This menu command will only be enabled if a protein sequence editing window or a reverse translation results window is the frontmost window on the desktop.
MacVector provides a way to find an oligo to use as a hybridization probe for a gene whose exact DNA sequence is not known, provided the amino acid sequence is known. The process used is as follows:
- reverse-translate the amino acid sequence;
- scan the resulting DNA sequence to find a region that is not very degenerate;
- use this region to make probes, thus minimizing the number of oligonucleotide probes that would have to be synthesized.
To use this functionality, a protein sequence window must be the active window.
Prior to the analysis, you may want to reduce the degeneracy of the resultant DNA sequence by eliminating certain codons from the genetic code used for the reverse translation. Your knowledge of the codon preferences of the organism of interest will be your best guide in eliminating codons.
1. Choose Analyze | Reverse Translation.
2. Type the base numbers that bracket the region you want to scan in the Region text boxes, or select a region from the features table drop-down menu at the right of the boxes.
3. Select the genetic code to be used from the genetic code drop-down menu.
4. Select the Probe list check box to generate a list of least-ambiguous oligonucleotide probes for the specified region.
The output lists the sequence of each probe, the corresponding amino acid sequence, the percent G+C content of the probe, and the dissociation temperature of the probe-DNA complex.
5. If you are generating a probe list, limit the range of probe sizes by entering values in the minimum and maximum text boxes.
6. Select the Annotated sequence check box to display the degenerate nucleic acid sequence that results from reverse translating the protein. The appearance of this display can be changed interactively by choosing Customize | Format Annotated Display. For more information, see the help topic "Formatting the annotated sequence display".
7. Select the New DNA check box to create a new DNA sequence window for the degenerate sequence created by reverse translating the protein sequence. Type a name for the new sequence in the text box.
8. Select OK to perform the screen.
If you have displayed an annotated sequence, the amino acid that results from translation is shown beneath the degenerate nucleic acid sequence. Note that this amino acid sequence may not match the original protein because certain degenerate codons could code for more than one amino acid. For example, serine has six codons, four of the form TCN and two of the form AGY. These two forms reduce to the single degenerate codon WSN. If this degenerate codon is then retranslated, it will be assigned the "unknown" amino acid X, since WSN can expand to any of the following:
- TCN = Ser
- ACN = Thr
- AGY = Ser
- AGR = Arg
- TGY = Cys
- TGA = End
- TGG = Trp.