Recursive PCR: a novel technique for total gene synthesis

Gene synthesis is an efficient and cost-effective alternative to molecular cloning for custom gene production, where the DNA is manufactured by assembling strings of oligos together.Gene synthesis is the process of chemically synthesizing double-stranded DNA molecules in vitro. The main concept of gene synthesis is to assemble custom oligos into long DNA molecules. For mote detail ,please click the link, discovery biology services or contact us. Email: marketing@medicilon.com.cn web:www.medicilon.com

In protein engineering applications involving mutagenesis and expression of proteins from recombinant DNA, synthetic genes offer many advantages over using cloned naturally occurring genes. By precisely specifying the nucleotide sequence, optimal codon usage for the expression host can be ensured and convenient restriction sites incorporated as required, facilitating cassette mutagenesis and sub cloning. The synthesis of even a relatively small gene has generally been considered a difficult and time consuming task, best left to specialist laboratories. Inthis paper, a new cost and labour saving PCR technique is described which greatly simplifies the process of gene synthesis and has the potential for the synthesis of significantly larger genes than currently established techniques. Synthetic genes are conventionally assembled by concatenation of shorter oligonucleotides. Generally, both DNA strands are completely synthesized as short overlapping oligonucleotides which are phosphorylated, annealed and ligated to generate the full-length product. The cost of the synthesis can be reduced by synthesizing oligonucleotides representing the partial sequence of each strand, and the gaps in the annealed product ‘filled in’ using DNA polymerase prior to ligation (Rink et al., 1984). In practice, both methods give a low yield of the full-length product and require amplification by cloning before any further manipula-tion of the synthesized gene. Recently, a PCR procedure has been described in which a 234base oligonucleotide was chemically synthesized and primers used to amplify any full-length molecules that resulted from the chemical synthesis (Barnet and Erfle, 1990). While this procedure effectively increases the length of sequence that can be synthesized directly at a useful yield, the length is still relatively short in comparison with even a moderately sized structural gene. Genescan also be assembled by the method of ‘splicing by over lapextension’ (Higuchi etal., 1988), in which PCR products are purified away from their amplifying primers and extended again steach other to produce a larger product. This product is simul-taneously amplified by the inclusion of smaller flanking primers. We have developed a PCR technique for gene synthesis which requires neither phosphorylation nor ligation, gives high yields and has the potential for the total synthesis of much larger genes than other established techniques. The cost of this method is relatively low because only oligonucleotides representing the partial sequence of each strand are chemically synthesized, as in the ‘DNA polymerase filling-in’ method. The oligonucleotides are mixed and subjected to PCR such that those overlapping at their 3' ends are extended to give longer double-stranded products. This is repeated for the double-stranded products until the full-sized gene is obtained. The full-length product is subsequently amplified by the outermost 5' oligonucleotides of each strand, which are present at a higher concentration than the internal oligonucleotides and act as primers for the amplification. The complete synthesis of the gene results from the mutual extension of two halves which themselves result from the mutual extension of two halves and so on. This is functionally identical to the technique of recursion in computer programming and we therefore describe this method as ‘recursive PCR’.

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