Comparative Study of Genomic DNA Extraction Protocols from Whole Blood for P53 Gene Polymorphism in Persons with and without Prostate Cancer
Keywords:DNA extraction, DNA purification, electrophoresis, enzymatic method, P53, PCa, PCR
In latest decades, genetic methods have developed into a potent tool in a number of life-attaching applications. In research looking at demographic genetic diversity, QTL detection, marker-assisted selection, and food traceability, DNA-based technologies like PCR are being employed more and more. These approaches call for extraction procedures that provide efficient nucleic acid extraction and the elimination of PCR inhibitors. The first and most important stage in molecular biology is the extraction of DNA from cells. For a molecular scientist, the high quality and integrity of the isolated DNA as well as the extraction method's ease of use and affordability are crucial factors. The present study was designed to establish a simple, fast and inexpensive method for DNA extraction from human peripheral blood (normal male n=2, age 24 years old, patient male (prostate cancer) n=2, age 65 years old) by comparing between them, and aimed to standardize a protocol of DNA extraction using five extraction protocols. The first method was the modified organic method by using sodium perchlorate instead of organic solvent (phenol, chloroform), sodium perchlorate advantage comes from its cheap price and low storage and shipping requirements, the second was the enzymatic method by using proteinase K, third method was done by using detergent, the fourth used phenol-chloroform; finally fifth one was salting out method. The result showed that the organic method gives a good DNA yield and needs relatively short time while the enzymatic method gives an excellent DNA purity which are more suitable for PCR by comparing five protocols using the spectrophotometer and Nanodrop technetium in addition to electrophoresis. Through the use of the five suggested procedures, the PCR multiplication of the P53 gene with the isolated DNA was effectively carried out. This indicates that, with the exception of the detergent approach, there were no significant inhibiting substances for Taq polymerase in the final solution.
Published Online First 20/08/2023
Banerjee PP, Banerjee S, Brown TR, Zirkin BR. Androgen action in prostate function and disease. Am J Clin Exp Urol. 2018; 6(2): 62.
Aboumarzouk OM, Jenkins B, Chlosta PL. Vasectomy and Seminal Vesicle Disorders. Blandy's Urology. 2019; Chap. 41: 817-824. https://doi.org/10.1002/9781118863343.ch41.
Henry GH, Loof N, Strand DW. OMIP-040: Optimized gating of human prostate cellular subpopulations. Cytometry. Part A: J Int Soci Ana Cyt. 2017; 91(12): 1147. https://doi.org/10.1002/cyto.a.23187.
Habib A, Jaffar G, Khalid MS, Hussain Z, Zainab SW, Ashraf Z, et al. Risk Factors Associated with Prostate Cancer. J drug deliv ther. 2021; 11(2): 188-193. https://doi.org/10.1016/j.envpol.2022.120041.
Rzhevskiy AS, Kapitannikova AY, Vasilescu SA, Karashaeva TA, Razavi Bazaz S, Taratkin MS, et al. Isolation of circulating tumor cells from seminal fluid of patients with prostate cancer using inertial microfluidics. Cancers. 2022; 14(14): 3364. https://doi.org/10.3390/cancers14143364.
Akoto T, Saini S. Role of exosomes in prostate cancer metastasis. Int J Mol Sci. 2021; 22(7): 3528. https://doi.org/10.3390/ijms22073528.
Rawla P. Epidemiology of prostate cancer. World J Oncol. 2019; 10(2): 63. https://doi.org/10.14740/wjon1191.
Costello AJ. Considering the role of radical prostatectomy in 21st century prostate cancer care. Nat Rev Urol. 2020; 17(3): 177-188. https://doi.org/10.1038/s41585-020-0287-y.
Kang R, Kroemer G, Tang D. The tumor suppressor protein p53 and the ferroptosis network. Free Radic Biol Med. 2019; 133(162-168. https://doi.org/10.1016/j.freeradbiomed.2018.05.074.
Gupta A, Shah K, Oza MJ, Behl T. Reactivation of p53 gene by MDM2 inhibitors: A novel therapy for cancer treatment. Biomed Pharmacother. 2019; 109(484-492. https://doi.org/10.1016/j.biopha.2018.10.155.
Zhang C, Liu J, Xu D, Zhang T, Hu W, Feng Z. Gain-of-function mutant p53 in cancer progression and therapy. J Mol Cell Biol. 2020; 12(9): 674-687. https://doi.org/10.1093/jmcb/mjaa040.
Al-Hilfy AA, Al-Malak MK, Al-Tomah MA. A Prevalence study of Entamoeba spp. in Basrah Province using Different Detection Methods. Baghdad Sci J. 2021; 18(4): 1163. https://doi.org/10.21123/bsj.2021.18.4.1163.
Dairawan M, Shetty PJ. The evolution of DNA extraction methods. Am J Biomed. 2020; 8(1): 39-46. https://doi.org/10.34297/AJBSR.2020.08.001234.
