We describe how these overlap sequences can be used for fusion DNA construction, in-frame gene fusion, and cloning in yeast. Overlap extension or fusion PCR is thought to be a simple and easy method to produce fusion DNA fragments without the need for restriction enzyme digestion and DNA ligation.

We used the same primers as described in the QuickChange Mutagenisis protocol. We use Q5 Polymerase (see Standard PCR). Purify reaction using PCR purification kit – elute in 30-35 μl. Determine amount (Nanodrop or agarose gel). below!) X°C – annealing temperature Y:YY – extension time. Extension time depends on amplicon length and complexity.

A PCR-fusion step involves the assembly of two or more PCR amplified DNA fragments into a fusion fragment flanked by attL1 / attL2 sites. The assembled DNA fragment is subsequently cloned directly into a Gateway destination vector using an LR Clonase recombination reaction.

Jan 1, 2020 · Overlap extension PCR is a valuable technique that is commonly used for cloning large complex fragments, making edits to cloned genes or fusing two gene elements together. After difficulties in utilizing this technique following existing methods, we …

Nov 17, 2011 · Here, we report a simple and effective PCR method namely, fusion primer and nested integrated PCR (FPNI-PCR). This method is based on the use of arbitrary degenerate nucleotides reflecting natural restriction sites or site-dependent nucleotides in the genomic DNA to design universal primers.

Polymerases with Sso7 displayed high tolerance to PCR inhibitors from tissues and DNA samples. This property has further enabled practical applications such as Direct PCR, which allows DNA amplification directly from tissue samples without the …

Jan 1, 2012 · In this protocol, we introduce reliable fusion PCR mediated by novel overlap sequences, many of these are short GC-rich sequences (Fig. 2). This approach cannot only be applied for conventional DNA fusion but also for in-frame gene fusion or gene splicing to produce chimeric proteins.

Apr 17, 2012 · Inverse fusion PCR cloning (IFPC) is an easy, PCR based three-step cloning method that allows the seamless and directional insertion of PCR products into virtually all plasmids, this with a free choice of the insertion site.

Jan 31, 2007 · We describe the use of fusion PCR to generate linear molecules consisting of two fragments amplified from A. nidulans genomic DNA flanking a selection cassette. The type of cassette used depends...

Gene splicing by fusion PCR is a versatile and widely used methodology, especially in synthetic biology. We here describe a rapid method for splicing two fragments by one-round fusion PCR with a dual-asymmetric primers and two-step annealing (ODT) method.