Evaluating the specificity of PCR primers is an essential step in PCR primer design. The MFEprimer-2.0 server allows users to check primer specificity against genomic DNA and mRNA/cDNA sequence databases quickly and easily. MFEprimer-2.0 uses a k-mer index algorithm to accelerate the search process for primer binding sites and uses thermodynamics to evaluate binding stability between each primer and its DNA template. Several important characteristics such as the sequence, melting temperature and size of each amplicon, either specific or non-specific, are reported on the results page. Based on these characteristics and the user-friendly output, users can readily draw conclusions about the specificity of PCR primers. Analyses for degenerate primers and multiple PCR primers are also supported in MFEprimer-2.0. In addition, the databases supported by MFEprimer-2.0 are comprehensive, and custom databases can also be supported on request. The MFEprimer-2.0 server does not require a login and is freely available at http://biocompute.bmi.ac.cn/CZlab/MFEprimer-2.0. Unlike MFEprimer 1.x versions, which use BLAST for primer binding sites search, MFEprimer-2.0 uses the k-mer index algorithm to speed up the primer binding sites search process. This is the speed problem I have to solve, while, the other question force me MUST to replace the BLAST. It's the "ACCURACY" problem. As we know that, BLAST is a famous program to find the homology sequence from a database by sequence similarity. However, the annealing process of primer and its target sequence is thermodynamics. They bind to each other just because they are stable in thermodynamics, not because they are matched in base pairs. For example, the mismatch "G-G" contributes as much as the Gibbs free energy of -2.2 kcal/mol to the duplex stability [SantaLucia 2004]. So the first step we have to do is to find all the possible binding sites with the k-mer index algorithm], and then to evaluate the binding stability using the Nearest-Neighbor model.