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Gene2Oligo: oligonucleotide design for in vitro gene synthesis
J.-M. Rouillard, W. Lee, G. Truan, X. Gao, X. Zhou and E. Gulari
Nucleic Acids Res. 2004 Jul 1;32(Web Server issue):W176-80
What is Gene2Oligo for?
Gene2Oligo is a program that divides a long sequence into a set of contiguous oligonucleotides corresponding to both DNA strands. Oligonucleotides from each strand overlap so they hybridize and be linked together to create the input sequence in vitro. We call "hybridization unit" the region of an oligonucleotide matching to a single complementary oligonucleotide. Thus, an oligonucleotide is made of two hybridization units. These oligonucleotides can be assembled together by using either Ligase Chain Reaction (LCR) or assembly PCR.
Application to the synthesis of the CYB5 gene
We have processed the CYB5 sequence with Gene2Oligo using a hybridization unit size of 20 nucleotides and the software optimized Tm option. The sodium and DNA concentrations were left unchanged. We have obtained a total of 21 oligonucleotides with hybridization unit sizes ranging from 16 to 22 oligonucleotides and Tm ranging from 62.5 to 69.6°C.
These oligonucleotides have been assembled into a full sized gene using either LCR or assembly PCR, followed by a PCR amplification (see methods). Here are the results:
Analysis of the synthetic CYB5 gene. 10 ul of each sample were analyzed on a 1% agarose gel. Lanes: L) 100 bp DNA ladder (NEB); 1) negative control, second PCR without template DNA; 2) LCR product before second PCR; 3) second PCR without primer after LCR; 4) second PCR after LCR; 5) assembly PCR product before second PCR; 6) second PCR without primer after assembly PCR; 7) second PCR after assembly PCR; 8) positive control, second PCR using yeast genomic DNA as template. The arrow indicates the 388 bp CYB5 PCR product.
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Here are some examples of input sequence: CYB5 and LacZ.
Here are the results for these sequences when using the oligo length priority mode, a hybridization unit size of 20 and the software optimized Tm: CYB5 and LacZ.
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Last modified: Fri Jul 1 09:09:11 EDT 2005