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Primo Melt 3.4: PCR Primer Design For DGGE And TGGE          Order
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How to use Primo Melt 3.4
Background

Denaturing Gradient Gel Electrophoresis (DGGE) and Temperature Gradient Gel Electrophoresis (TGGE) are frequently used to identify single base mutations in DNA sequences. As DNA enters a gradient gel, the double-stranded DNA molecules become partially melted, and their gel-electrophoretic mobility decreases. However, if the double-stranded DNA molecules become completely melted into single strands, their mobility increases. As a result only mutations in the lowest melting domain can be identified by DGGE or TGGE. To prevent the complete melting of double-stranded DNA, a stretch of GC-rich sequences (GC-clamp) is frequently added to one-end of the DNA sequences.

Primo Melt allows users to identify the lowest melting domain and to design PCR primers for amplifying the domains. Users have a choice of adding a GC-clamp. The batch model can be used for multiple sequences.

Usage

1. Copy and paste (Ctrl-c Ctrl-v or Apple-c Apple-v) your DNA sequences into the input window. Numbers and blanks will be ignored. Degenerate codes are OK.
2. Click the "Melt Map" radio button and then click on Go. A melt map with melting domains illustrated in red bars will appear in the top-right. Click on the map will show the position in the sequence and melting temperature. Parameters for the lowest melting domain will be shown in the results window.
3. Exam the melting map, select one or more melting domains to PCR. Change the 5' and 3' ranges so the primers will cover the melting domains.
4. Select desired parameters (e.g., GC-clamp, primer Tm, etc.), and click on Go. If no GC-clamp is added, the PCR product will contain more than one melting domain, and the melting temperature difference between two of the domains will be greater than 3 degrees.

Batch mode

To use the batch mode, select the batch mode checkbox and input multiple sequences 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.

Behind the scene

1. Melting temperature calculations

Melting curves are based on the model proposed by Poland (Biopolymers, Vol.13, 1859-1871 (1974)) and modified by Fixman and Freire (Biopolymers, Vol.16, 2693-2704 (1977)). Stacking parameters are from SantaLucia (Proc. Natl. Acad. Sci. Vol. 95, p1460-1465 (1998)). The SantaLucia model has a salt concentration of 1 M. A correction ( -16.2*log(0.04) ~ 23 degrees) due to lower salt concentration in the gel running buffer (~40 mM for 1xTAE) is subtracted after the melting curve has been computed.

More information about helix melting can be found at the Poland site.

2. PCR primer design

If no GC-clamp is added, the PCR product must have at least two melting domains and the melting temperature difference between the highest and lowest melting domains must be greater than 3 degrees. If GC-clamp is selected, it will be added to the end with higher melting temperature.

For more details about PCR primer design, please visit user instructions for Primo Pro.



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