PCR fusion

Usually introduction of a point mutation is desired to occur in the middle of the target gene and not at the N- or C-terminal end, for which the primers are normally designed. To ensure that both DNA strands will carry the mutation, a fusion PCR can be performed. 

Fusion PCR was a successful but not very efficient method because it involved two successive PCR reactions, which can introduce errors into the gene. Amplification of the fusion product can be difficult when the overlapping region of the mutation features a low melting temperature. 

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Figure 10.1 Fusion PCR, an early method to introduce point mutations in site-directed mutagenesis.

Although the concept of fusion PCR is simple and appears to be easy, fusion PCR has not been used frequently thus far. Fusion PCR often proved difficult in constructing large DNA fragments of greater than 2 kb (11). Even if PCR conditions and length of the overlap sequences are modified, some of the selected authentic overlap sequences have resulted in failed fusion or only yielded low-fusion DNA products (12). These unsatisfactory results and the consequently limited adoption of this method have suggested that the approach per se is afflicted with problems. We expected that this... 

Fig, 1. Fusion PCR by overlap extension. DNA fragment 1 and 2 contain an overlap sequence at the 3 and 5 end, respectively. PCR allows annealing of the sequence that overlaps between fragment 1 and 2, followed by DNA polymerase extension from the 3 end of the overlap sequence. Primers [arrowy at the both ends of the fused fragment produce fusion DNA after the extension. 

Fig. 2. Sets of overiap sequences for fusion PCR. Each set of primers, such as 15C and 15G, should be used together to amplify the two fragments to be fused, one at the 5 end and another at the 3 end. Type 1 sequence is used for fusions that allow mutations in 15C/15G region (see Note 2). Type 2 sequence is useful for in-frame fusion owing to its low mutation frequency. Type 3 sequence Is used for GC-rich templates that sometimes prevent amplification with GC-rich primers.

Reliable fusion PCR can be achieved with the appropriate design of overlap annealing sequences. Three types of overlap sequences are used for different purposes (Fig. 2). Type 1 is the 15C/15G sequence. This sequence produces reliable and reproducible results even if it is shorter than 15 nucleotides (12), but then might lead... 

Fig. 3. Fusion PCR between GAUOp-yEGfP and transformation marker gene using the 15C/15G overlap sequence. Thick bars with arrows indicate the 15C and 15G sequences in the primers. PCR of fragment 1 and 2 annealing at 47°C and 55°C, respectively, with 2-min extension. Fusion PCR 60°C annealing with 3-min extension. One microliter of ten-microliter reaction mixture was subjected to agarose gel electrophoresis.

After fusion PCR, 1 pL of sample is mixed with gel loading buffer and subjected to agarose gel electrophoresis (Fig. 3). 

When the DNA concentration of the fragments was not adjusted, the fusion PCR failed (see No adjust lane in Fig. 4), but when it was adjusted, the correct band of 2.5 kb could be detected, indicating that DNA concentration of the fragments needs to be adjusted for multiple-fragment fusion. 

Construction of a Chimeric Gene by In-Frame Fusion PCR in Yeast... 

Low PCR amplification, or none at all, from certain GC-rich templates, such as mammalian and virus genes, was experienced with primers containing Type 1 and 2 sequences. This suggests that primer with a high GC content anneal to several places on the templates, resulting in weak amplification. For such templates, primers with Type 3 sequences are more effective for the conventional amplification and fusion PCR. 

DNA concentrations in conventional PCR are approximately 1-5 ng/pL. In this concentration range, DNA can be used directly for fusion PCR of two fragments without further adjustment. 

Cha-aim K, Fukimaga T, Hoshida H, Akada R (2009) Reliable fusion PCR mediated by GC-rich overlap sequences. Gene 434 43 9... 

Szewczyk, E., Nayak, T, Oakley, C.E., Edgerton, H., Xiong, Y, Taheri-Talesh, N., Osmani, S.A., Oakley, B.R., 2006. Fusion PCR and gene targeting in Aspergilus nidulans. Nature Protocols 1,3111-3120. 

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