Comparison of Four Different DNA Extraction Methods and their Compatibility with Present-day STR Based Capillary Electrophoresis Typing

Neha Gupta¹, Rajesh Kumar² and Kunwar Veer Vikram Srivastav^3*
*Correspondence: Kunwar Veer Vikram Srivastav, Forensic Scientific Assistant, DNA Division, State Forensic Science Laboratory, Rajasthan, Jaipur, India, Tel: +919792067065, Email:

Keywords

PCIA • AutoMate express TM • EZ1 advanced XL • Chelex DNA extraction methods • Capillary electrophoresis

Introduction

Short Tandem Repeats (STRs) based forensic DNA typing technology is being internationally used in solving a diverse range of forensic cases and presently, forensic DNA tests employ Polymerase Chain Reaction (PCR) and Capillary Electrophoresis (CE) based fragment analysis methods to detect length variation in STRs. The quality of STRs DNA profiles from challenging samples depends upon the yield and quality of DNA and yield of DNA is basically dependent upon the method used for DNA extraction [1,2]. DNA extraction is the first and most important step in any Forensic DNA analysis. Human DNA can be extracted from all the nucleated cells such as hair, tissue, blood etc. The DNA extraction of forensic sample is a critical step in the analysis process and plays a significant role in the success of next applications. The wide range of samples processed in forensic casework, which contains varying amounts of DNA, require a robust DNA extraction method [3-5]. DNA was first extracted by Friedrich Miescher who named it "nuclein" in 1869 and currently it is a routine procedure in forensic casework as well as molecular biology [4]. There are various methods of extraction of DNA. The Phenol-chloroform Method (PCIA) of DNA extraction is a well-established procedure of forensic DNA fingerprinting laboratories. It is known to be laborious compared with alternative approaches like magnetic beads based DNA extraction and it involves toxic reagents, such as phenol, which requires special safety precautions in the laboratory (laminar flow fume hood). But, phenol–chloroform extraction method is still the gold standard choice for samples containing very little amounts of DNA or samples suspected to contain Polymerase Chain Reaction (PCR)-inhibiting substances. Although this method involves intensive manual interaction-the sample is handled through at least three generations of reaction tubes—the DNA recovery of the phenol-chloroform extraction is known to be relatively high [6]. Chelex method of extraction was introduced in 1991 to the forensic DNA community. Chelex is an ion-exchange resin that is added as a suspension to the samples and it is composed of styrene divinylbenzene copolymers containing paired iminodiacetate ions that act as chelating groups in binding polyvalent metal ions such as magnesium. By removing the magnesium from the reaction, DNA destroying enzymes known as nucleases are inactivated and the DNA molecules are thus protected. Chelex extraction procedures for recovering DNA from bloodstains or semen containing stains are not effective for RFLP typing because Chelex denatures double-stranded DNA and yields single-stranded DNA from the extraction process. Thus, it can only be followed by PCR-based analysis [7]. Now-a-days, DNA extraction instruments are routinely used for obtaining DNA from various forensic samples [8]. Magnetic beads are more suitable for automation than conventional column methods; that is why magnetic beads are used in small and large scale DNA extraction instruments. The Bio Robot EZ1 (QIAgen, Hilden, Germany) is widely used in forensic DNA laboratories because of its ease of use, less time taking and flexibility [8-10]. Recently, the AutoMate Express TM (Applied Biosystems, Foster City, CA) was developed, it has the same ease of operation as the EZ1 series but it takes more time than EZ1. The magnetic particles are much smaller in comparison with other commonly used magnetic particles, which results in a larger surface area with a higher DNA binding capacity [9]. In our previous study (Table 1), we compared PCIA and chelex method of extraction for their efficiency in extracting DNA from bloodstains on various surfaces [11].

Present study involves comparative analysis of performance and DNA yield of four different DNA extraction methods- EZ1 advanced XL, AutoMate Express TM, PCIA and Chelex method on broad range of samples commonly encountered in forensic caseworks.

Materials and Methods

In the present study, triplicate of sixteen variety of routine casework and simulated samples including Semen (liquid), Nail Clipping, Chest Hair, Blood stain on T-Shirt (Terrycot), Blood stain on Gauge, Pubic Hair, Blood stain on Baniyan (cotton) Semen Stain on cloth, Blood Stain on Inner (wool) Hair (from canal), Half Singed Hair, Blood (liquid), Saliva stain (paper), Cigarette butt (with filter), Cigarette butt (without filter) and Aspermic semen were subjected DNA extraction in equal amount using PCIA, Automate express TM, EZ1 advanced XL and Chelex DNA extraction methods as per standard recommendations (Figure1) [12-15] .

