Comparative analysis of human DNA extraction methods and mitochondrial DNA HV1 and HV2 haplogroup determination
The high integrity of extracted DNA is necessary for the determination of human haplogroups, based on the mitochondrial DNA (mtDNA) marker sequences. The study aims were to isolate the total DNA from selected human samples and establish a protocol that gives the best yield and proper purity of DNA m...
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| Format: | Article |
| Language: | English |
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University of Kragujevac, Faculty of Science
2020-01-01
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| Series: | Kragujevac Journal of Science |
| Subjects: | |
| Online Access: | https://scindeks-clanci.ceon.rs/data/pdf/1450-9636/2020/1450-96362042073N.pdf |
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| author | Nikezić Aleksandra G. Blagojević Stefan Z. Ćupurdija Maja Đ. Planojević Nevena S. Jovankić Jovana V. Rakobradović Jelena D. Vidanović Dejan S. Arsenijević Dejan D. Marković Snežana D. |
| author_facet | Nikezić Aleksandra G. Blagojević Stefan Z. Ćupurdija Maja Đ. Planojević Nevena S. Jovankić Jovana V. Rakobradović Jelena D. Vidanović Dejan S. Arsenijević Dejan D. Marković Snežana D. |
| author_sort | Nikezić Aleksandra G. |
| collection | DOAJ |
| description | The high integrity of extracted DNA is necessary for the determination of human haplogroups, based on the mitochondrial DNA (mtDNA) marker sequences. The study aims were to isolate the total DNA from selected human samples and establish a protocol that gives the best yield and proper purity of DNA material for further analysis. Furthermore, human hypervariable regions - HV1 and HV2, located on mtDNA were sequenced to define human haplogroups. For extraction, samples of buccal mucosa from ten human volunteers were taken and three different protocols were used (a method with ammonium acetate, a salting-out method and GeneJET Genomic DNA Purification Kit (Thermo Fisher Scientific)). Concentration and purity of DNA were measured on BioPhotometer. Sequencing was performed by Sanger method on 3130 Genetic Analyzer (Applied Biosystems) with commercial BigDye™ Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems). Sequences were processed using Chromas and MEGA software and compared with sequence databases with BLAST tool on NCBI. Haplogroups and mutations were determined in the MITOMAP online database. Results of extraction indicated that the Purification Kit gives the highest concentration of DNA (11.86±0.63) and low purity (1.17±0.02), while a method with ammonium acetate gave the best purity (1.67±0.17). Sequencing obtained on DNA extracted with a commercial kit, gave 5 different human haplogroups U2e, T1a, J1c, H2a, K. In conclusion, it is recommended to use the Purification Kit for obtaining high integrity DNA that can be used for effective sequencing. Determination of haplogroups and haplotypes can reveal our ancestry and have important implications in molecular systematics, phylogenetics and phylogeography. |
| first_indexed | 2024-04-14T05:38:58Z |
| format | Article |
| id | doaj.art-fe674862f02c4476a2d23c11d1ffc61d |
| institution | Directory Open Access Journal |
| issn | 1450-9636 2466-5509 |
| language | English |
| last_indexed | 2024-04-14T05:38:58Z |
| publishDate | 2020-01-01 |
| publisher | University of Kragujevac, Faculty of Science |
| record_format | Article |
| series | Kragujevac Journal of Science |
