Extraction, Sequencing and Analysis of Human mtdna Taken from a U.S. Population

Transcription

1 BIO 316 (Genetics Lab) 12/4/12 Lab Section: Tuesday 5:45 Dr. Braun Extraction, Sequencing and Analysis of Human mtdna Taken from a U.S. Population Regan Robinson-Barnes

2 Abstract Exploring human mitochondrial DNA has allowed us to understand human origins with a high level of certainty. Analysis of mtdna has become accessible to individuals for information about personal ancestry. Although personally extracted mtdna was unviable, it was possible to obtain useful information from using sequences obtained from a BLAST search. Mitochondrial DNA sequences from Croatia, Italy, Albania, Iran, Nepal, Siberia, Newfoundland and Zambia were used and comparisons were made with a polymorphism table and phylogenetic tree. The results of the phylogeny support the out of Africa theory because Zambia shares a common ancestor with all other locations many years in the past. The other locations are also arranged by increasing relatedness from Zambia as geographical distance from Eurasia increases. Introduction Mitochondrial DNA (mtdna) is an extremely useful tool in understanding the origin and evolution of species. Mitochondrial DNA is a separate circular genome, found in the mitochondria of a cell. A vertebrate mitochondrial genome is approximately 17 kbp and 38 genes (Carr, et.al, 2006). mtdna can be used to create phylogenetic trees which are useful in showing the relatedness of mtdna sequences and tracing lineages back to a common ancestral group. Variation among mtdna sequences has arisen from the colonization of different geographical regions; these first colonizers became the founders of different mtdna lineages (Torroni, et al., 1998). At the root of the human phylogenetic tree is the mitochondrial Eve, who gave rise to the multiple haplogroups identified today (Torroni, et al., 2006). Mitochondrial DNA is not only used on living human populations, due to its ability to be extracted from ancient remains mtdna can be used to study the dispersal of early humans (Adachi, 2004). Many companies have made mtdna analysis open to the public as a way to trace their ancestry. This is done by sequencing the individual s mtdna and comparing the sequence to its known origins. A similar process was performed in the experiment described in this paper. This experiment deals with mtdna extracted from mouth cells of an individual in an upstate New York population. The extracted sequence was edited using the BioEdit program. Using nucleotide BLAST, a DNA sequence database, similar sequences were found. The extracted mtdna was aligned with eight sequences from difference geographical locations and polymorphisms

3 were recorded. With immigration incredibly accessible to the majority of the world, it is not surprising that many different haplogroups have mixed and are living far from their origin. From this data, a phylogenetic tree showing the bootstrap values of relatedness between Albania, Croatia, Iran, Nepal, Newfoundland, Siberia and Zambia was created using the program MEGA. Materials and Methods DNA Extraction One 15 ml conical tube was filled with 10 ml of sterile water. The 10 ml of water was then poured into my mouth and sloshed vigorously for ~1 minute and then spit back into the conical tube. The cap was then replaced and the tube was allowed to stand for ~20 minutes. Because cells had not adequately settled they were spun in a centrifuge at 4000G for 4 minutes. The pellet was then transferred to a 1.5 ml tube using a sterile transfer pipet. The 1.5 ml tube was placed in the microcentrifuge and spun at full speed for 5 minutes. Without disturbing the pellet, the supernatant was poured back into the 15 ml conical tube. To the 1.5 ml tube, 100 μl of Chelex with which the cells were thoroughly mixed. The tube was placed in a Dri-Block set at 100 C for 10 minutes and then transferred to ice for 3 minutes. The tube was then centrifuged at maximum speed for 5 minutes. DNA Quantification using the NanoDrop The NanoDrop was used to quantify the amount of DNA extracted and determine the amount of DNA to be added to the PCR reaction. Using the formula C S V S = C F V F the volume of DNA needed was calculated. 20- V S was used to determine the amount of water needed. Polymerase Chain Reaction To a 1.2 ml tube, 30 μl of master mix, 18 μl of water and 2 μl of DNA were added and gently mixed. The tube was then placed in the thermocycler at 95 C for 15 minutes; 30 cycles of: 94 C for 1 minute, 58 C for 1 minute, 72 C for 1 minute and a final extension of 72 C for 5 minutes, followed by a 10 C hold. Sequencing mtdna The PCR product was transferred into a 1.5 ml tube and 225 μl of PB1 buffer was added and sample was mixed. To a spin column, the entire 270 μl was pipetted and the column was centrifuged at high speed for 1 minute. The clear liquid in the tube was discarded and the column was washed with 750 μl of PE buffer and

