This option allows you to design primers to amplify a specific region. To use this option, change the Task to Precise. If this is on but set to zero, Geneious will not limit primer distances but will try not to reuse the same primer in different pairs. The number of bases specified here is the minimum distance between the 3′ ends of primers of the same direction in different pairs. If you have chosen to return more than one pair of primers and you do not want the same primer used in different pairs, open the Characteristics panel and check Minimum Primer Distance. Setting this to 1 will give you only the primer pair which was considered best by the set parameters. The final option in this section is Number of Pairs to Generate which specifies how many candidate pairs of primers and DNA probes to generate and is compulsory. Warning: Setting these options can cause the primer design process to take considerably longer to complete. Setting this will mean primer pairs that have a product size close to this will be chosen over those that do not. Optimal Product Size: Specifies the preferred size of the product. The product size is the distance in bp between the beginning of the forward primer to the end of the reverse primer. Product Size: Specifies the range of sizes which the product of a primer pair can have. Target Region: Specifies which region of the sequence you wish to amplify and unless the advanced options allow otherwise, the forward and reverse primers must fall somewhere outside this region. This must surround the target region and allows you to choose a small region on either side of the target in which primers must lie. Included Region: Specifies the region of the sequence within which primers are allowed to fall. All of these are expressed in base pairs from the beginning of the sequence and are as follows: If you have selected a region in the sequence before opening the primer dialog then this region will automatically be used for Included Region and Target Region. Most options are optional and can be enabled or disabled with the associated check boxes beside them. These options allow you to specify what part of a sequence you wish to amplify. This option will design standard PCR primers according to the region input options you select. Design for Sequencing allows you to tile primers across a sequence in one or both directions to facilitate Sanger sequencing of the selected molecule or region. For both of these options, Generic or Cloning primers can be designed. It also allows you to design a probe to match a pair of primers. Design with Existing can design a partner primer to match an existing one, for example a reverse primer for a forward or vice versa. You can specify if you wish to design with or without a matching probe. Three options are available for primer design: Design New, Design with Existing, or Design for Sequencing.ĭesign New designs a pair of forward and reverse primers. In Geneious Prime 2023 onwards, primers can also be tested for specificity against an off-target database during primer design. The Primer Design dialog allows you to set options for where your PCR primers should sit, what size product to return and characteristics such as primer length and melting temperature. Geneious Prime uses Primer3 to design PCR primers. A popup menu will appear showing the operations valid for your current selection. To use any one of these primer operations simply select the appropriate nucleotide sequences and either select Primers from the Tools menu or right-click (Ctrl+click on Mac OS X) on the document(s) and select Primers. Characteristics can also be determined for any number of primer sized selections made in the Sequence View. In addition, Geneious Prime can determine the primer characteristics for a primer sized sequence and convert it into a primer. 5′ extensions consisting of restriction enzymes or arbitrary sequence may also be added to primer documents. A PCR product can be extracted from a sequence that has been annotated with both a forward and a reverse primer. PCR Primers and DNA or hybridisation probes can be designed for or tested on existing nucleotide sequences or alignments. Geneious Prime provides several operations for designing and working with PCR Primers and DNA or hybridisation probes.
0 Comments
Leave a Reply. |
Details
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |