We are going to design some primers to the first exon 4 in the TNF-alpha sequence.
Begin by opening the molecule, if it is not already open. Now select the exon 4 by clicking either on the name or on the arrow corresponding to the sequence.
To design the primers, choose Find PCR primers from the Analyze menu.
This will open the primer selection dialog box. (Note the mac version is different from this, but the options are similar, just in different locations).
There are lots of options available for designing the right primers:
Clicking the More >> button allows you to add further details to the primers, such as tag sequences that you want at the 5' or 3' end of each primer. This is useful if you wish to add restriction sites at the end of the primers to allow subcloning of the PCR product into another vector.
These are the basic operations, and if you click OK, you will get a list of primers. However there are many advanced options that you can set and are worth examining for your particular use of the oligos.
| Amplicon | The amplicon box allows you to set properties of the PCR product, notably first and last nucleotides, minimum %GC and so on. |
| Structure | The structure dialog allows you to control some aspects of the primer structures, including the presence of hairpin loops, nucleotide repeats, and palindromes. Later we will learn how to examine the primers for these features. |
| Pairs | The pairs dialog allows you to set some parameters between the primers, such as the difference between the Tm of the two primers, and dG and %GC differences. |
| Similarity | Controls how similar the primers are to the template. Generally Best Fit is suitable for most applications. |
| 3' end | Under the 3' end you can limit the nucleotides at the 3' end of the primer. Often G/C pairs are better at the 3' end as they have stronger bonds than A/T pairs, and so you may not wish A/T pairs at the immediate 3' end. |
| Uniqueness | The uniqueness will ensure that the primers do not anneal elsewhere in your molecule. |
| Qualities | The qualities dialog allows you to emphasize some of the criteria over others. For example, a high Tm may be more important than the Tm difference. You can give each criteria a score between 1 and 10, where 1 is least important and 10 is critically important. |
| Filters | The filters allow you to include, or exclude, specific features from the feature map. |
When you have worked through these selections, you may click OK to begin finding the primers.
N.B. It is very easy while trying to optimize primer conditions to produce conditions for which no primers could ever be made, and vectorNTI will not warn you about this. The Mac version (at least) will continue a futile attempt to keep finding primers under these conditions, and will search for ever (or at least overnight-I clicked Stop Search in the morning and it reported the primers that it had found back to me). If the search is taking too long and not getting anywhere, click Stop Search and review your conditions.
Results Analysis
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The analysis results are in the PCR products folder in the left text pane. Typical results will look like this (not, only one folder is expanded): The PCR product lengths are shown in the folder names, but the folders contain a lot of information about the primers. For each PCR product the region of the sequence that will be amplified is shown. The thermodynamic information for that PCR product is also shown. For each primer, the sequence, similarity, thermodynamic information, and sequence data is shown. This can be used by the investigator (that would be you!) to determine which of the primer pairs selected by vectorNTI is most suitable for the desired use. Several different primer pairs, and resulting PCR products are shown, and each has an arbitrary score that considers all of the criteria used to design the primers. The PCR products are usually ordered with highest scoring products at the top of the list. Note: If you design new PCR products without saving the results of this analysis first, all results will be lost. To save the oligos you can either save them to the oligo database or to the temporary oligo list by right clicking on the oligo. To save the PCR product you can save it to the DNA database of your choice by clicking on the PCR product name |
To find any PCR product in the sequence and graphical views, right click on the folder containing the PCR product name, and choose Find PCR product from the pull down menu. The PCR product will be highlighted on both graphical and sequence panes.
From this same menu you can also save the PCR product in your DNA sequence database (always a good idea so that you can find it again quickly). When you save the product you will be prompted to fill in information about the product (eg name etc), however the source of the product (in this case the TNF-alpha molecule) will be autmatically remembered. You can also simultaneously save it to the database and open a new window to perform more analysis.
You can also do similar tricks with the oligo sequences. Right click (mac: CTRL-click) on the oligo name and choose save to database. This will save your oligo sequence to the Oligo database. You can also add it to a list of temporary oligos which you can view from Oligo List from the List menu.
You can also analyze the oligo from this menu.
Oligo Analysis
The analysis window is shown here:
Click Analyze to display the results of the analysis (as shown above). This will display several results from the analysis:
| Mol. Wt | Molecular weight of the oligo |
| %GC | The percentage of G and C in the oligo. |
| Therm. Tm | The melting temperature (temperature at which 50% of the oligo is a duplex) calculated by the nearest-neighbors method. This is useful for short oligos, < ~35 bp. |
| %GC Tm | The %GC Tm field shows the melting temperature calculated by the %GC method. This is useful for long oligos, > ~35-40 bp. |
| dG | The free energy values of the entire oligonucleotide. |
| 3' End dG | The free energy of the 3' end of the oligo. |
| dH | The enthalpy of the entire oligo. |
| dS | The entropy of the entire oligo. |
The dialog also has two panels on the left that will display palindromes and repeats within each primer.
Click on the Dimers & Hairpin Loops... box to view the dimers and hairpin loops in the primer. An example (from a different primer) is shown below:
This primer has 10 dimers and 5 hairpin loops, though only one is shown at a time. Click on the >> button to scroll through the list of either dimers or hairpin loops. When designing primers, you want to minimize the number of dimers/hairpin loops as these will bind to themselves, and result in amplification of only a short region of the primer and not amplification of your target gene.
Other Primer Analysis
VectorNTI allows you to easily design other primers. There are several selections in the Analyze menu, all of which work essentially as described above.
| Amplify Selection | Will design primers that flank the selection, allowing you to clone a complete region |
| Amplify Features | Will design primers (you can set how many per feature) for each of the features in your selection. To use this function, select a region of the sequence and then select Amplify Features. Click on the features tab, and select which features you want to amplify. For example, you can amplify all the CDS, or just those CDS regions that you choose from the list. Then click OK, and it will return several primers for each feature. Use the Find PCR product for each PCR product to identify which feature it amplifies. |
| Sequencing Primers | You can select a large region of sequence, and this will be broken into smaller fragments. The default is 300 bp, but you should choose a length shorter than your sequence run. Primers will be designed for each fragment along the region that you have selected. |
| Hybridization Probes | Will design short probes that anneal to the sequence for hybridizations (Southerns, Northerns, etc). |