| spelling | doaj.art-fe674862f02c4476a2d23c11d1ffc61d2022-12-22T02:09:32ZengUniversity of Kragujevac, Faculty of ScienceKragujevac Journal of Science1450-96362466-55092020-01-0120204273831450-96362042073NComparative analysis of human DNA extraction methods and mitochondrial DNA HV1 and HV2 haplogroup determinationNikezić Aleksandra G.0Blagojević Stefan Z.1Ćupurdija Maja Đ.2Planojević Nevena S.3Jovankić Jovana V.4Rakobradović Jelena D.5Vidanović Dejan S.6https://orcid.org/0000-0003-0192-5120Arsenijević Dejan D.7Marković Snežana D.8https://orcid.org/0000-0002-3892-8977University of Kragujevac, Faculty of Science, Department for Biology and Ecology, Kragujevac, SerbiaUniversity of Kragujevac, Faculty of Science, Department for Biology and Ecology, Kragujevac, SerbiaUniversity of Kragujevac, Faculty of Science, Department for Biology and Ecology, Kragujevac, SerbiaUniversity of Kragujevac, Faculty of Science, Department for Biology and Ecology, Kragujevac, SerbiaUniversity of Kragujevac, Faculty of Science, Department for Biology and Ecology, Kragujevac, SerbiaInstitute for Oncology and Radiology of Serbia, Belgrade, SerbiaVeterinary Specialized Institute "Kraljevo", Kraljevo, SerbiaUniversity of Kragujevac, Faculty of Science, Department for Biology and Ecology, Kragujevac, SerbiaUniversity of Kragujevac, Faculty of Science, Department for Biology and Ecology, Kragujevac, SerbiaThe high integrity of extracted DNA is necessary for the determination of human haplogroups, based on the mitochondrial DNA (mtDNA) marker sequences. The study aims were to isolate the total DNA from selected human samples and establish a protocol that gives the best yield and proper purity of DNA material for further analysis. Furthermore, human hypervariable regions - HV1 and HV2, located on mtDNA were sequenced to define human haplogroups. For extraction, samples of buccal mucosa from ten human volunteers were taken and three different protocols were used (a method with ammonium acetate, a salting-out method and GeneJET Genomic DNA Purification Kit (Thermo Fisher Scientific)). Concentration and purity of DNA were measured on BioPhotometer. Sequencing was performed by Sanger method on 3130 Genetic Analyzer (Applied Biosystems) with commercial BigDye™ Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems). Sequences were processed using Chromas and MEGA software and compared with sequence databases with BLAST tool on NCBI. Haplogroups and mutations were determined in the MITOMAP online database. Results of extraction indicated that the Purification Kit gives the highest concentration of DNA (11.86±0.63) and low purity (1.17±0.02), while a method with ammonium acetate gave the best purity (1.67±0.17). Sequencing obtained on DNA extracted with a commercial kit, gave 5 different human haplogroups U2e, T1a, J1c, H2a, K. In conclusion, it is recommended to use the Purification Kit for obtaining high integrity DNA that can be used for effective sequencing. Determination of haplogroups and haplotypes can reveal our ancestry and have important implications in molecular systematics, phylogenetics and phylogeography.https://scindeks-clanci.ceon.rs/data/pdf/1450-9636/2020/1450-96362042073N.pdfdna ancestrydna isolationhv1hv2mtdna haplogroups |
| spellingShingle | Nikezić Aleksandra G. Blagojević Stefan Z. Ćupurdija Maja Đ. Planojević Nevena S. Jovankić Jovana V. Rakobradović Jelena D. Vidanović Dejan S. Arsenijević Dejan D. Marković Snežana D. Comparative analysis of human DNA extraction methods and mitochondrial DNA HV1 and HV2 haplogroup determination Kragujevac Journal of Science dna ancestry dna isolation hv1 hv2 mtdna haplogroups |
| title | Comparative analysis of human DNA extraction methods and mitochondrial DNA HV1 and HV2 haplogroup determination |
| title_full | Comparative analysis of human DNA extraction methods and mitochondrial DNA HV1 and HV2 haplogroup determination |
| title_fullStr | Comparative analysis of human DNA extraction methods and mitochondrial DNA HV1 and HV2 haplogroup determination |
| title_full_unstemmed | Comparative analysis of human DNA extraction methods and mitochondrial DNA HV1 and HV2 haplogroup determination |
| title_short | Comparative analysis of human DNA extraction methods and mitochondrial DNA HV1 and HV2 haplogroup determination |
| title_sort | comparative analysis of human dna extraction methods and mitochondrial dna hv1 and hv2 haplogroup determination |
| topic | dna ancestry dna isolation hv1 hv2 mtdna haplogroups |
| url | https://scindeks-clanci.ceon.rs/data/pdf/1450-9636/2020/1450-96362042073N.pdf |
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