Product size: 80 - 120 bp**
Product Tm: Min. 68; Opt. 71; Max. 75†
Primer size: Min. 24; Opt. 27; Max. 33‡
Primer Tm: Min. 60; Opt. 68; Max. 74§
Primer GC%: Min. 35; Opt. 44; Max. 60
(**) tHDA works most efficiently with a product size around 100 bp. Successful tHDA amplifications were achieved with a product as short as 85 bp and as long as 129 bp.
(†) The Tm of an amplicon with a product size around 100 bp and a G + C content around 40% is approximately 71°C from the calculation of Primer3. Successful tHDA amplifications were achieved with a product Tm as low as 68 °C and as high as 77°C.
(‡) The optimal primer size may be set at 26 bases when the G + C content of target sequence is larger than 45%. Successful tHDA amplifications were achieved with a primer size as short as 22 bases and as long as 32 bases.
(§) The optimal primer Tm may be set at 64 - 66°C when the G + C content of target sequence is smaller than 37.5% and at 70 - 72°C when the G + C content of target sequence is larger than 45%. Successful tHDA amplifications were achieved with a primer Tm as low as 60 °C and as high as 75 °C.
1) Amplicons (regions to be amplified) containing a G + C content of approximately 40% are preferable (the G + C content of amplicons and target sequences can be calculated using the Oligonucleotide Properties Calculator program found at: (http://www.basic.northwestern.edu/biotools/oligocalc.html).
2) Primer sets obtained using Primer3 with an average G + C content closest to the G + C content of the corresponding amplicons are preferable.
3) To obtain ideal amplification performance, test several amplicon regions for each target gene and design several primer sets for each selected region. The efficiency of the tHDA reactions may vary dramatically for different amplicons as well as for different primer sets within the specific region. Using primers optimized for PCR reactions may not guarantee similar performance in tHDA reactions.
4) Once a set of primers yields a positive amplification result, serial primer sets longer or shorter than this set may be analyzed to generate optimal primers for increased amplification efficiency.
5) Due to the sensitivity of tHDA to changes in salt concentrations in the reaction, the use of desalted primers is recommended. Successful tHDA reactions were achieved using salt-free primers synthesized by Operon Biotechnologies.