PrecisePrimer: an easy-to-use web server for designing PCR primers for DNA library cloning and DNA shuffling

PrecisePrimer is a web-based primer design software made to assist experimentalists in any repetitive primer design task such as preparing, cloning and shuffling DNA libraries. Unlike other popular primer design tools, it is conceived to generate primer libraries with popular PCR polymerase buffers proposed as pre-set options. PrecisePrimer is also meant to design primers in batches, such as for DNA libraries creation of DNA shuffling experiments and to have the simplest interface possible. It integrates the most up-to-date melting temperature algorithms validated with experimental data, and cross validated with other computational tools. We generated a library of primers for the extraction and cloning of 61 genes from yeast DNA genomic extract using default parameters. All primer pairs efficiently amplified their target without any optimization of the PCR conditions.


Supplementary materials S1 Melting temperature calculation
Our program implements the popular nearest neighbour thermodynamic based algorithm using thermodynamic parameters from Breslauer [1] and SantaLucia [2].
Where ∆H and ∆S are respectively the free enthalpy and the entropy of the primer-template dimer, ∆H init ∆S init respectively the initiation enthalpy and entropy, ∆H term and ∆S term the termination enthalpy and entropy of the primer dimer. N N is the nucleotide pair in the primer-template dimer, ∆H N N and ∆S N N the corresponding enthalpy and entropy. The melting temperature is given by classical thermodynamics (3) [2].
Where R is the universal gas constant and K is the equilibrium thermodynamic constant. T m 0 is the melting temperature in Kelvin at 1 M NaCl concentration.
Where %DM SO is the percentage of DMSO in the solution, and T m f inal the final melting temperature in Celsius.

S3 Validation graphs
The different combination of algorithms were assayed on experimental data from this article [5]. The results are shown on Figure S1. Figure S1: Validation curve of the algorithms on experimental data, with the 6 combinations of algorithms proposed.

S2
The algorithms proposed are capable of reproducting the NEB melting temperature calculation. The set here were random sequences calculated with the NEB calculator available on-line. The melting temperature calculation is compared to the one provided by our calculator. There are only two plots because the algorithms either use SantaLucia [2] thermodynamic table with Owczarzy [4] salt correction for all the polymerases but for the Phusion polymerase which uses Breslauer thermodynamic table [1] and Schilkraut [3] salt correction. The results are shown on Figure S2. Figure S2: Validation curve of the algorithms on random sequences calculated with NEB tool compared with Pre-cisePrimer algorithms.
The empirical formula from this paper [5] was also implemented in PrecisePrimer code. The Figure S3 shown its validation curve. Figure S3: Validation curve of the empirical formula from [5]

S4 Experimental validation
We designed 96 primers using the default parameters of PrecisePrimes, except for the optimal melting temperature that was set at 60 • C for higher specificity of the primer on the genomic target, using the algorithm for NEB Q5 polymerase buffer. We amplified 61 different coding sequence fragments from yeast genomic extract (we reused the forward or reverse primer for some of them). On the first try we got 59 amplicons showing the correct size on agarose gel ( Figure S4-a). The amplicon Flo1p-2 shows multiple band on gel because the primers turned to match several time on the gene due to the presence of repeated motifs.
We repeated the PCR for the two amplicons that did not worked in the first place, in order to check for pipeting errors or any other factor that may have affected the reaction. On the exact same conditions, the two PCR gave a band at the expect size on the gel ( Figure S4-b).
Overall, all primers we designed successfully amplified their target sequence. We repeated the amplification of the two genes that didn't worked on the first round in the exact same conditions. The amplicon size is correct showing that the failure of the first PCR was due to experimental mistake and not to the primer design.
The primers used to amplify these genes are shown in Table S1.