Quantitative PCR

To assess DNA yields, we used Quantifiler® DNA Quantification Kit (Thermo Fisher Scientific, USA) using Real-Time PCR 7000 system (Thermo Fisher Scientific, USA).

Results and Discussion

The average value of DNA yield (ng/μl) from Semen(liquid), Nail Clipping, Chest Hair, Blood stain on T-Shirt (Terrycot), Blood stain on Gauge, Pubic Hair, Blood stain on Baniyan (cotton) Semen Stain (cotton), Blood Stain on Inner (woolen), Hair(from canal), Half Singed Hair, Blood (liquid), Saliva stain (paper), Cigarette butt (with filter), Cigarette butt (without filter) and Aspermic semen were 536.7909, 88.58139, 3.956289, 128.9829, 250.4594, 12.17523, 181.4622, 318.9606, 97.20501, 1.539087, 3.936004, 199.3071, 2.859168, 0.121715, 0.200838 and 0.825395 (ng/ μl) respectively from the Phenol- Chloroform Isoamyl Alcohol (PCIA) extraction method. The average value of DNA yield (ng/μl) from Semen(liquid), Nail Clipping, Chest Hair, Blood stain on T-Shirt(Terrycot), Blood stain on Gauge, Pubic Hair, Blood stain on Baniyan (cotton) Semen Stain (cotton), Blood Stain on Inner (woolen), Hair (from canal), Half Singed Hair, Blood(liquid), Saliva stain (paper), Cigarette butt (with filter), Cigarette butt(without filter) and Aspermic semen were 184.5211, 58.35163, 1.324459, 87.30354, 87.08136, 5.115998, 101.5098, 89.57643, 48.35943, 0.661917, 1.414296, 118.0767, 0.602129, 0.008866, 0.543836 and 0.008429 (ng/μl) respectively from the Automate Express. The average value of DNA yield (ng/μl) from Semen(liquid), Nail Clipping, Chest Hair, Blood stain on T-Shirt (Terrycot), Blood stain on Gauge, Pubic Hair, Blood stain on Baniyan (cotton) Semen Stain (cotton), Blood Stain on Inner (woolen), Hair(from canal), Half Singed Hair, Blood(liquid), Saliva stain (paper), Cigarette butt (with filter), Cigarette butt(without filter) and Aspermic semen were 101.9156, 30.42272, 1.039988, 59.65596, 89.38024, 2.206983, 73.34391, 75.17647, 62.31758, 0.591762, 2.485101, 68.19788, 0.527838, 0.010279, 0.099436 and 0.398694(ng/μl) respectively from the EZ1. The average value of DNA yield (ng/μl) from Semen (liquid), Nail Clipping, Chest Hair, Blood stain on T-Shirt (Terrycot), Blood stain on Gauge, Pubic Hair, Blood stain on Baniyan (cotton) Semen Stain (cotton), Blood Stain on Inner (woolen), Hair (from canal), Half Singed Hair, Blood (liquid), Saliva stain (paper), Cigarette butt (with filter), Cigarette butt (without filter) and Aspermic semen were 10.85961, 0.795589, 0.346774, 10.16022, 1.902488, 0.373759, 14.64154, 6.954396, 5.615016, 0.389568, 0.282296, 22.7131, 0.016886, 0.001343, 0.00287 and 0.049771(ng/ μl) respectively from the Chelex method.

The highest average yield of DNA (536.7909 ng/μl) was obtained from liquid semen whereas the lowest average yield of DNA (0.121715 ng/μl) was obtained from cigarette butt (with filter) with the use of the PCIA method. From the Automate Express, the highest average yield of DNA (184.5211 ng/μl) was obtained from liquid blood whereas the lowest average yield of DNA (0.008429 ng/μl) was obtained from the cigarette butt (with filter). From the EZ1, the highest average yield of DNA (101.9156 ng/μl) was obtained from liquid blood whereas the lowest average yield of DNA (0.010279 ng/μl) was obtained from the cigarette butt (with filter). In the Chelex method, the highest average yield of DNA (22.7131 ng/μl) was obtained from liquid blood whereas the lowest average yield of DNA (0.001343 ng/μl) was obtained from the cigarette butt (with filter). Comparatively higher DNA yield was observed in DNA extraction using PCIA method for all the samples.