Barazesh A, Sarkari B, Ebrahimi S, Hami M. DNA extraction from hydatid cyst protoscolices: comparison of five different methods. Vet World. 2018; 11(2): 231. https://doi.org/10.14202%2Fvetworld.2018.231-234.
Mardan‐Nik M, Saffar SS, Biabangard‐Zak A, Asghari M, Saljoughian S, Tajbakhsh A, et al. A method for improving the efficiency of DNA extraction from clotted blood samples. J Clin Lab Anal. 2019; 33(6): e22892. https://doi.org/10.1002/jcla.22892.
Barbier FF, Chabikwa TG, Ahsan MU, Cook SE, Powell R, Tanurdzic M, et al. A phenol/chloroform-free method to extract nucleic acids from recalcitrant, woody tropical species for gene expression and sequencing. Plant Methods. 2019; 15(1): 1-13. https://doi.org/10.1186/s13007-019-0447-3
Sedlar K, Vasylkivska M, Musilova J, Branska B, Provaznik I, Patakova P. Phenotypic and genomic analysis of isopropanol and 1, 3-propanediol producer Clostridium diolis DSM 15410. Genomics. 2021; 113(1): 1109-1119. https://doi.org/10.1016/j.ygeno.2020.11.007.
Kellner MJ, Koob JG, Gootenberg JS, Abudayyeh OO, Zhang F. SHERLOCK: nucleic acid detection with CRISPR nucleases. Nature protocols. 2019; 14(10): 2986-3012. https://doi.org/10.1038/s41596-019-0210-2.
Aboul-Maaty NA, Oraby HA. Extraction of high-quality genomic DNA from different plant orders applying a modified CTAB-based method. Bull Natl Res Cent. 2019; 43(1): 1-10. https://doi.org/10.1186/s42269-019-0066-1.
Marotz CA, Sanders JG, Zuniga C, Zaramela LS, Knight R, Zengler K. Improving saliva shotgun metagenomics by chemical host DNA depletion. Microbiome. 2018; 6(1): 1-9. https://doi.org/10.1186/s40168-018-0426-3.
Singh UA, Kumari M, Iyengar S. Method for improving the quality of genomic DNA obtained from minute quantities of tissue and blood samples using Chelex 100 resin. Biol Proced Online. 2018; 20(1): 1-8. https://doi.org/10.1186/s12575-018-0077-6.
Liu X, Ren J, Luo N, Guo H, Zheng Y, Li J, et al. Comprehensive DNA methylation analysis of tissue of origin of plasma cell-free DNA by methylated CpG tandem amplification and sequencing (MCTA-Seq). Clin Epigenetics. 2019; 11(1): 93. https://doi.org/10.1186/s13148-019-0689-y.
Guha P, Das A, Dutta S, Chaudhuri TK. A rapid and efficient DNA extraction protocol from fresh and frozen human blood samples. J Clin Lab Anal. 2018; 32(1): e22181. https://doi.org/10.1002/jcla.22181.
Roulston JE, John SB, eds. Molecular diagnosis of cancer: methods and protocols. Vol. 97. Springer Science and Business Media, 2004. https://doi.org/10.1385/1592597602
Frazer Z, Yoo C, Sroya M, Bellora C, DeWitt BL, Sanchez I, et al. Effect of different proteinase K digest protocols and deparaffinization methods on yield and integrity of DNA extracted from formalin-fixed, paraffin-embedded tissue. J Histochem Cytochem. 2020; 68(3): 171-184. https://doi.org/10.1369/0022155420906234.
Al-Ani SF, Al-khafaji AA, Ali OA. Optimum Concentration Of DNA Extracted From Human Peripheral Blood. Al-Anbar Med J. 2009; 7(1): 30-33.
Barbaro A, Staiti N, Cormaci P, Saravo L. DNA profiling by different extraction methods. Int Congr Ser. 2004: 562-564, Elsevier. https://doi.org/10.1016/S0531-5131(03)01647-9.
Mohammadpour A. Evaluation of a modified salt-out method for DNA extraction from whole blood lymphocytes: A simple and economical method for gene polymorphism. Pharm Biomed Res. 2018; 4(2): 28-32. http://pbr.mazums.ac.ir/article-1-194-en.html
Khosravinia H, Murthy HN, Parasad DT, Pirany N. Optimizing factors influencing DNA extraction from fresh whole avian blood. Afr J Biotechnol. 2007; 6(4): 481-486. https://www.ajol.info/index.php/ajb/article/view/56247
Taylor D, Richards K, Morris D. Rapid recovery of Echinococcus granulosus following ‘successful’albendazole therapy in a gerbil model. J Helminthol. 1989; 63(4): 349-352. https://doi.org/10.1017/S0022149X00009251.
Al-azawy A. A rapid and non-enzymatic method for genomic DNA extraction from whole blood and some other mammalian tissues. J. Tikrit Univ for Agri. Sci. 2012; 12(4): 165-169.
Nasiri H, Forouzandeh M, Rasaee M, Rahbarizadeh F. Modified salting‐out method: high‐yield, high‐quality genomic DNA extraction from whole blood using laundry detergent. J Clin Lab Analys. 2005; 19(6): 229-232. https://doi.org/10.1002/jcla.20083.
