DNAMAN provides functions to characterize oligonucleotides, design PCR/Sequencing primers and analyze possible mispriming sites of a primer on the target sequence.
Primer analysis starts with loading an oligonucleotide sequence into memory. You may use the oligo sequence in memory for many functional analyses.
You may enter sequence directly from keyboard or retrieve an oligo sequence from database. For keyboard input, choose the Primer | Load Primer | From Input command to open the Input Primer dialog box, then type a nucleotide sequence. For retrieve from database, choose the Primer | Load Primer | From Oligo Database command to open the Oligo Database box. Select one of the available databases and double click the record you want to load it into memory.
By choosing the Primer | Self-complementarity command, you can searches for the most possible self-complementarity conformation (Hairpin structure) of the oligonucleotide.
A dialog box appears for the selection parameters of hairpin structure. The first parameter is the minimum Paired Bases (PB) and the second is the maximum deltaG (DG). If PB is the factor to determine the structure, you may set maximum DG to 80 kcal/mol. On the other hand, set minimum PB to 2 if DG is the value to filtrate the hairpin conformation.
The structure stability is filtrated according the free energy. The free energy is calculated using the nearest-neighbor method. A qualified hairpin structure will be displayed in a graphic window. If many structures are found according to the filtration parameters, DNAMAN displays the one with lowest free energy.
The hairpin structure is draw in a graphic window. If the unpaired bases (loop) between the complementary sequence are more than 4 bases, the unpaired bases will be indicated with ":::".
You may modify the graphic content as described in Chapter V.
Choosing the Primer | Melting Temperature command opens the Melting Temperature dialog box. The oligo sequence in memory is displayed in the Oligo sequence box. DNAMAN calculates the length, GC% and the molecular weight of the primer according to the sequence and displays the results in the dialog box.
The melting temperature of an oligonucleotide is calculated with three methods:
1)Thermodynamic Tm:
The Tm is calculated using the nearest-neighbor thermodynamic values method (Breslauer et al). This method is less accurate for longer oligos. The formula for the Tm is
Tm=dH/(dS-Rln(Cd))-273.15+16.6*(log10(Cs))
Where dH is the enthalpy, dS is the entropy, R is 1.987 cal K-1 mol-1, Cd is the DNA concentration, and Cs is the salt concentration.
2)Hybridisation Tm:
This method is generally used for DNA or RNA hybridization, especially in presence of high salt and formamide. It is more accurate with longer oligos. The Tm is calculated using the following formula:
for DNA:DNA hybridization:
Tm=81.5+16.6*(log10[Na+])+0.41*[%(G+C)]-0.63*(%Formamide)-500/L-1.5(%Mismatch)
for DNA:RNA hybridization:
Tm=79.8+18.5*(log10[Na+])+0.58*[%(G+C)]+11.8*[%(G+C)]2-0.5*(%Formamide)-820/L-1.5(%Mismatch);
for RNA:RNA hybridization:
Tm=79.8+18.5*(log10[Na+])+0.58*[%(G+C)]+11.8*[%(G+C)]2-0.35*(%Formamide)-820/L-1.5(%Mismatch);
* where L is the length (bases) of the oligonucleotide.
3)GC+AT Tm: the estimated Tm is the sum of the contribution of each base: 2°C for A and T and 4°C for G and C.
There are several parameters involved in Tm calculation.
DNA concentration is used only in thermodynamic Tm. You may increase oligo DNA concentration in order to increase Tm.
Salt concentration affects thermodynamic Tm and hybridization Tm. In the [Na+][mM] box, you may type a suitable salt concentration of your experiments. The value must be an integer greater than 0.
Hybridization type is required for Hybridization Tm. In the DNA/RNA box, enter the hybridization type:
D:D for DNA and DNA hybridization
D:R for DNA and RNA hybridization
R:R for RNA and RNA hybridization.
Formamide concentration affects only the hybridization Tm. You may enter an integer in the "Formamide (%)" box.
Mismatch number also affects only the hybridization Tm. You may type in the "Mismatch" box a mismatch number of base pairs between the oligo and DNA.
With all parameters entered, click the Show Tm button to display the melting temperatures. You may change the sequence in the Oligo sequence box and then click the Show Tm button to recalculate the length, GC%, MW and Tm of the oligo.
Click the Report button to display the analysis results in a text window. You may save it as a text document.
Example:
Oligo:
5'-GACTGCCACTTCCTCGATGAAGG-3'
SEQ Primer 1: 23 bp;
Composition: 5 A; 7 C; 6 G; 5 T; 0 OTHER
Percentage: 21% A; 30% C; 26% G; 21% T; 0% OTHER
Molecular Weight (kDa): ssDNA: 7.06
Hybridization type: DNA:DNA
Salt concentration: 50 mM
Formamide: 0 percent
Mismatch: 0 bp
Thermo Tm = 60.6 °C %GC Tm = 57.0 °C GC+AT Tm = 72.0 °C
DNAMAN searches for the complementary sequences between the oligonucleotide and both strands of the current sequence. The two longest complementary sequences will be shown in a text window, if the paired bases in continuous are equal to or greater than 10 bp.
Primer-EXAMPLE1(1-886) complementarity.
First complementarity in continuous: 21 bp
5'-TGGGGACCAACGTGTGATGGC-3' Primer
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3'-ACCCCTGGTTGCACACTACCG-5' (376) Strand -
No second possible complementarity
You may examine the complementarity of two oligo sequences with DNAMAN. To perform this function, the second primer should be loaded into the DNAMAN memory by choosing the Primer | Two Primer Complementarity command.
