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HT Gene Amplification
Melting Temperature Calculation
The melting temperature (Tm) calculations are based on nearest neighbor method as described by Breslauer et al., (1986) (1) and two options of thermodynamic data are available for calculating cloning primers: from Breslauer et al., (1986) and from Sugimoto et al., (1996)Data Input
Input data must be a flat text containing coding region sequences of desired target protein genes in the FASTA format. For better usage, use descriptors (header of FASTA sequence) with a maximum of 20 characters. More than 20 characters will make hard to understand output file, although this is irrelevant if you open it with a CSV reader (like MS Excel or GNumeric).Optimal Tm
The primers will be designed according to the optimal Tm selected by the user. The program uses a maximum Tm variation of 5 degrees or 4 bases from the optimal value and the maximum Tm difference of 5 degrees or 4 bases between forward and reverse primers.Primer Concentration
This feature specifies the concentration of primers to be used in Tm calculations. The concentration must be the same as used in PCR reactions. The default value for this field is 500nM, which is the recommended primer concentration for most PCR reactions.Monovalent Cation Concentration
In this field the user can define the monovalent cation concentration for Tm calculations according to the polymerase buffer composition. As compositions of most buffers are proprietary, the standard value of 50mM is set as default.Append Extra Sequence to Forward and Reverse Primers
In these fields the user can append extra sequences at the 5' ends of generated primers containing LIC sequences, recombination sites or restriction sites for downstream cloning. Note that the application does not check for the existence restriction sites within the gene sequence that would interfere in restriction enzyme/ligation methodologies. Thus, we recommend the use of a sequence-independent cloning methodology like LIC, PIPE, Gateway™ or In-Fusion™. The appended sequences are NOT taken into account for Tm calculation.Output Data
Output data from both mutagenesis and cloning tools are given as CSV text containing the GeneID (first uninterrupted characters from FASTA header), primers sequences, lengths and Tm.HT Mutagenesis
Fig 1: Types of mutation by inverse PCR method
Mutagenic primers designed by HTP-OligoDesigner are based on inverse PCR method (3). The cloning plasmid or expression vector containing the gene of interest is used as template for a PCR reaction. Forward (F) and reverse (R) primers anneal back to back to the plasmid, which is entirely amplified by a proofreading polymerase that produces consistently blunt-ended DNA, like Pfu DNA polymerase or Phusion® High-Fidelity DNA Polymerase. After amplification, PCR product is circularized by blunt-end ligation with T4 DNA ligase and, for this reason, primers must be synthesized with 5´end phosphorylation or PCR product must be phosphorylated by T4 Polynucleotide Kinase. Circularized PCR product is then transformed into E. coli competent cells. This method generates up to 75% of mutated DNA copies in the third PCR cycle resulting in nearly 100% of molecules being mutated at a specific site after 30 cycles (3). HTP-OligoDesigner is capable of designing primers to introduce different types of mutation by inverse PCR method (Fig. 2).
Melting Temperature Calculation
The melting temperature (Tm) calculations are based on nearest neighbor method as described by Breslauer et al., (1986) (1) and two options of thermodynamic data are available for calculating cloning primers: from Breslauer et al., (1986) and from Sugimoto et al., (1996)Data Input
Input data must be a flat text containing coding region sequences of desired target protein genes in the FASTA format. For better usage, use descriptors (header of FASTA sequence) with a maximum of 20 characters. More than 20 characters will make hard to understand output file, although this is irrelevant if you open it with a CSV reader (like MS Excel or GNumeric). The desired mutations, deletions or insertions for each sequence must be indicated into the sequences by the following code: The modification must be enclosed in parenthesis. The base(s) to be changed and the new base(s) must be separated by a point| Point Mutation: | TG(C.A)TTA | replaces C for A in TGCTTA, that becomes TGATTA |
| Deletion: | TG(C.)TTA | deletes C in TGCTTA, that becomes TGTTA |
| Insertion: | TGC(.GAT)TTA | inserts GAT in TGCTTA, that becomes TGCGATTTA |
Choose Design Method
This feature allows the user to choose if primer design will be “By Size” or “By Tm”. In “By Size” option, the number of nucleotides of the forward primer (which contains the mutation) will be fixed and the reverse primer will be calculated to have the same Tm as the forward. In “By Tm” option, both forward and reverse primers will be designed according to the selected Tm. The program uses a maximum Tm variation of 5 degrees or 4 bases from the optimal value and the maximum Tm difference of 5 degrees or 4 bases between forward and reverse primers.Primer Concentration
This feature specifies the concentration of primers to be used in Tm calculations. The concentration must be the same as used in PCR reactions. The default value for this field is 500nM, which is the recommended primer concentration for most PCR reactions.Monovalent Cation Concentration
In this field the user can define the monovalent cation concentration for Tm calculations according to the polymerase buffer composition. As compositions of most buffers are proprietary, the standard value of 50mM is set as default.Output Data
Output data from both mutagenesis and cloning tools are given as CSV text containing the GeneID (first uninterrupted characters from FASTA header), primers sequences, lengths and Tm.
Gustavo Machado Alvares de Lima
PhD in Biomolecular Physics
Cesar Moises Camilo
PhD in Biochemistry