There are three methods for introducing templates into the fluorescent quantitative PCR gene amplifier:
1. Enter directly with the keyboard:
a. Click the New Sequence floating command in the file menu, or directly click the New Sequence command in the toolbar to enter the sequence display window;
b. At this time, you can enter the DNA sequence;
c. If necessary, Oligo provides a base playback function, which reads out bases while typing to prevent input errors. Click "Readback on" in the Edit menu.
2. Use copy and paste: when our sequence already exists as a TXT file or other file formats that oligo cannot directly open, such as word files. html format, this feature is very useful. After copying the sequence in the corresponding file and pasting it in the sequence display window, oligo will automatically remove non-base characters. After the sequence input or paste is completed, click the Accept floating command in the Accept / Discard menu to enter the primer design mode.
3. If the sequence has been saved in Seq format or FASTA, GenBank format, oligo can directly open the sequence file.
Click the "Open" floating command in the File menu to find the desired file and open it.
After entering the primer design mode, oligo generally pops up three windows, namely the 6-base frequency window, the base annealing temperature window and the base stability window within the sequence. The annealing temperature window is the main window of our primer design. The other two windows play an auxiliary role in the design process. For example, the 6-base frequency window allows us to intuitively see the frequency of the designed primers in the genome of the corresponding species. If our template is genomic DNA or mixed In the case of DNA, this information becomes useful, and the internal stability window can show whether the stability of the 5 'end of the primer is slightly higher than the 3' end, etc.
1. Search for common primer pairs:
Take Mouse 4E (cDNA sequence) as an example. Our goal is to use Mouse 4E (2361 bp) as a template to design a pair of primers to amplify 600-800 bp long PCR products.
1. Click the "For Primers and Probes" command in the "Search" menu to enter the primer search dialog;
2. Since we are designing a pair of PCR primers, both the positive and negative strand check boxes must be selected, and Compatible pairs.
In the default state of Oligo, the requirements for this primer pair are: a, no dimer; b, the 3 'end is highly specific, the GC content is limited, d, and the primers caused by the removal of errors are removed.
3. The remaining work is to determine the position of the upstream and downstream primers, the length of the PCR product and the primer design parameters.
â‘ Click: "search Ranges" button, a "Search Ranges" dialog box pops up. Enter the range of the upstream primer: 1-2000, the position of the downstream primer: 100-2300; the length of the PCR product is 600-800bp.
â‘¡ Click the "Paramaters" button to enter the "Search Parameters" dialog box. The dialog box is divided into three pages, namely: different settings, parameters and more parameters.
â‘¢ In the "General Settings" window, it provides us with a very intuitive method of setting primers, with six levels from high to low, and finally there is a user customization option.
â‘£When we are not particularly clear about the meaning of the various parameters of the primers and how much they should be set, we can directly set Very high / High to complete the setting of the primer design parameters.
⑤When we select "Automatically Change String", Oligo will search during the primer search: if the primer pair cannot be found in the high level setting, it will automatically lower the rating to search, and know that the primer pair is found. When designing the reverse PCR primer pair, select the "Inverse PCR" check box.
â‘¥We can also change the length of the primer to suit the set Tm value or PE? (Prime Effitions, priming efficiency). The maximum number of primer pairs selected can also be limited.
⑦In the "Parameters" window, we only need to change the length of the primer, and make corresponding changes according to the test requirements, such as 23nt. Other parameters use Oligo's default values, generally do not need to be changed. In the "More Parameters" window, there is generally no need to make any changes, just use Oligo's default values.
4. Click "Confirm"-"Ok" to enter the search window. After clicking "OK" again, Oligo automatically completes the search for the primer pair and a search result window appears. The number of primer pairs obtained is shown.
5. Click "Ok", the "PrimerPairs" window appears, which lists the simple information of 7 pairs of primers, primer position, product length, optimal annealing temperature and GC content. Click the "Sort" button, you can arrange in order of product length, optimal annealing temperature and GC content from small to large or from large to small.
6. Click any pair of primers, and a window called "PCR" will pop up next to it. In the window, we can see the approximate position of the primers in the template, and the optimal annealing temperature (this temperature can be directly applied to our actual The second annealing step in PCR). In addition, the Tm value of the primer, the GC content, and the initiation efficiency. At the same time, the difference between the Tm value of the product and the Tm value of the primer and the difference of the Tm value between the primers are calculated. Generally we try to keep the former within 20 degrees and the latter within 5-6 degrees. When you click on different primer pairs, the content of the PCR window changes accordingly.
7. If you want to know the detailed information of the primers, such as the formation of dimer, hairpin structure, composition, Tm value and error triggering site, etc., just click the corresponding command in the "Analyze" menu to get the relevant information. information. For example, click "Duplex Formation", we can get the formation of dimer between upstream primers, downstream primers and upstream and downstream primers. Similarly, "Hairpin Formation", "Composition and Tm", "False Priming Sites" and other commands can display relevant information. The results of all these analyses help us to select the best primer pair from the multiple pairs of primers obtained by Oligo search.
It should be noted that â‘ If there are too many primer pairs in one search, we can appropriately increase the selected conditions and specify a more suitable search range to reduce the number of primer pairs; â‘¡Primers should generally not be at the 3 'end or away from 3 'The end is too close to form a dimer. At the same time, the absolute value of the dimer's own energy should be as small as 10; In the sequencing reaction, the efficiency of error initiation should be strictly controlled below 120 points.
8. The detailed information of each pair of primers in Oligo can be directly exported as a text file:
First click "Print / Save Options" in the "File" menu, select "Selected" in the general settings of the dialog box that appears; select the information to be saved in "Analyze I", and select PCR in "Analyze II" .
Click the OK button to exit, then click the "File" menu again, "Data Save as" in the Save floating command, select the path and file name.
2. Design of sequencing primers In oligo, the design process of test primers is mostly similar to that of ordinary primers.
Taking Mouse 4E as an example, suppose we want to design a sequencing primer at the position of 600-800bp.
Open-Search
In the "Search" window, select "Sequence Rrimer", and remove the negative chain Search. In the "Search Range" window, enter 600-800bp for the positive chain
Select "very high" in "Parameters" and change the primer length to 18bp
The result is 11 sequencing primers, which is the same as ordinary primers. According to its detailed information, you can choose the best sequencing primer.
3. Design of the probe:
The design of the probe is basically the same as the design of the sequencing primer. Just select the probe design in the "Search" window and change the length of the probe.
4. Evaluation of primer pairs:
The primers we found in various literatures are often difficult to repeat the experimental results of others if PCR is performed directly. Then I thought, is there a problem with the quality of the primer? Can you use software to do some analysis on these problem primers? The answer is yes.
1. Click the New command in the File menu;
2. In the "Edit New Sequence" window, use the keyboard to enter the upstream primer;
3. If the first position of the primer is not 1, you can enter a new 5 'position number in the "Edit" window, such as 20;
4. Click the Accept command in the Accept / Discard menu;
5. If the length of the primer sequence is different from the current primer, you can change the current primer length from the "Change" menu;
6. Select the current sequence as the upstream primer (click the "upper" button);
7. Select the "Lower Primer" command from the Edit menu and enter the sequence of the downstream primer in the Edit Lower window;
8. At the upper corner of the Edit window, enter the corresponding 5 'position;
9. Select the "Accept and Quit" command;
If you want the program to give the best annealing temperature, enter the length of the PCR product and the percentage of GC content in the dialog box at this time. The GC in mammalian cDNA sequences is about 44%.
10. Click OK to complete various analyses in the "Analyze" menu.
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