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About Primo Diverse 3.2: Online
Primo Diverse 3.2 is designed to PCR amplify the most diverse regions of a gene family using one pair of degenerate primers. The primers will be chosen from the most conserved regions (i.e., least degenerate), yet the products are highly degenerate, allowing users to identify the products by hybridization, sequencing, or other methods.
Figure 1. Differences between Primo Degenerate and Primo Diverse. Primers are selected from conserved regions. For Diverse, the PCR products are divergent, i.e., highly degenerate.
About Primo Degenerate 3.2: Online
Degenerate PCR is frequently used to clone unknown genes in a family. Primo Degenerate 3.2 designs PCR primers based on a single peptide sequence or multiple alignments of proteins or nucleotides. For degenerate primers, the probability of binding to the target is proportional to the effective concentration of the specific primer (i.e., inversely to the fold of degeneracy). Most programs and models (e.g., 2 °C for each A and T, 4 °C for each C and G) did not take this into account thus will predict a much too high melting temperature. By using the Nearest Neighbor Tm model, Primo automatically adjusts Tm for degenerate primers, resulting in better primer design.
How to calculate melting temperature for degenerate primers
For normal PCR primer design, please use
Primo Pro 3.2. Primo Pro 3.2 introduces an option for reducing PCR noise by lowering the probability of random
primering on non-target DNA sequences. This option is kept in
Primo Multiplex. For example the primer TGCACTACCTGCTGCTGCAC for
the p53 gene looks perfectly OK because it meets most criteria we commonly use for designing
PCR primers. This primer may have high background amplification because the 3’-prime 8 nucleotides
appears in 4800 unique genes in human transcriptome. Primo Pro analyzed in advance
transcriptomes of different species to mark sequences that are over-represented, thus allow users to
select primers with few over-represented sequences at the 3’-end. As a result of reduced random
priming, we expect improved PCR amplification efficiency and cleaner PCR products, especially for
RT-PCR reactions.
Primo Pro 3.2 also introduces a batch mode option for high throughput
PCR primer design. By selecting the batch mode, users can input
multiple sequences and design PCR primers for multiple sequences. Batch
mode is adapted for multiplex primer design in Primo Multiplex.
A lot of calculations have been done in advance for Primo Pro.
That is why Primo Pro runs amazingly fast in your web browser.
But to take full advantage of Primo Pro, you would need to install it on your computer.
For example, the online Primo does not allow you to chose a species other than human
because limitations in passing large number of data on the web. Note it searched
only transcribed sequences and each species has a unique pattern of
over-represented sequences. Unlike proteomes of closely related species,
transcriptomes of close relatives usually don't share over-represented
sequences, probably because untranscribed regions are less conserved
and/or many species have young transposons.
The stand-alone version of Primo Pro 3.2 and Primo Multiplex 3.2 currently has
transcriptome data for 11 species. More species will be added in the near future.
Copy the sequences in the example into the input window.
On Windows you may need to use Ctrl-C (copy) and Ctrl-V (paste).
On Macs you may need to use Apple-C (copy) and Apple-V (paste).
Numbers and white spaces will be ignored.
Select the "Diverse PCR" or "Degenerate PCR" checkbox, and appropriate options for the input sequence (Single protein, Multiple proteins, or
multiple nucleotides) and click on "Go" to start.
The Nearest Neighbor Tm formula is suggested for designing degenerate
primers since it will automatically correct for the effects of
primer degeneracy. Users may select the AT=2 CG=4 formula to disable
this feature. As explained in how to calculate
melting temperature for degenerate primers, degeneracy results
in lowered melting temperature.
2. Melting temperature and annealing temperature
Melting temperature is the temperature at which 50% of the oligo and its
perfect complement are in duplex. PCR annealing temperature
a few degree (4-6) lower than the melting temperature is usually used to increase
the probability of primer binding. There are two options for calculating the
melting temperature. The first uses the simple rule of 2 degree for each A or T
and 4 degree for each C or G.
Melting temperature = 4 * Number of G or C + 2 * Number of A or T.
The second "Nearest N" predicts melting temperature using the "Nearest Neighbor" model (Jhon SantaLucia, Proc. Natl. Acad. Sci. Vol. 95, p1460-1465 (1998)).
The cation concentration is assumed to be 50 mM and the primer concentration is assumed
to be 200 nanomolar. The "Nearest N" is presented because it is more
accurate and other formulae can be viewed as approximations of the
"Nearest N".
The "Nearest N" formula has a correction for primer concentration.
If lower or higher primer concentration is used, the rule-of-thumb is
that for each two fold increase or decrease in the primer concentration, the melting temperature should increase or decrease by 1 degree. (Click here to see the relationship between primer concentration and melting temperature.)
For degenerate primers, the effective concentration is lower because of
degeneracy. Users don't need to adjust for the lowered effective concentration if using the "Nearest N" formula, Primo has already taken that into account. Users are suggested to select the "Nearest N"
formula if template sequences consist of degenerate codes.
