| Prim-SNPing User Manual | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Prim-SNPing implements four primer design tools, namely general primer design, natural primer design, mutagenic primer design and PCR-CTPP primer design. Figure1 shows the head banner of this system, from which users can select the desired function. The selections are (1) Introduction, (2) General PD, (3) Natural PD, (4) Mutagenic PD, (5) CTPP PD, (6) User Manual and (7) Contact US. They will be described in detail below.
(1) Introduction Introduction describes the functions the system contains, and how to apply these functions to different areas. (2) General PD General PD is used for general primer design. In a first step, users select from two different input modes, Sequence Input or File Input, as shown in Figure2.
When selecting Sequence Input, a text input box will appear into which the sequence can be typed (Fig.3). File Input gives the user the option of entering the sequence from a file on their computer Fig.4).
Users can select one of the methods by which they receive the results: (1) Online (2) By email (3) Both online and by email (Figure 5). When selecting the email option an email address must be entered.
In order to design primers further, General PD provides advanced options by which the results of the primer design can be adjusted further by users. It is divided into two parts, Primer design constraint and Annealing check as shown in Figure6. Primer design constraints of General PD include primer length (default is 18 bps ~ 26 bps), primer length difference (default is 5 bps), Tm (melting temperature) (default is 50.0 ¢XC ~ 62.0 ¢XC), Tm difference (default is 5 ¢XC), Na+ (molar sodium concentration) (default is 0.5 M), GC proportion (default is 40% ~ 60%) and PCR length (default is 100 bps¡Ó 20 bps).
General PD online output: Near the top of the General PD output window a Sort by tm-diff and a Blast for specificity button can be found. Figure 7 shows the results for 10 primers without these options. After clicking on the Sort by tm-diff button the primers with the smallest tm-diff are listed at the top. When the Blast for specificity button is clicked on, the results contain a new column. Figure 8 shows the results after sorting by tm-diff and blasting. For the 10 primer sets the tm-diff is now 0 ¢XC.
When clicking on the "Blast" button, the system utilizes the NCBI netblast to blast the primer. A user can judge the specificity of the primer based on the blast results. (shown in Fig.9)
(3) Natural PD Natural PD is used for natural primer design. It mainly designs primer sets from SNP sequences. First, users can select the input mode (Sequence Input or File Input) shown in Figure 10 (same as Figure 2).
Selecting Sequence Input will open an rs# input box and a text input box for sequence input (Figure 11). The rs# input box allows for inputting rs# of three organisms, Human, Mouse and Rat. The user can select the desired SNP flanking length for primer design. Through the "put in" button, the system will find SNP flanking sequences of the rs# for the desired length, and put these into the text input box below. Selecting File Input will open a file box for file selection. (Figure 12)
Users can select one of the methods by which they receive the results: (1) Online (2) By email (3) Both online and by email. (Figure 13) When selecting the email option an email address must be entered.
In order to design primers further, Natural PD provides advanced options by which the results of the primer design can be adjusted further by users. It is divided into two parts, Primer design constraint and Annealing check, shown in Figure14. Primer design constraints of Natural PD include primer length (default is 18 bps ~ 26 bps), primer length difference (default is 5 bps), Tm (melting temperature) (default is 50.0 ¢XC ~ 62.0 ¢XC), Tm difference (default is 5 ¢XC), Na+ (molar sodium concentration) (default is 0.5 M), GC proportion (default is 40% ~ 60%), PCR length (default is 100 bps¡Ó 20 bps) and Ratio for RE-digested RFLP length (default is 3 : 1). The Ratio for RE-digested RFLP length is shown in Figure 15.
Natural PD online output is as follows: Near the top of the General PD output window a Sort by tm-diff and a Blast for specificity button can be found. Figure 16 shows the results for 10 primers without these options. After clicking on the Sort by tm-diff button the primers with the smallest tm-diff are listed at the top. When the Blast for specificity button is clicked on, the results contain a new column. Figure 17 shows the results after sorting by tm-diff and blasting. The tm-diff became smaller after blasting.