4 centrifuged again for 1 minute. The clear liquid was discarded and the column was dry spun in the centrifuge for 1 minute. The spin column was placed in clean 1.5 ml tube and 30 μl of EB buffer was added to the column and allowed to incubate at room temperature for 1 minute. The column was centrifuged at high speed for 1 minute and the PCR product in the 1.5 ml tube was saved. DNA sequencing As described above, the DNA was quantified using the NanoDrop and the amount of water and DNA needed was calculated. A 0.2 ml tube was obtained and to it 1 μl DI water and 4 μl of DNA, 1 μl of primer and 4 μl master mix was added and the solution was mixed. The tube was then placed in the thermocycler at 40 cycles of 96 C for 20 seconds, 50 C for 20 seconds and 60 C for 4 minutes; followed by a 10 C hold. Analysis of Sequencing Data After sequencing the mtdna using a CEQ 8000, BioEdit was used to edit the mtdna sequence. BLAST was then used to find similar sequences on the database and finally MEGA5 was used to construct a phylogenetic tree. Results The original sample of mtdna extracted from personal cheek cells was unviable due to thermocycler malfunction. A replacement sequence was obtained from an unknown individual to complete the PCR reaction which was also unsuccessful. Another sequence of unknown origin was obtained and compared to 8 mtdna sequences. This loaned sequence also proved unviable as is was not able to be aligned to the 8 other mtdna sequences and was removed. The polymorphisms in the 8 sequences are shown below in table 1. The relatedness of the different regions is shown in figure 1 with the corresponding bootstrap values. At 146bp all locations contain thymine except Iran which contained cytosine. At 189bp, all locations show the nucleotide adenine except Zambia which contains guanine. Zambia also shows polymorphisms at 195bp and 200bp of cytosine and guanine respectively. Newfoundland shows a polymorphism at 199bp and 311bp both of which were cytosine. Nepal contains a polymorphism at 312bp of thymine and lastly Siberia has one polymorphism at 491bp cytosine.

5 The phylogenetic tree shown in figure 1 provides the bootstrap values to quantify the level of relatedness of each location. According to the phylogeny, the common ancestor to between Zambia and the other locations was the furthest in the past. The most closely related is shown to be Italy and Croatia with a bootstrap value of 23. The bootstrap value of Albania s relatedness to Italy and Croatia is 64, followed by Iran s bootstrap value of 42, Nepal with a bootstrap value of 66 and finally Siberia with 75. Table 1: This table shows the polymorphisms identified in the sequences of mtdna when compared to the Albanian sequence. Empty cells show an unknown nucleotide. Region Albania C A T C A T T A T T Croatia C A T C A T T A C T Iran T A C C A T T A C T Nepal T A T C A T T A T T T Newfoundla T G T T A T C A C C T nd Siberia T G T C A T T A T C C Zambia T G T T G C T G T C T Italy C G T C A T T A T C T Figure 1: Phylogenetic tree of human mtdna sequences from Croatia, Italy, Albania, Iran, Nepal, Siberia, Newfoundland and Zambia. Discussion The results obtained from this experiment largely support the out of Africa hypothesis. Although many of the collected mtdna samples from our New York state population were unviable, useful information was obtained by compiling the mtdna sequences from the BLAST search. The out of Africa hypothesis of the origin of humans states that all humans share a common ancestor in Africa (Carr, et al, 2006). The modern human whom evolved approximately 150,000 years ago in Africa split off and colonized Eurasia approximately 100,000 years ago (Templeton, 1997). Although our bootstrap values were all less than 80 and therefore

6 statistically insignificant, the organization of the phylogenetic tree (fig. 1) is feasible when compared to the polymorphisms shown in table 1. Since the modern human originated in Africa, populations in Zambia would have had the most time to acquire mutations which differentiate their mtdna from other populations. This is supported by Zambia being the least related to the other populations in the phylogenetic tree. The location of Newfoundland in the phylogeny is unexpected; however a study conducted by Carr et al. in 2008 offers a possible explanation. According to this study, the Newfoundland colony was settled by western England and southeastern Ireland and some French peoples between 1592 and Some of these French individuals share the mitochondrial DNA characteristics of Eurasian populations. Since the first modern humans who migrated out of Africa went to Eurasia, it makes sense that some of these characteristics still exist in the Newfoundland population. It seems that other location s relatedness can be explained by sheer geographic location with decreasing relatedness as distance from Eurasia increases.

7 References Adachi, N., Umetsu, K., Takigawa, W., & Sakaue, K. (2004, February 2). Phylogenetic analysis of the human ancient mitochondrial DNA. Journal of Archaeological Science, doi: /j.jas Carr, S. M., Marshall, H., Duggan, A. T., Flynn, S. C., Johnstone, K. A., Pope, A. M., & Wilkerson, C. D. (2006, December 15). Phylogeographic genomics of mitochondrial DNA: Highly-resolved patterns of intraspecific evolution and a multi-species, microarray-based DNA sequencing strategy for biodiversity studies. Comparative Biochemistry and Physiology, Part D(3), doi: /j.cbd Templeton, A. R. (1997, December). Out of Africa? What do genes tell us? Current Opinion in Genetics & Development, 7(6), Torroni, A., Achilli, A., Macaualy, V., Richards, M., & Bandelt, H. (2006, June). Harvesting the fruit of the human mtdna tree. Trends in Genetics, 22(6), doi: /j.tig Torroni, A., Bandelt, H., D'Urbano, L., Lahermo, P., Moral, P., Sellitto, D., & Rengo, C. (1998, May). MtDNA Analysis Reveals a Major Late Paleolithic Population Expansion. The American Journal of Human Genetics, 62(5),