MacVector provides functionality for screening a nucleic acid sequence for likely sequencing primers or hybridization probes of up to about 40 nucleotides.
To use this functionality, a nucleic acid sequence window must be the active window.
1. Choose Analyze | Primers | Sequencing Primers/Probes.
The Find Sequencing Primers/Probes dialog box is displayed.
2. In the Region to scan panel, enter the base numbers that bracket the region you want to scan by typing them in the text boxes, or by selecting a region from the features table drop-down menu at the right of the boxes.
3. Choose the strand to scan from the strand drop-down menu.
4. Type the required range of primer/probe lengths in the length text boxes. The range between these values must be 20 or less.
Note that the Tm of primers longer than 35 base pairs has NOT been empirically tested.
5. Type the required percentage range of primer/probe G+C content in the percent G+C text boxes.
6. Type the required range of primer/probe Tm values in the Tm (°C) text boxes.
7. Type the IUPAC code for the two nucleotides that you want to appear at the 3' end of the primer/probe in the 3' dinucleotide text box.
8. Use the primer vs primer (any) drop-down menu to specify the maximum number of consecutive bonds of any type that you will allow the primer/probe to form with itself (hairpin formation) or with another primer/probe (dimer formation).
9. Use the primer vs primer (G-C) drop-down menu to specify the maximum number of consecutive G-C bonds that you will allow the primer/probe to form with itself (hairpin formation) or with another primer/probe (dimer formation).
10. Use the 3' end vs 3' end drop-down menu to specify the maximum number of consecutive bonds that you will allow for the formation of " dimers".
This is important for sequencing primers; for hybridization probes, choose the largest value.
11. Use the primer vs sequence drop-down menu to specify the maximum number of consecutive bonds you will allow between the primer/probe and the sequence that will be present in the sequencing or hybridization reaction. There are two associated options with this:
- for a sequencing primer, you would usually select the radio button that specifies 3' end only
- for a hybridization probe, you would usually select the radio button that specifies the entire primer.
12. In the over the region text boxes, type the base numbers that bracket a comparison region, or select a region from the features table drop-down menu to the right of the boxes.
This is the region of the sequence that you wish to compare with the potential primer/probe to eliminate those that bind to alternate sites.
13. Choose the strand to use in the comparison from the strand drop-down menu.
14. Type in the sum of the concentrations of the two primers at the start of the amplification reaction in the total initial primer conc. (µM) text box.
15. Type in the monovalent cation (Na and K) concentration of the reaction mixture in the monovalent cation conc. (mM) text box.
16. Select OK to perform the screen.
Alternatively, select Defaults to restore the default settings, or Cancel to close the dialog box without performing the screen.
At the end of the screen, the Primers/Probes Display dialog box is displayed.
The Primers/Probes Display dialog box contains statistical information about the scan for primers/probes: how many were considered, how many eliminated and the reasons for rejection. If none were found, look at these statistics to determine the most common cause of rejection. This can help you decide which setup parameters to modify.
The Primers/Probes Display dialog box provides a number of options for displaying the results of the screen.
1. The Primers/Probes Display dialog box is displayed on completion of each analysis. To display this dialog box at other times, for example to change the display parameters, choose Analyze | Primers | Sequencing Primers/Probes when any Primers/Probes display result window is active.
2. Select the Tm (°C) checkbox to restrict the displays to those primers/probes whose Tm's lie within the range specified in the text boxes.
3. Select the percent G+C checkbox to restrict the displays to those primers/probes whose G+C percentage lies within the range specified in the text boxes.
4. Select the list of primers/probes checkbox to see a list of the primers or probes and information about them: Tm, G+C content, length, and location.
5. Select the map of primers/probes checkbox to see a graphical representation of the sequencing primers or hybridization probes.
6. Select OK to display the results.