You may use your browser's save function to save the web page in your local computer. Other saving options will be implemented later.

All the primers in PrimerBank were designed using a program called uPrimer. Great care has been given to avoid primer mispriming to other known genes in a genome. Here is a list of criteria for gene specific primer design:

1. The primer length range: 19 - 23 nt, with the optimal length at 21 nt.
2. The primer GC percentage range: 35% - 65%.
3. The delta G value for the five 3’ end-bases is at least -9 kcal/mol.
4. The primer Tm range: 60 - 63 °C, determined by the Nearest Neighbor Method.
5. The PCR product length range is 100 - 250 bp. If this requirement cannot be satisfied, alternative ranges will be used.
6. The default number of primer pairs designed for each sequence is 3.
7. No primer is designed from low-complexity regions.
8. A primer does not contain 6 or more contiguous same nucleotides.
9. A primer does not contain any ambiguous nucleotide.
10. No repetitive 15-mer from other gene sequences in the genome (for both strands) anywhere in a primer.
11. No repetitive 13-mer from non-coding RNA sequences (for both strands) anywhere in a primer.
12. The global BLAST score for any primer is less than 30 (equivalent to 15-mer perfect match).
13. The maximum Tm for the 3’ end perfect match to other gene sequences does not exceed 46 °C; does not exceed 42 °C when compared to non-coding RNA sequences (Tm determined by the Nearest Neighbor Method).
14. For primer secondary structure (the primer-primer self-annealing)
    1. No repetitive 5-mer is allowed anywhere when a primer sequence is compared to its complementary strand.
    2. The four 3’-end bases should be unique when compared to the primer’s complementary strand.
15. The forward and reverse primers should not anneal to each other. The filter setting is the same as in the primer secondary structure filter.
16. For sequence secondary structure (the primer-sequence self-annealing)
    1. No repetitive 9-mer is allowed when a primer sequence is compared to the complementary strand of its cognate sequence.
    2. The BLAST score is less than 18 when a primer sequence is compared to the complementary strand of its cognate sequence.

All the primers in PrimerBank have melting temperatures (Tm) of 60 - 63 °C. A higher annealing temperature results in more specific priming. Previous studies indicated sufficient priming should occur at primer Tm. Therefore, an annealing temperature of 60 °C is recommended for all PrimerBank primers. Non-specific PCR products are likely to occur at lower annealing temperature. We have tested a few hundred primers under 60 °C annealing temperature and the PCR experiments worked very well.

The primer Tm values are calculated using the Nearest Neighbor Method with the up-to-date thermodynamic parameters. Tm values are also dependent on primer and salt concentrations. Higher concentrations of primer and salt usually lead to higher Tm. The Tm values included in PrimerBank are for 0.25 uM primer, 1.5 mM Mg²⁺, 50 mM Na⁺, and 0.8 mM dNTP, which are typically used in PCR experiments. Other common PCR conditions only affect the Tm slightly.

上一页  [1] [2] [3] 下一页