Comparing mitochondrial DNA (mtDNA) to nuclear DNA sequences provides valuable insights into evolutionary relationships and human history. This article outlines how to perform such comparisons using the National Center for Biotechnology Information (NCBI) database and BLAST (Basic Local Alignment Search Tool).
Accessing and Filtering Mitochondrial DNA Sequences
The NCBI Nucleotide database houses a vast collection of DNA sequences. To specifically find human mitochondrial DNA, use the following search terms:
human[organism] AND mitochondrion[title]Further refine your search by selecting “RefSeq” under “Source databases” in the filter menu. RefSeq sequences are high-quality, curated, and annotated by NCBI staff, ensuring reliable data for comparison. You’ll find three human mitochondrial genome Reference Sequences: modern human (Homo sapiens), Neanderthal (Homo sapiens neanderthalensis), and Denisovan (Homo sp. Altai).
Comparing Sequences Using BLAST
To compare these sequences, utilize BLASTn (Nucleotide BLAST). In the NCBI results page, click “Run BLAST” under “Analyze these sequences” in the right-hand menu. The accession numbers of the selected RefSeq sequences will automatically populate the Query Sequence box.
Enable the “Align two or more sequences” option. To compare the modern human mtDNA (NC_012920.1) to Neanderthal (NC_011137.1) and Denisovan (NC_013993.1) sequences, move the latter two accession numbers to the Subject Sequence box. Input a job title and click “BLAST,” retaining default settings.
Analyzing the Results
The results will display two comparisons: modern human mtDNA against Neanderthal and Denisovan mtDNA, respectively. You’ll observe high similarity percentages (around 99% for Neanderthal and 98% for Denisovan).
To delve into specific differences:
- Navigate to the “Alignments” tab and select “Pairwise with dots for identities” in the “Alignment view” dropdown.
- Check the “CDS feature” box.
Each comparison presents a pairwise alignment where dots represent identical bases and red letters highlight differences. Focus on the Coding Sequence (CDS) regions, displayed in four lines: amino acid translation for modern human, modern human sequence, ancient human sequence, and amino acid translation for the ancient human.
For instance, note the two additional amino acids (Methionine and Proline) at the beginning of the modern human protein sequence compared to Neanderthal. This stems from a Thymine (T) at position 3308 in the modern human sequence, contrasting with Cytosine (C) in the Neanderthal sequence. Another difference lies at position 3334, where Adenine (A) in the modern human results in Isoleucine (I), while Guanine (G) in the Neanderthal leads to Valine (V). These variations can be further analyzed for their impact on protein structure and function, determining if they are conservative (minimal change) or non-conservative (significant change) mutations. Examine the Denisovan sequence at these same positions (3308 and 3334) for any variations.
Determining Evolutionary Relationships
To visualize the evolutionary connections:
- Go to the “Description” tab and click “Distance tree of results.”
- In the displayed cladogram, navigate to “Tools” > “Layout” and select “Slanted Cladogram.” This visualization clarifies which ancient hominin, Neanderthal or Denisovan, shares a closer evolutionary relationship with modern humans based on mtDNA comparisons.
