Reliable fusion PCR using Taq polymerases and pGEM-T easy vectors |
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Authors: | An Ruisheng Bai Xiaodong Grewal Parwinder S |
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Institution: | (1) Department of Entomology, The Ohio State University, 1680 Madison Ave, Wooster, Ohio 44691, USA |
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Abstract: | We describe a reliable method for the production of fusion PCR products that can be used to transform the wild-type bacteria
to replace target genes for mutagenesis studies. The relevant gene fragments and selective cassette are first amplified separately
by PCR using primers that produce overlapping ends. As economic Taq DNA polymerase is disappointed in producing overlap ends
due to adding an extra 3′-end ‘A’ base which potentially blocks the successful fusion of the amplified fragments, we use a
new primer design strategy to overcome this disadvantage by introducing an additional ‘A’ base in the overlap primers. The
amplified gene fragments were then separately cloned into a pGEM-T easy vector and re-amplified with the aid of a universal
primer T7/SP6. This procedure enables performing nested PCR with the outmost primers in the fusion reaction to reliably splice
the gene fragments into a single molecule with all sequences in the desired order. |
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Keywords: | |
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