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Primo Profile 3.2 Help: PCR Profile Thousands of Genes

About Primo Profile 3.2: Online

Primo Profile 3.2 is designed to select a few degenerate primers for the purpose of PCR amplifying most of the genes in a set of hundreds or thousands of genes. The goal is to allow users to profile gene expression of a selected set of genes with only a few PCR reactions. The technique is similar to Differential Display and may customaries Differential Display for genes that have low expression levels or lack polyA tails.

Giving the sequences of a set of hundreds or thousands of genes, Primo Profile first finds all potential primer sequences that satisfy the pre-set criteria. Next it analyzes the primer sequences to find common features and select a few degenerate primers that will match a significant fraction of the input genes. It takes advantage of the fact that certain short sequences are over-represented in the transcribed sequences. Note in Primo Pro, primers with these over-represented sequences are eliminated to reduce background PCR amplification.

Primo Profile has several advantages: (1) Compared to Differential Display which uses random 5' primers, the primers designed using Primo Profile have more uniform characteristics such as GC-content and melting temperature. Consistent primer characteristics should allow most genes to be amplified with similar efficiency. (2) The degeneracy is also lower compared to random primers of the same length. This allows longer primers to be used. Longer primer length should improve overall PCR efficiency. (3) Primo Profile can be used for design 5' as well as 3' primers. The 3' primers may allow us to profile mRNA independent of the polyA tails.

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.

Browser requirements:

Primo Pro 3.2 Online and Primo Profile 3.2 Online runs on the following Java-enabled browser:
  • PC: Internet Explorer 5.5 or higher and Netscape 4.08 or higher.
  • Mac (OS 9.*): Internet Explorer 5.0
  • Mac (OSX10): Early version of OSX10 IE5.1 has a bug. Copy and paste will kill the browser. The bug should have been fixed in later versions of Mac IE.

    If you use one of the above browsers 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, and 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:

    Input sequences by copy and paste. Sequences should be in the following format:
    >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
    
    

    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.

    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.

    For the stand-alone version, save sequences in the above format into a file, and load the file into the program. The online version is limited by the size of the clip board and is also slower. Stand-alone versions will be needed if you want to design degenerate primers for hundreds or thousands of genes.

    Select the appropriate options and click on "Go" to start.

    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.

    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 degrees (4-6) lower than the melting temperature is usually used to increase the probability of primer binding. For simplicity, only the simple rule of 2 degree for each A or T and 4 degree for each C or G is provided in Primo Profile.

       Melting temperature = 4 * Number of G or C + 2 * Number of A or T.

    3. Primer length, degeneracy, and random primer sequence length

    Users need to find the optimal combination of primer length and degeneracy for their experiments. In general, longer primer length is preferred for PCR reactions. Increase the primer length increases degeneracy or decreases the percentage of genes the primers will match. The random primer sequence length is another way of saying the degeneracy , i.e., the equivalent number of random nucleotides to generate the approximate degeneracy. These parameters are used as a starting point for the program. The actual degeneracy for the predicted primers may differ.

    Additional sequences (e.g., linkers with restriction sites) may be added at the 5' end of the predicted primers, which may increase PCR efficiency for the following up amplifications.

    4. # Primers

    The selected number of primers will be predicted. Combinations of multiple primers will increase the probability for detecting a particular gene.

    5. Anchor position

    Predicted primers will have lower degeneracy at the anchor position (5'-end or 3'-end) and increased degeneracy towards the other end.

    6. % match

    % match is an estimate of the probability that a gene in the input set will be amplified using the predicted primer. This is an over-estimate since it assumes primers with a few mismatch will work fine. In reality, primers with perfect match may not work because of other experimental factors.

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