DNAMAN searches for complementary sequences between two primers. The results indicate the continuous and discontinuous complementary sequences between the two primers.
Two primers complementarity.
Max complementarity in continuous: 4 bp, free energy=-3.40 Kcal/mol
5'-TGGGGACCAACGTGTGATGGC-3'
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3'-CTTAATTCAATTACCAGGGGGT-5'
Max complementarity in discontinuous: 7 bp
5'-TGGGGACCAACGTGTGATGGC-3'
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3'-CTTAATTCAATTACCAGGGGGT-5'
You may use DNAMAN to select PCR primers that satisfy your requirements to amplify template DNA. Many parameters are designed to filtrate PCR primers, such as the range of PCR product size, primer lengths, Tm, GC compositions and salt concentration. Other parameters are used for rejection of "low quality" primers.
Choose the Primer | Design PCR Primers command to open the Design PCR Primer dialog box. There are three steps to setup parameters to design primers.
Step 1: Primer filtration
Primers are selected according to the physical properties.
- Range of PCR product size. The default range is from 400 to 600 bp.
- Sequence region for the sense primer
- Sequence region for the antisense primer
- Range of primer length. The default range is from 18 to 21bases.
- Range of primer Tm. The default range is from 62 to 65 °C.
- Range of GC content. The default range is from 40% to 60 %.
- DNA primer concentration. This parameter is used to calculate Tm. The default value is 50 nM.
- Salt concentration. This parameter is used to calculate Tm. The default value is 50 mM.
- Check the Shortest primers only option if too many primers are found after selection.
Parameters to disqualify primers:
- 3’ end dimer. A single primer may form dimers if the nucleotides at the 3' end are self-complementary. A high quality primer should have least length of the 3' end dimer structure. The default length is 3 bases.
- PolyN. PolyN may hybridize to non-specific regions of genomic DNA. High quality primer should have minimum consecutive identical nucleotides. The default number is 3 base.
- Hairpin structure. Hairpin structures reduce the potential hybridization of primer to target DNA. This parameter defines the maximum nucleotides to form a hairpin structure. The default number is 3 base pairs.
- 3’ unique bases. The longer the 3' unique bases are, the lower the mispriming possibility will be. This parameter defines the minimum length of nucleotides at the 3' end complementary with DNA. The default length is 6 bases.
If the PCR product will be used as hybridization probe of Southern blot, check the Product for hybridization option. There are four parameters to determine the thermodynamic properties of the PCR product.
Click the Next button to start the analysis. DNAMAN will select primers according to the parameters.
Step 2: Refinement and pair selection.
In the second page, all primers meeting the above criteria are displayed in two list boxes: Sense primers and Antisense primers. If the primers are used for DNA sequencing, it is not necessary to perform the next steps. You may press the Export List button to display the primer list in a text window.
To improve the quality of PCR primers, you may disqualify some primer pairs with the following criteria:
- Primer-Primer interaction. High number of complementary bases may result in hybridization of the two primers with them-self. The primer-primer complementary bases should be as few as possible. The default number is 7.
- Primer Tm difference. High difference makes the efficiency of hybridization difficult to control. Amplification is better performed when the difference is lower. The default difference is 2 degrees.
- Mispriming analysis on target sequence. Mispriming may result in amplification of unexpected PCR products. Check this option will increase the primer quality. You can set up a cut-off score for the analysis. Primers with high score of mispriming will be disqualified. See the next section for information on mispriming analysis
- Restriction analysis. Restriction analysis may eliminate some undesirable primers. You have options to keep primers with restriction sites or keep primers without any restriction site.
Click the Next button to enter the final step.
Step 3: Final.
All qualified primers are listed in one box. You can set the order of the list according to primer positions, product sizes, sum of paired Tms, Tm differences or mispriming scores. Clicking any pair of primers shows its characteristics. Press the Finish button to report the list of primers in a text window.
Mispriming analysis is designed to find all possible annealing sites of a primer on the default DNA sequence. This function is useful in primer selection for PCR and DNA sequencing. A weight matrix is used to differentiate the importance of primer positions. Since the matches of a primer to target DNA at 3’ end is more important than 5’ end for PCR amplification, more weight is given to 3’ end. In order to increase the specificity of PCR primers, you should always check if there are secondary annealing sites between selected primers and target DNA.
Choose the Primer | Load Primer command to load an oligo sequence from Input or from a database. Choose the Primer | Mispriming Analysis command to set parameters for mispriming analysis. The following parameters are used to find out mispring sites:
- Score Matrix section. 1) Score of Perfect Match. The default is 2. 2) Score of Mismatch. The default is -2. 3) Score of G-T pair. The default is 1.
- Position Weight Matrix section. 1) Length from 3’ end. The length of position weight matrix beginning from 3' end. The default is 9. 2) Weight from 1 to. Set a maximum weight for the 3’ end, the default is 10. In the default case, the first base at 3’ end weighs 10. The second base weighs 9… The ninth base weighs 2. All other base weighs 1.
- Gap penalty. You may set gap open and extension penalties. The default value of both penalties is -2.
- Scan for both strands of oligo. Check this option if you want to perform mispriming analysis for both strands.
- Cut-off score can be determined automatically if you check this option. You may also set your own score as well. The default cut-off score is 90.
Press the OK button to perform the analysis.
The results are shown in a text window. A list of possible binding sites of the primer is displayed in the order of score. High score sites are shown first and low score last. No site or only the perfect match site will be reported, if no secondary binding site is found. It is highly recommended not to use primers with secondary binding sites for PCR and sequencing works.