The function of the "Blast" button is the same as in General PD.
(4) Mutagenic PD Mutagenic PD is used for the mutagenic primer design. It also designs primer sets from SNP sequences. First, users select the input mode (Sequence Input or File Input) shown in Figure18 (same as Figure 2 and Figure 10).
Selecting Sequence Input will open an rs# input box and text input box for sequence input (Figure 19). The rs# input box allows for inputting rs# of three organisms, Human, Mouse and Rat. The user can select the desired SNP flanking length for primer design. Through the "put in" button, the system will find SNP flanking sequences of the rs# for the desired length, and put these into the text input box below. Selecting File Input will open a file box for file selection. (Figure 20)
Users can select one of the methods by which they receive the results: (1) Online (2) By email (3) Both online and by email. (Figure 21) When selecting the email option an email address must be entered.
In order to design primers further, Mutagenic PD provides advanced options by which the results of the primer design can be adjusted further by users. It is divided into two parts, Primer design constraint and Annealing check, shown in Figure14. Primer design constraints of Mutagenic PD include primer length (default is 18 bps ~ 26 bps), primer length difference (default is 5 bps), Tm (melting temperature) (default is 50.0 ¢XC ~ 62.0 ¢XC), Tm difference (default is 5 ¢XC), Na+ (molar sodium concentration) (default is 0.5 M), GC proportion (default is 40% ~ 60%), PCR length (default is 100 bps¡Ó 20 bps) and the range for mutagenic bases from SNP (default is 5 bps).
Mutagenic PD online output is as follows: Near the top of the General PD output window a Sort by tm-diff and a Blast for specificity button can be found. Figure 23 shows the results for 10 primers without these options. After clicking on the Sort by tm-diff button the primers with the smallest tm-diff are listed at the top. When the Blast for specificity button is clicked on, the results contain a new column. Figure 24 shows the results after sorting by tm-diff and blasting. We observe that the tm-diff of all primer sets is 0 ¢XC except for the tenth when 10 records are shown.
The function of the "Blast" button is the same as in General PD.
(5) CTPP PD CTPP PD is used for PCR-CTPP primer design. It also designs primer sets from SNP sequences. First, users select the input mode (Sequence Input or File Input) shown in Figure25 (same as Figure2, Figure10 and Figure18).
Selecting Sequence Input will open an rs# input box and text input box for sequence input (Figure 26). The rs# input box allows for inputting rs# of three organisms, Human, Mouse and Rat. The user can select the desired SNP flanking length for primer design. Through the "put in" button, the system will find SNP flanking sequences of the rs# for the desired length, and put these into the text input box below. Selecting File Input will open a file box for file selection. (Figure 27)
Users can select one of the methods by which they receive the results: (1) Online (2) By email (3) Both online and by email. (Figure 21) When selecting the email option an email address must be entered.
In order to design primers further, CTPP PD provides advanced options by which the results of the primer design can be adjusted further by users. It is divided into two parts, Primer design constraint and Annealing check, showed in Figure14. Primer design constraints of CTPP PD include primer length (default is 18 bps ~ 26 bps), primer length difference (default is 5 bps), Tm (melting temperature) (default is 50.0 ¢XC ~ 62.0 ¢XC), Tm difference (default is 5 ¢XC), Na+ (molar sodium concentration) (default is 0.5 M), GC proportion (default is 40% ~ 60%), PCR length (default is 100 bps¡Ó 20 bps).
CTPP PD online output is as follows: Near the top of the General PD output window a Sort by tm-diff and a Blast for specificity button can be found. Figure 30 shows the results for 10 primers without these options. After clicking on the Sort by tm-diff button the primers with the smallest tm-diff are listed at the top. When the Blast for specificity button is clicked on, the results contain a new column. Figure 31 shows the results after sorting by tm-diff and blasting. The tm-diff became smaller after blasting.
The function of the "Blast" button is the same as General PD. (6) User Manual The User Manual introduces how to use this system. (7) Contact Us Should you have any further questions, plesase contact us.
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