Pyracantha coccinea'da etkili DNA geri kazanım yönteminin belirlenmesi ve adli botanikte kullanımı

Öz

Adli Botanik disiplini bitki materyallerini kullanarak olay yeri, fail veya kurban arasındaki bağlantıyı ortaya çıkarmayı hedeflemektedir. Bitkilerde bulunan ikincil metabolitlerin varlığı farklı bitki türlerinde değişiklik gösterdiğinden DNA izolasyon yöntemleri her bitki türüne hatta her bitki dokusuna özel olarak ayarlanmalıdır. Çalışmamızda, Türkiye’de yetişen Ateş Dikeni (Pyracantha coccinea M.Roem.) bitkisinin meyveleri için sıvı nitrojen kullanılmadan en uygun DNA izolasyon metodunun belirlenmesi amaçlanmıştır. Çalışmamızda, ateş dikeni bitkisinin meyveleri taze (12 adet) ve kurutulmuş (12 adet) olarak iki gruba ayrılmış ve her analizde tek meyve kullanılarak farklı homojenizasyon yöntemlerine tabi tutulmuştur. DNA izolasyonu tamamlanan örneklerin DNA miktarları ölçülerek hangi metodun optimum sonuç verdiği belirlenmiştir. En yüksek DNA miktarı 4,80 ng/µl olarak havan ve boncuklu doku öğütücü ile homojenize edilen kurutulmuş meyvelerden elde edilirken, en düşük DNA miktarı ise 0,13 ng/µl olarak yalnızca mutfak robotu ile homojenize edilen taze meyvelerden elde edilmiştir. Çalışmamızın bulguları hem taze hem de kurutulmuş meyveler için, homojenizasyonun havan ve boncuklu doku öğütücü ile gerçekleştirilmesi halinde bitki materyallerinden yüksek miktarda DNA geri kazanımı olacağı yönündedir. Yaptığımız çalışma, Ateş Dikeni bitkisinin dahil olabileceği adli vakalarda DNA izolasyonu için optimizasyon sunmaktadır.

Anahtar Kelimeler

• Adli bilimler
• adli botanik
• bitki DNA izolasyonu
• homojenizasyon metotları
• Pyracantha coccinea (ateş dikeni)

Determination of efficient DNA recovery method in Pyracantha coccinea for use in forensic botany

Öz

Forensic Botany aims to uncover the connection between crime scenes, perpetrators, or victims using plant materials. Since the presence of metabolites in plants varies among different species, DNA isolation methods should be adjusted specifically for each plant species and even each plant tissue. In this study, the objective was to determine the most suitable DNA isolation method for the fruits of firethorn (Pyracantha coccinea M.Roem.) growing in Türkiye without using liquid nitrogen. In the study, the fruits of Firethorn were divided into two groups as fresh (12 pieces) and dried (12 pieces), which each fruit was subjected to different homogenization methods using a single sample for each analysis. After completing the DNA isolation of the samples, the DNA quantities were measured to determine which method yielded optimal results. The highest DNA quantity of 4.80 ng/µl was obtained from dried fruits homogenized using a mortar and tissue lyser with beads, while the lowest DNA quantity of 0.13 ng/µl was obtained from fresh fruits homogenized only with a kitchen blender. The findings of the study indicate that for both fresh and dried fruits, homogenization with a mortar and tissue lyser with beads would result in a high recovery of DNA from plant materials. This study provides an optimization for DNA isolation in forensic cases where the Firethorn plant may be involved.

Anahtar Kelimeler

• Forensic Sciences
• Forensic Botany
• Plant DNA Extraction
• Homogenization Methods
• Pyracantha Coccinea (Firethorn)

Destekleyen Kurum

Uskudar University

Etik Beyan

No acknowledgment and/or disclaimers applicable

Teşekkür

The authors would like to thank Üsküdar University for the laboratory opportunity

