For any sequencing primer, MacVector can generate useful information about its properties and binding characteristics. This enables you to assess sequencing primers from sources other than the MacVector screening function. The calculated characteristics include:
- length and G-C percentage of each sequence
- average Tm, derived from the reverse complement of the primer
- self-dimer formation
- hairpin formation
- self-duplex formation
- binding site information.
To use this functionality, a DNA sequence must be the active window.
1. Choose Analyze | Primers | Test Sequencing Primers/Probes.
The Sequencing Primer dialog box is displayed.
2. Enter the primer sequence into the Primer text box, using the standard IUPAC one-letter codes. The sequence order must be 5' to 3'.
3. If required, select the Parameters button to adjust the parameters used to control the tests that generate the characteristics. See "To modify the sequencing primer test parameters", below , for further details.
4. You can restrict the search for unwanted binding sites to a certain region of the active sequence you are testing primers against, by typing in the base numbers that bracket the region in the Region text boxes, or by selecting a region from the features table drop-down menu at the right of the text boxes.
5. Do one of the following:
- select Apply to run the analysis and display results in the information text box. This leaves the Sequencing Primer dialog box displayed.
- select OK to close the Sequencing Primer dialog box and display a text window of the same name that duplicates the results shown in the information text box. It also shows aligned sequences and product sequences.
NOTE: If a text window of the correct name is already open, then the contents will simply be updated. Because the contents are only updated when the OK button is clicked, it is possible to display information in the text window that is out of step with the PCR Primer Pair dialog information boxes.
The characteristics are calculated from a series of tests, and you can modify the test parameters. The parameters are accessible from the Sequencing Primer dialog box, and include several parameters used on the Find Sequencing Primers/Probes dialog box.
NOTE: The shared parameters are linked, so changes on one dialog will be reflected in the other.
1. Choose Analyze | Primers | Test Sequencing Primer/Probe.
The Sequencing Primer dialog box is displayed.
2. Select the Parameters button.
The Sequencing Primer Parameters dialog box is displayed.
3. Select Allow Ambiguous residues to let primer sequences contain characters other than A, C, T, and G.
4. Use the Mismatches drop-down menu to specify the maximum number of residues by which the primer can diverge from the target sequence.
5. Use the 3'-end clamp drop-down menu to specify the number of residues at the 3' end that must bind with the target.
This is used to reduce the number of potential binding sites.
6. 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 to form with itself (hairpin formation) or with another primer (dimer formation).
7. 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 to form with itself (hairpin formation) or with another primer (dimer formation).
8. 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 "primer dimers."
Primer dimers can lead to amplification of the primers alone instead of the required product.
9. 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.
10. Type in the monovalent cation (Na and K) concentration of the reaction mixture in the monovalent cation conc. (mM) text box.
NOTE: Steps 9 and 10 only affect the Tm calculation.
11. Select OK to return to the PCR Primer Pair dialog box.
Alternatively, select Defaults to restore the default settings, or Cancel to close the dialog box without saving the parameters.