Conclusion

The yield of DNA from Semen(liquid), Nail Clipping, Chest Hair, Blood stain on T-Shirt(Terrycot), Blood stain on Gauge, Pubic Hair, Blood stain on Baniyan (cotton) Semen Stain (cotton), Blood Stain on Inner (woolen), Hair(from canal), Half Singed Hair, Blood (liquid), Saliva stain (paper), Cigarette butt (with filter), Cigarette butt(without filter) and Aspermic semen encountered in various crimes like murder, rape etc. was observed highest in Phenol Chloroform Isoamyl Alcohol (PCIA) method than the Automate Express, EZ1 and Chelex method. The observations of the study strongly suggest that for the forensic samples, where chance of getting DNA is low, the Phenol Chloroform Isoamyl Alcohol (PCIA) method should be preferred but if the quality and quantity sample is good then we can use Automate express and EZ1 in the place of PCIA.

References

1. Dubey, Ishwar Prasad, R. K. Kumawat, I. P. Tripathi, and Pankaj Shrivastava. "A pilot study of DNA yield from bloodstains on various surfaces using Phenol chloroform isoamyl alcohol (PCIA) and Chelex DNA extraction methods." GSC Biol Pharm Sci 13 (2020): 124-127.

Google Scholar, Crossref

2. Shrivastava, Pankaj, Toshi Jain, and V. Ben Trivedi. "DNA fingerprinting: a substantial and imperative aid to forensic investigation." Eur J Forensic Sci 3 (2016): 23-30.

Google Scholar, Crossref

3. Montpetit, Shawn A., Ian T. Fitch, and Patrick T. O'Donnell. "A simple automated instrument for DNA extraction in forensic casework." J Forensic Sci 50 (2005): 1-9.

Google Scholar, Crossref, Indexed at

4. Bharatha, Ambadasu, Sajja Suguna, Nandal DH Suresh and Kamble Rahul Kunkulol. "Genomic DNA isolation from human whole blood samples by non-enzymatic salting out method." Int J Pharm Sci 6 (2014): 198-9.

Google Scholar

5. Xavier, Catarina, Mayra Eduardoff, Barbara Bertoglio, and Christina Amory, et al. "Evaluation of DNA extraction methods developed for forensic and ancient DNA applications using bone samples of different age." Genes 12 (2021): 146.

Google Scholar, Crossref

6. Köchl, Silvano, Harald Niederstätter, and Walther Parson. "DNA extraction and quantitation of forensic samples using the phenol-chloroform method and real-time PCR." Forensic DNA Typing Protocols (2005): 13-29.

Google Scholar, Crossref, Indexed at

7. Butler, John M. “Forensic DNA typing: biology, technology, and genetics of STR markers.” Elsevier (2005).

Google Scholar

8. Scherczinger, C. A., M. T. Bourke, C. Ladd, and H. C. Lee. "DNA extraction from liquid blood using QIAamp." J Forensic Sci 42 (1997): 893-896.

Google Scholar, Indexed at

9. Fujii, Koji, Shota Inokuchi, Tetsushi Kitayama, and Hiroaki Nakahara, et al. "A comparison of DNA extraction using a uto m ate e xpress™ and EZ 1 advanced XL from liquid blood, bloodstains, and semen stains." J Forensic Sci 58 (2013): 981-988.

Google Scholar, Crossref, Indexed at

10. Anslinger, Katja, Birgit Bayer, Burkhard Rolf, and Wolfgang Keil, et al. "Application of the BioRobot EZ1 in a forensic laboratory." Leg Med 7 (2005): 164-168.

Google Scholar, Crossref, Indexed at

11. Montpetit, Shawn A., Ian T. Fitch, and Patrick T. O'Donnell. "A simple automated instrument for DNA extraction in forensic casework." J Forensic Sci 50 (2005): 1-9.

Google Scholar, Crossref

12. Kishore, Ram, W. Reef Hardy, Vince J. Anderson, and Nick A. Sanchez, et al. "Optimization of DNA extraction from low‐yield and degraded samples using the BioRobot® EZ1 and BioRobot® M48." J Forensic Sci 51 (2006): 1055-1061.

Google Scholar, Crossref, Indexed at

13. Willard, Jeanne M., Dennis A. Lee, and Mitchell M. Holland. "Recovery of DNA for PCR amplification from blood and forensic samples using a chelating resin." Forensic DNA Profiling Protocols (1998): 9-18.

Google Scholar, Crossref, Indexed at

14. Applied Biosystems. “PrepFilerTM ExpressTM and PrepFilerTM Express BTATM Forensic DNA extraction kits.” User Guide (2021).
15. QIAgen. “EZ1® DNA investigator handbook, 4th Edition Hilden, Germany: QIAgen, 2009.” (2021).