To compare melting temperatures calculated using the two formulae, type or
copy the primer sequence into the "2nd primer" field, and mouse-click on the
text field. The melting temperature will be calculated using the selected
formula. Click here to see the melting temperatures calculated using both formulae
for some commonly used PCR and sequencing primers.
For degenerate nucleotides, an average is used.
3. Primer-primer dimer
4. Random background priming
5. Diversity score
Each sequence starts with an info line with a > sign. The description
of the sequence may be followed by the optional 5' and 3' range for
forward or reverse primers. The three fields are separated by "tab."
If the range value starts with a minus sign, then the counting will be from
the 3'-end of the sequence. If both ranges are missing in the info line,
the input value from the Primo interface will be used. If only one range is present in
the info line, that value will be used for designing forward or reverse primer only. It will be ignored for selecting pairs of forward and reverse primers.
Contact us if a species you are interested in is not on this list, we might be able to help.
Browser requirements:
Primo Pro 3.2 Online and Primo Multiplex 3.2 Online runs on the following Java-enabled browser:
If you use one of the above browser and you can't run Primo Pro 3.2, please
make sure Java is enabled in your browser. For Internet Explorer, go to
Tools/Internet Options, click on Security Settings, scroll down to
find Microsoft VM, deselect "Disable Java".
Mac OSX 10 IE5.1 has a bug, it does not allow copy/paste into a Java
text field, thus you will not be able to import a new sequence.
Attempting copy/paste may kill your browser. Stand-alone versions
don't require IE to run, thus don't have this problem.
How to use:
Behind the scene:
1. Code for Degenerate Oligos
A A C C
G G T T
U T M (AC)
R (AG) W (AT)
S (CG) Y (CT)
K (GT) V (ACG)
H (ACT) D (AGT)
B (CGT) N (ACGT)
Any other letters will be ignored, so you can paste in a nucleotide
sequences with spaces and numbers.
If you select to "Check primer-primer dimers", Primo Pro 3.2 eliminates primers
that might form primer-primer dimers such as the following:
P1: 5'-...GGCGATCG-3'
||||||
3'-GCTAGCGG...-5' : P1 or
P1: 5'....AGGGCCC-3'
|| |
3'-CGCGAAT...-5' : P2
The following single-base pair is also not allowed:
P1: 5'-...AGGTCGCG-3'
|
3'-CGGATT...5' : P2
Primo Pro has blacklisted a large number of over-represented sequences of 8 nucleotides by
analyzing transcriptome data from a number of species. Note there will be
a unique list for each species. A primer candidate will be compared to the
blacklist for matches in the last 10 nucleotides. Any one with a match will be
eliminated from the candidate pool. The following three primers will be
eliminated:
Black list oligo: 5'-TGCTGCAC-3'
Primer 1: 5'-TGCACTACCTGCTGCTGCAC-3'
Primer 2: 5'-GCACTACCTGCTGCTGCACC-3'
Primer 2: 5'-GCACTACCTGCTGCTGCACCG-3'
A primer with missmatch at the last two nucleotides is eliminated because of
high probability there will be sequences that match the primer and exonuclease
activities of DNA polymerases.
Diversity score (0-4) is approximately the average number of degeneracy
for each position of the multiple alignments. Higher the score, higher the degeneracy, i.e., diversity of the amplified sequences.
Multiplex PCR
To use the multiplex PCR, select the "Multiplex PCR" checkbox and input
multiple sequences in the following format (the same format is used
in Primo Pro 3.2 for batch submission):
>seq1 0-100 -100-0
1 ggccgggcgc ggtggctcac gcctgtaatc ccagcacttt gggaggccga ggcgggtgga
61 tcacctgagg tcaggagttc gagaccagcc tggccaacat ggtgaaaccc cgtctctact
121 aaaaatacaa aaattagccg ggcgtggtgg cgggcgcctg taatcccagc tactcgggag
181 gctgaggcag gagaatcgct tgaacccggg aggcggaggt tgcagtgagc cgagatcgcg
241 ccactgcact ccagcctggg caacaagagc gaaactccgt ctcaaaaaaa a
>seq2
1 accgcagcgg acagcgccaa gtgaagcctc gcttccctcc cgcggcgacc agggcccgag
61 ccgagagtag cagttgtagc tacccgccca gaaactagac acaatgtgcg acgaagacga
121 gaccaccgcc ctcgtgtgcg acaatggctc cggcctggtg aaagccggct tcgccgggga
181 tgacgcccct agggccgtgt tcccgtccat cgtgggccgc ccccgacacc agggcgtcat
241 ggtcggtatg ggtcagaaag attcctacgt gggcgacgag gctcagagca agagaggtat
Species available in the stand-alone version
Species Name Common Name Arabidopsis thaliana Arabidopsis Zea mays Corn Bos taurus Cow Drosophila melanogaster Drosophila Xenopus laevis Frog Homo sapiens Human Mus musculus Mouse Rattus norvegicus Rat Oryza sativa Rice Danio rerio Zebrafish