using silica beads (QIAEX II beads from Qiagen, CA). PCR Amplification of Mitochondrial and Nuclear DNA Total DNA from the pig bones was used as a template for PCR amplification with primers to the SW 24 locus [11]. For human studies, nuclear markers Amelogenin [12] and mitochondria (HVI and HVII) were evaluated. In addition, the samples were amplified using the AmpFLSTR Profiler Plus ID (Applied Biosystems, Foster Cit y, CA) multiplex accordi ng to the manufacturer’s instructions. PCR products were electrophoresed on agarose gels and capillary instruments (Beckman CEQ 8000 and ABI Prism 310 genetic analyzer).

Journal of Forensic Identification 432 / 60 (4), 2010

Results and Discussion Acoustic Disruption of Cells The acoustic parameters were originally optimized using pig bone. Bone samples were initially pulverized into smaller pieces in the CryoPrep. The CryoPrep is fundamentally a hammer and anvil system controlled by a microprocessor. The controlled repeatable mechanic impact to the sample is delivered via an electronic solenoid. The CryoPrep uses an interlocking glass or plastic film tube (TT1) that allows cryo pulverization in an enclosed space, unlike the traditional mortar and pestle method of bone disr uption. In addition, the plastic f ilm-based tube (Kapton) can withstand low temperatures of cryogenic freezing. Before the CyroPrep treatment, the TT1 tube is attached to a borosilicate tube, which ensures easy transfer of the bone fragments to the transfer tube for acoustic disruption in the next step. The most important benefits from this system are the ease of use and minimal contamination risk. Bone fragment samples were then subjected to ultrasonic energy using the Covaris E 200 instrument (Covaris, Woburn, MA). The instrument sends acoustic energy wave packets from a dish-shaped transducer that converges and focuses on a small localized area containing bone fragements within a tube (Figure 1). This technique, unlike sonication, provides noncontact controlled isothermal mechanical energy. The high-intensity transducer provides shockwaves ideal for tissue disr uption. Acoustic energy traveling through a medium causes compression and expansion. In liquids, tiny bubbles form and collapse when acoustic energy is removed by a process called cavitation. The physical forces caused by the collapse provide the intense force for disrupting tissues. Following ult rasonic t reat ment, total ext racted DNA as measured using a spectrophotometer indicated differences in the amount of DNA obtained depending on acoustic parameters (duty cycle and amplitude). Duty cycle is defined as the percentage of the time that the transducer is creating acoustic waves. Initial results using pig bone indicated that higher duty cycles (5% and 20%) resulted in too much acoustic energy being delivered to the tube, resulting in lower yield (Figure 2). The optimum duty cycle for human DNA was therefore set at 2%. The amplitude of the waveform created by the acoustic transducer is proportional to the voltage applied to it. When the amplitude Journal of Forensic Identification 60 (4), 2010 \ 433

was varied between 250 mV and 500 mV, the highest yield of DNA was obtained at 500 mV (Table 1). All further studies were carried out at these acoustic parameters. DNA Extraction and Amplification The extraction included the use of hexadecyltimethylammomium bromide (CTAB) and polyvinylpyrrolidone (PVPP) previously used for removing polyphenolics and polysaccharides while extracting DNA from algae [13], thus controlling inhibition of PCR. DNA from pig bones was amplified using SW 24 primers (Figure 3). DNA was also obtained from three different human bone samples and amplified using STR primers. DNA profiles were obtained using the Profiler Plus amplification kit (Table 2). Conclusions Successful extraction of DNA was achieved using acoustic energy using both pig and human bones. Additionally, the DNA obtained was successfully amplified using pig and human specific primers, respectively. We conclude that acoustic energy is another method for obtaining DNA from bone samples. Acknowledgment This project was suppor ted by the Lucas grant from the Forensic Science Foundation (American Academy of Forensic Science). We would like to thank Dr. Alberte for useful comments and suggestions. We would like to thank Arthrex Inc. (Naples, FL) for the donation of the human bone samples. For further information, please contact: Dr. Sulekha Coticone Department of Chemistry and Mathematics Florida Gulf Coast University 10501 FGCU Blvd Ft Myers, Fl 33965 [email protected] (239) 590-7528


