Assembly PCR oligo maker: a tool for designing oligodeoxynucleotides for constructing long DNA molecules for RNA production

Abstract

We describe a computer program, Assembly PCR Oligo Maker, created to automate the design of oligodeoxynucleotides for the PCR-based construction of long DNA molecules. This program is freely available at http://publish.yorku.ca/~pjohnson/AssemblyPCRoligomaker.html and has been specifically designed to aid in the construction of DNA molecules that are to be used for the production of RNA molecules by in vitro synthesis with T7 RNA polymerase. The input for Assembly PCR Oligo Maker is either the desired DNA sequence to be made or an RNA sequence. If RNA is the input, the program first determines the DNA sequence necessary to produce the desired RNA molecule. The program then determines the sequences of all the oligodeoxynucleotides necessary for a two-step assembly PCR-based synthesis of the desired DNA molecule. The oligodeoxynucleotide sequences outputted are designed to have a uniform melt temperature and are checked for regions of overlap outside of the desired priming regions necessary for the PCR reaction. The validity of the program was verified experimentally by synthesizing a 191-nt long DNA molecule using the DNA sequences suggested by the program.

Figure 1

The assembly PCR method for constructing long DNA molecules. (a) In the first PCR step a pool of oligodeoxynucleotides anneal and are (b) elongated to produce a full-length DNA molecule. In addition to the full-length product, a host of shorter molecules also results. (c) In the second PCR step the desired full-length molecule is selectively amplified from the mixture using primers specific for the desired full-length product.

Figure 2

Interface of the Assembly PCR Oligo Maker program. The user enters the target sequence in the top window, presses the ‘Determine Oligos’ button and the output appears in the boxes below. The oligodeoxynucleotide sequences for the first PCR step are found in the ‘Assembly Oligo’ box, and the two oligodeoxynucleotide sequences for the second PCR step are found in the two ‘Flanking Primers’ boxes. User controlled settings and information about the program are accessible under the File and Help menus.

Figure 3

The construction of a 191-nt DNA molecule using the oligodeoxynucleotides determined by the Assembly PCR Oligo Maker program. (a) The sequence of the 191-nt DNA target to be produced. (b) The DNA sequences reported by Assembly PCR Oligo Maker program. Sequences for both steps of the assembly PCR reaction are reported. (c) Diagram showing how the four oligodeoxynucleotides anneal to produce the full-length dsDNA product (d) Agarose gel showing the results of the first (lane 2) and second (lane 3) PCR steps. The desired 191-nt molecule is visible after the second PCR step. DNA length markers are shown in lane 1.