Open the browser and go to the home page of ORF Finder. Figure 1 shows the ORF finder main page at NCBI.
For info on accessing the ORF Finder please go to simulator tab.
Figure 1: Home page of ORF finder
Here one can see a text field to enter the accession number of the query sequence, a text box to enter the query sequence in FASTA format and a button to run the ORF finder.
FASTA format is a simplest sequence format which starts with a ‘>’ symbol followed by the sequence ID, other comments and computationally represented protein sequence.
Enter a computerized nucleotide sequence in the box provided (in FASTA format).
Figure 5: Copied FASTA sequence of hemoglobin is pasted into the box provided.
There is a drop down menu to select a genetic codon dictionary. It contains 20 different codon dictionaries that contains codons for different organisms and organelles.Select any from the list which you want for the search method (Figure 6).
Figure 6: Select any codon dictionary from the drop down menu provided.
In the genetic codes list, one can see many codon dictionaries. It differs in the triplets those codes for amino acids. The first one is the "standard" which is the default codon (Figure 7).
Figure 7: Genetic code list
For example, the standard code AUG code for methionine. But in Vertebrate Mitochondrial Code and Yeast Mitochondrial Code, AUA codes for methionine.
Now press the ORF finder button to get the result .The result shows the all the possible six reading frame present in the entered sequence query (Figure 8). One can see that the ORF is listed according to their size and the graphical representation of the sequence.
Figure 8: All the possible six reading frame present in the entered sequence query
Click on the green region which represent the ORF in the sequence, to see the ORF. Once you click, it will turn into purple color indicating that the particular ORF is selected (Figure 9). The selected ORF is also indicating in the list. It also displays the length and location of the selected ORF.
Figure 9: Select an ORF
One can see the sequence of the selected ORF which actually codes for the protein. The user can find the start codon, stop codon and the total number of the amino acids from the sequence. Now click on Accept button(Figure 10).
Figure 10: Accept the selected ORF
The accepted region will turn into light green color indicates that the selected ORF is accepted. This is to indicate that the user wants to study further about the particular ORF (Figure 11).
Figure 11: Accepted ORF turn into light green color
User can also perform a BLAST search for the particular ORF that we selected. Select the appropriate program and database. Then click on the BLAST button (Figure 12).
Figure 12: BLAST search for the a particular ORF
BLAST (Basic local alignment search tool) is a pair wise sequence alignment tool which compares nucleotide or protein sequences, finds the local similarity between sequences and calculates the statistical significance of matches. BLAST can be used to find the functional and evolutionary relationships between the sequences. There are different BLAST programs for different comparisons (Table 1).
Table 1: Different BLAST programs for different comparisons
Now in the resulting page click on the ‘View Report’ button (Figure 13).
Figure 13: View Report shows the BLAST results.
This following (Figure 14) is the BLAST resulting page. You can see the alignment scores and description here.
After submitting the query sequence for sequence similarity search, the result page will appear along with the information like Query id, Description, Molecule type, Length of sequence, Database name and BLAST program. It shows the putative conserved domains that have been detected while undergoing sequence similarity search.
The query sequence represented as a numbered red bar below the color key. Database hits are shown below the query (red) bar according to the alignment score. Among the aligned sequences, the most related sequences are kept near to the query sequence. User can find more description about these alignments, by dragging the mouse to the each colored bar.
Figure 14: BLAST results
The alignment is preceded by the sequence identities, along with the definition line, length of the matched sequence, followed by the score and E-value. The line also contains the information about the identical residues in alignment (identities), number of positivity’s, number of gaps used in the alignment. Finally it shows the actual alignment, along with the query sequence on the top and database sequence below the query. The number on either sides of the alignment indicates the position of amino acids/nucleotides in sequence.
This experiment uses: NCBI database, www.ncbi.nlm.nih.gov/, ORF finder, http://www.ncbi.nlm.nih.gov/projects/gorf/