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Holland, M. M.; Cave, C. A.; Holland, C. A.; Bille, T. W. D evelo p me nt of Q u a l it y H ig h T h r ou g hput DNA Analysis Procedure for Skeletal Samples to Assist with the Identification of Victims from the World Trade Center Attacks. Croat. Med. J. 2003, 44 (3), 264-272. Yang, D. Y.; Eng, B.; Waye, J. S.; Dudar, J. C.; Saunders, S. R. Improved DNA Extraction from Ancient Bones Using Silica-based Spin Columns. Am. J. Phys. Anthropol. 1998, 105 (4), 539-43. Kreader, C. A. Relief of Amplification Inhibition in PCR with Bovine Serum Albumin or T4 Gene 32 Protein. Appl. Environ. Microbiol. 1996, 62 (3), 1102-1106. McKeown, B. J. An Acetylated ( Nuclease-free) Bovine Serum Albumin in a PCR Buffer Inhibits Amplification. Biotechniques 1994, 17 (2), 246-248. Wilson, I. G. Inhibition and Facilitation of Nucleic Acid Amplification. Appl.Environ. Microbiol. 1997, 63 (10), 37413751. Fisher, D. L.; Holland, M. M.; Mitchell, L.; Sledzik, P. S.; Wilcox, A. W.; Wadhams, M.; Weedn, V. W. Extraction, Evaluation and Amplification of DNA from Decalcified and Undecalcified United States Civil War Bone. J. For. Sci. 1993, 38 (1), 60-68. Hoff-Olsen, P.; Mevåg, B.; Staalstrøm, E.; Hovde, B.; Egeland, T.; Olaisen, B. Extraction of DNA from Decomposed Human Tissue: An Evaluation of Five Extraction Methods for Short Tandem Repeat Typing. For. Sci. Int. 1999, 105 (3), 171-183. Höss, M.; Pääbo, S. DNA Extraction from Pleistocene Bones by a Silica-based Purification Method. Nucleic Acids Res. 1993, 21 (16), 3913-3914. Lucas, M.; Cardoni, A.; McCulloch, E.; Hunter, G.; MacBeath, A. Applications of Power Ultrasonics in Engineering. Applied Mechanics and Materials, 2008, 13-14, 11-20 Miller, S. A.; Dykes, D. D.; Polesky, H. F. A Simple Salting out Procedure for Extracting DNA from Human Nucleated Cells. Nucleic Acids Res. 1988, 16 (3), 1215. Archibald, A. L.; Haley, C. S.; Brown J. F.; Couperwhite, S.; McQueen, H. A.; Nicholson, D.; Coppieters, W.; Van de Weghe, A.; Stratil, A.; Winterø, A. K. The PiGMaP Consortium Linkage Map of the Pig (Sus scrofa) Mamm. Genome 1995, 6 (3), 157-175. Sullivan, K. M.; Mannucci, A.; Kimpton, C. P.; Gill, P. A Rapid and Quantitative DNA Sex Test: Fluorescence-based PCR A nalysis of X-Y Homologous Gene A melogenin. Biotechniques 1993, 15 (4), 636-641. Coyer, J. A.; Steller, D. L.; Alberte, R. S. A Field-compatible Method for Extraction of Finger print-quality DNA from Macrosystis pyrifera (Phaeophyceae) J. Phycol. 1995, 31 (1), 177-180. Journal of Forensic Identification 60 (4), 2010 \ 435

Covaris parameters

yield (mg/g of bone)*

2%, 250 mV, 50 cycles/burst

6


10.5


35

* All data is within 10% standard error obtained from three replicate samples.

Table 1 Quantitation of DNA using different acoustic parameters.

Human Bone Sample A

Human Bone Sample B

Human Bone Sample C

Locus

Alleles

Alleles

Alleles

D3S1358

16

16

15

D3S1358

18

18

17

vWA

17

17

15

vWA

18

19

17

FGA

20

23

20

FGA

22

25

21

Amelogenin

X

X

X

Amelogenin

X

Y

Y

D8S1179

13

12

14

D8S1179

13

14

15

D21S11

30

29

29 30

D21S11

31.2

30

D18S51

15

15

10

D18S51

15

16

19

D5S818

11

12

9

D5S818

13

12

11

D13S317

11

11

9

D13S317

11

11

12

D7S820

9

10

11

D7S820

10

11

12

Table 2 STR amplification of DNA obtained from treatment of human bone.


Figure 1 Adaptive Focused Acoustics (AFA) schematic representation provided courtesy of Covaris, Inc. Acoustic energy wave packets from a dish-shaped transducer converge and focus to a small, localized area.

Figure 2 Optimization of parameters of acoustic energy for the extraction of genomic DNA from pig bone. Lane1, standard marker. Lanes 2, 3 (2% 500 mV); lanes 4, 5 (5 % 500 mV); lanes 6, 7 (20% 500 mV). Journal of Forensic Identification 60 (4), 2010 \ 437

Figure 3 Amplification of pig DNA from bone extracted using Covaris with SW 24 primers.
