Comparative analysis of different DNA extraction methods and their genotyping efficiency in Mexican population
PDF (Español (España))
HTML (Español (España))

Keywords

DNA extraction
genotyping
SNP
polymorphisms
PCR
genetic variability
Mexico.

Abstract

Genetic variability studies have presented inconsistencies between populations, mainly because methods of deoxyribonucleic acid (DNA) extraction and genotyping techniques show high variability between them, leading to an erroneous assignment of genotypes. The objective of this study is compare different techniques for DNA extraction and genotyping of polymorphisms. To perform this study, DNA from 10 blood samples corresponding to mestizo Mexicans was analyzed and purified by methods of phenol-chloroform-isoamyl alcohol (PCA), salting out gradient (SG), sucrose gradient (SCG), DNAzol ® method and DNeasy Blood & Tissue Kit ®. GSTT1*0 and GSTM1*0 polymorphisms were genotyped by multiplex PCR, CYP1A1*2C by PCR-RFLP's assay and AhR Arg554Lys by real-time PCR. Results shown that amount, purity and integrity of DNA were evaluated. Different methods results showed significant differences (p < 0.001). Molecular analyses showed that PCA method presents inhibitors for PCR reaction because it was not possible to amplify fragments in multiplex PCR and endpoint. Although, there was amplification in real time PCR. SG and SCG methods amplified all samples in three types of PCR and the results were concordant between them. While in PCR - RFLP analysis, samples extracted by DNAzol ® and DNeasy ® amplified only 80% of samples with no concordant results (50% for DNAzol ® and 80% for DNeasy ®). SG extraction methods and SCG showed higher DNA recovery, with optimal quality parameters for molecular analyses by PCR.  


https://doi.org/10.15174/au.2016.1078
PDF (Español (España))
HTML (Español (España))

References

Abdel-Rahman, S. Z., el-Zein, R. A., Anwar, W. A., & Au, W. W. (1996). A multiplex PCR procedure for polymorphic analysis of GSTM1 and GSTT1 genes in population studies. Cancer Letters, 107(2), 229-233.


Baena, J. A., Ramos, A. J., Gómez, C. J., & Gómez, D. E. (2013). Comparación de métodos de extracción de ADN en tejidos parafinados y utilidad para amplificación por PCR. Revista Colombiana de Biotecnología, 15(1), 172-179.


Blanco-Jarvio, A., Martínez, L. A., & Bautista, G. A. (2014). Optimización de un protocolo de extracción de DNA total para la amplificación de marcadores moleculares funcionales específicos de organismos desnitrificantes. CICIMAR Oceánides, 29(2), 37-44.


Caboux, E., Lallemand, C., Ferro, G., Hemon, B., Mendy, M., & Biessy, C. (2012). Sources of pre-analytical variations in yield of DNA extracted from blood samples: Analysis of 50 000 DNA samples in EPIC. PLoS One, 7(7), e39821-e39821.


Cascorbi, I., Brockmöller, J., & Roots, I. (1996). A C4887A polymorphism in exon 7 of human CYP1A1: population frequency, mutation linkages, and impact on lung cancer susceptibility. Cancer Research, 56(21), 4965-4969.


Daly, A., Steen, V., Fairbrother, K., & Idle, J. (1996). CYP2D6 multiallelism. Methods in Enzimology, 272, 199-201.


Dong, L., Potter, J., White, E., Ulrich, C., Cardon, L., & Peters, U. (2008). Genetic susceptibility to cancer: the role of polymorphism in candidate genes. The Journal of the American Medical Association, 299(20), 2423-2434.


Eguiarte, L. E., Souza, V., & Aguirre, X. (2007). Ecología molecular. México: Secretaría de Medio Ambiente y Recursos Naturales (Semarnat) / Comisión Nacional para el Conocimiento y uso de la Biodiversidad (Conabio).


Gaedigk, A., Freeman, N., Hartshorne, T., Riffel, A. K., Irwin, D., Bishop, J. R., Stein, M. A., Newcorn, J, F., Montané, L. K., Cherner, M., & Leedert, J. S. (2015). SNP genotyping using TaqMan® technology: the CYP2D6*17 assay conundrum. Scientific Reports, 19(5), 1-9.


Garte, S. (1998). The role of ethnicity in cancer susceptibility gene polymorphism: the example of CYP1A1. Carcinogenesis, 19(8), 1329-1232.


Green, M., & Sambrook, J. (2012). Molecular Cloning. A laboratory manual (4th ed.). New York: Cold Spring Harbor Laboratory Press.


He, H. R., You, H. S., Sun, J. Y., Hu, S. S., Ma, Y., Dong, Y. L., & Lu, J. (2014). Glutathione S-transferase gene polymorphisms and susceptibility to acute myeloid leukemia: meta-analyses. Japanese Journal of Clinical Oncology, 44(11), 1070-1081.


International HapMap Project (2015). Recuperado el 20 de abril de 2015 de http://hapmap.ncbi.nlm.nih.gov/


Lahiri, D. K., & Nurnberger, J. (1991). A rapid non-enzymatic method for the preparation of HMW DNA from blood for RFLP studies. Nucleic Acids Research, 19(19), 5444.


Monien, B. H., Schumacher, F., Herrmann, K., Glatt, H., Turesky, R. J., & Chesne, C. (2014). Simultaneous detection of multiple DNA adducts in human lung samples by Isotope-Dilution UPLC-MS/MS. Analytical Chemistry, 87(1), 641-648.


National Center for Biotechnology Information (2015). Basic Local Alignment Search. Recuperado el 5 de junio de 2015 de https://blast.ncbi.nlm.nih.gov/Blast.cgi


Osorio-Cadavid, E., Ramírez, M., López, W. A., & Mambuscay, L. A. (2009). Estandarización de un protocolo sencillo para la extracción de ADN genómico de levaduras. Revista Colombiana de Biotecnología, 15(1), 125-131.


Pérez-Morales, R., Méndez-Ramírez, I., Moreno-Macías, H., Mendoza-Posadas, A. D., Martínez-Ramírez, O. C., Castro-Hernández, C., Gonsebatt, M. E., & Rubio, J. (2014). Genetic susceptibility to lung cancer based on candidate genes in a sample from the Mexican Mestizo population: A case-control study. Lung, 192(1), 167-73.


Saitou, M., & Ishida, T. (2015). Distributions of the GSTM1 and GSTT1 null genotypes worldwide are characterized by latitudinal clines. Asian Pacific Journal of Cancer Prevention, 16(1), 355-361.


Spink, B. C., Bloom, M. S., Wu, S., Sell, S., Schneider, E., Ding, X., & Spink, D. C. (2014). Analysis of the AHR gene proximal promoter GGGGC-repeat polymorphism in lung, breast, and colon cancer. Toxicology and Applied Pharmacology, 282(1), 30-41.


Thompson, R. E., Duncan, G., & McCord, B. R. (2014). An investigation of PCR inhibition using Plexor®-Based Quantitative PCR and Short Tandem Repeat Amplification. Journal of Forensic Sciences, 59(6), 1517-1529.


Zhang, L., Zhao, J., Cui, G., Wang, H., & Wang, D. W. (2015). Genotyping on ALDH2: Comparison of four different technologies. PLoS One, 10(3), e0122745-e0122754.