Kaynakça

1. Akguc N, Ozyigit İİ, Yasar U, Leblebici Z, Yarcı C (2010). Use of Pyracantha coccinea Roem. as a possible biomonitor for the selected heavy metals. International Journal of Environmental Science and Technology 7(3): 427-434.
2. Aquila I, Ausania F, Serra A, Di Nunzio C, Boca S, Capelli A, Magni P, Ricci P (2014). The role of forensic botany in crime scene investigation: Case report and review of literature. Journal of Forensic Sciences 59: 820-824. https://doi.org/10.1111/1556-4029.12401.
3. Couch J, Fritz P (1990). Isolation of DNA from plants high in polyphenolics. Plant Molecular Biology Reporter 8: 8-12.
4. Craft KJ, Owens JD, Ashley MV (2007). Application of plant DNA markers in forensic botany: Genetic comparison of Quercus evidence leaves to crime scene trees using microsatellites. Forensic Science International 165(1): 64-70. https://doi.org/10.1016/j.forsciint.2006.02.035
5. Ding SX, Li JC, Hu K, Huang ZJ, Lu, RS (2022). Morphological characteristics and comparative chloroplast genome analyses between red and white flower phenotypes of Pyracantha fortuneana (Maxim.) Li (Rosaceae), with implications for taxonomy and phylogeny. Genes 13(12): 2404.
6. Guillemaut P, Maréchal-Drouard, L (1992). Isolation of plant DNA: A fast, inexpensive, and reliable method. Plant Molecular Biology Reporter 10: 60-65. https://doi.org/10.1007/BF02669265
7. Huang CJ, Chu FH, Huang YS, Hung YM, Tseng YH, Pu CE, Chao CH, Chou YS, Liu SC, You YT, Hsu SY, Hsieh H C, Hsu CT, Chen MY, Lin TA, Shyu HY, Tu YC, Chen CT (2020). Development and technical application of SSR-based individual identification system for Chamaecyparis taiwanensis against illegal logging convictions. Scientific Reports 10(1): 22095.
8. Intron Biotechnology (2012). i-genomic Plant DNA Extraction Mini Kit Protocol. Intronbio.com Website: https://intronbio.com:6001/inc/downfile.php?filename=F1523839442.pdf&orgfilename=IDR-RP0014_i-genomic%20Plant.pdf&filedir=product_protocol [accessed 20 August 2024].

9. Leitch I, Johnston E, Pellicer J, Hidalgo O, Bennett M. (2019). Plant DNA C-Values Database. cvalues. Science.kew.org Website: https://cvalues.science.kew.org/ [accessed 20 August 2024].
10. QIAGEN Sample & Assay Technologies (2009). TissueLyser LT Handbook. Qiagen.com/us Website: https://www.qiagen.com/us/resources/download.aspx?id=eb067bcc-0638-4f75-bf00-b54094d858fe&lang=en [accessed 20 August 2024].
11. QIAGEN Sample & Assay Technologies (2019). Multiplex PCR Handbook. Qiagen.com/us Website: https://www.qiagen.com/at/resources/download.aspx?id=a541a49c-cd06-40ca-b1d2-563d0324ad6c&lang=en&srsltid=AfmBOoqTpC4_TFwlF5UBTu7wnA6Xqf0RjHuDXmfl2zrh_x3_u5YfaCbZ [accessed 20 August 2024].
12. Sahu SK, Thangaraj M, Kathiresan K (2012). DNA extraction protocol for plants with high levels of secondary metabolites and polysaccharides without using liquid nitrogen and phenol. ISRN Molecular Biology 2012: 1-6. https://doi.org/10.5402/2012/205049.
13. Sigma Aldrich. (2023). Plant DNA Extraction & WGA Amplification Protocol. Sigmaaldrich.com/TR Website: https://www.sigmaaldrich.com/TR/en/technical-documents/protocol/genomics/dna-and-rna-purification/extraction-protocol-plant-tissues# [accessed 20 August 2024].
14. Thermo Fisher Scientific Inc. (2022). Qubit™ dsDNA HS Assay Kit (Invitrogen) User Guide. Thermofisher.com Website: https://www.thermofisher.com/document-connect/document-connect.html?url=https://assets.thermofisher.com/TFS-Assets%2FLSG%2Fmanuals%2FQubit_dsDNA_HS_Assay_UG.pdf [accessed 20 August 2024].
15. Uchiyama K, Fujii S, Ishizuka W, Goto S, Tsumura Y. (2013). Development of 32 EST-SSR markers for Abies firma (Pinaceae) and their transferability to related species. Applications in Plant Sciences, 1: 1200464. https://doi.org/10.3732/apps.1200464.
16. Wickneswari R, Rajanaidu N (2011). Genetic identification of oil palm: A tool for the sustainable management of palm oil. Journal of Plantation Research 25(3): 305-312.