Shaik M, Alladi A, Vedamurthy A, Devaraju K, Kamate M, Kruthika-Vinod TP. Large-scale Extraction of DNA by Using Salting-out Principle for Dried Blood Spots to Screen Multiple Mutations in GCDH Gene. Iranian J Sci Tech, Transactions A: Science. 2022; 46(1): 33-40. https://doi.org/10.1007/s40995-021-01225-x.
Wilson IG. Inhibition and facilitation of nucleic acid amplification. Appl Environ Microbiol. 1997; 63(10): 3741-3751.
Aidar M, Line SRP. A simple and cost-effective protocol for DNA isolation from buccal epithelial cells. Braz Dent J. 2007; 18(2): 148-152. https://doi.org/10.1590/S0103-64402007000200012.
Brassard D, Geissler M, Descarreaux M, Tremblay D, Daoud J, Clime L, et al. Extraction of nucleic acids from blood: unveiling the potential of active pneumatic pumping in centrifugal microfluidics for integration and automation of sample preparation processes. Lab on a Chip. 2019; 19(11): 1941-1952. https://doi.org/10.1039/C9LC00276F.
Chacon-Cortes D, Haupt LM, Lea RA, Griffiths LR. Comparison of genomic DNA extraction techniques from whole blood samples: a time, cost and quality evaluation study. Mol Biol Rep. 2012; 39(5): 5961-5966. https://doi.org/10.1007/s11033-011-1408-8.
Khosravinia H, Ramesha K. Influence of EDTA and magnesium on DNA extraction from blood samples and specificity of polymerase chain reaction. Afr J Biotechnol. 2007; 6(3): 184-187.
Tao R, Wang S, Zhang J, Zhang J, Yang Z, Sheng X, et al. Separation/extraction, detection, and interpretation of DNA mixtures in forensic science (review). Int J Legal Med. 2018; 132(5): 1247-1261. https://doi.org/10.1007/s00414-018-1862-0.
Jikuzono T, Horikawa A, Ishikawa T, Hirokawa M, Sugitani I, Inui T, et al. Proteinase K treatment improves RNA recovery from thyroid cells fixed with liquid-based cytology solution. BMC Res Notes. 2018; 11(1): 822. https://doi.org/10.1186/s13104-018-3914-4.
Yang JL, Wang MS, Cheng AC, Pan KC, Li CF, Deng SX. A simple and rapid method for extracting bacterial DNA from intestinal microflora for ERIC-PCR detection. World J Gastroenterol: WJG. 2008; 14(18): 2872. https://doi.org/10.3748%2Fwjg.14.2872.
Goldenberger D, Perschil I, Ritzler M, Altwegg M. A simple" universal" DNA extraction procedure using SDS and proteinase K is compatible with direct PCR amplification. Genome Res. 1995; 4(6): 368-370. https://doi.org/10.1101/gr.4.6.368.
Manuja A, Manchanda SK, Kumar B, Khanna S, Sethi RK. Evaluation of different methods of DNA extraction from semen of buffalo (Bubalus bubalis) bulls. Buffalo Bull. 2010; 29(2): 109-114.
Shamas S, Zafar M, Irum S, Khan L, Sadia H, Roshan S, et al. Prevalence of Diabetes Mellitus in Hepatitis C Patients in Wazirabad Tehsil of Gujranwala District of Pakistan: hepatitis C in Diabetic patients, Baghdad Sci J. 2020; 17(4): 1154. https://doi.org/10.21123/bsj.2020.17.4.1154.
Peker N, Couto N, Sinha B, Rossen JW. Diagnosis of bloodstream infections from positive blood cultures and directly from blood samples: recent developments in molecular approaches. Clin Microbiol Infect. 2018; 24(9): 944-955. https://doi.org/10.1016/j.cmi.2018.05.007.
Phua TJ. The etiology and pathophysiology genesis of benign prostatic hyperplasia and prostate cancer: a new perspective. Medicines. 2021; 8(6): 30. https://doi.org/10.3390/medicines8060030.
Ng KL. The Etiology of Prostate Cancer. In Prostate Cancer 2021, pp 17-27, Exon Publications. https://doi.org/10.36255.
Matthews HK, Bertoli, C, de Bruin, R A. Cell cycle control in cancer. Nat Rev Mol Cell Biol. 2022; 23(1): 74-88. https://doi.org/10.1038/s41580-021-00404-3.
Wan J, Zhang J, Zhang J. Expression of p53 and its mechanism in prostate cancer. Oncol Lett. 2018; 16(1): 378-382. https://doi.org/10.3892/ol.2018.8680.
Gesztes W, Schafer C, Young D, Fox J, Jiang J, Chen Y, et al. Focal p53 protein expression and lymphovascular invasion in primary prostate tumors predict metastatic progression. Sci Rep. 2022; 12(1): 1-12. https://doi.org/10.1038/s41598-022-08826-5.
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