![]() Second, the primers do not to overlap in their 3'-end, and, hence, are shorter and should perform better in PCR (Figure 2B). First, whereas traditional exponential amplification WHOPS uses two unique primers for each mutation (Figure 2A), uracil-excision offers the opportunity to reuse one primer when several mutations need to be introduced in one location (Figure 2B). The method offers several advantages compared to previous WHOPS protocols (Figure 2A-E). Uracil-excision can be used in several ways as a simple, versatile and large-insertion compatible site-mutagenesis WHOPS method. In the typical PCR-based approach, template carry-over is inhibited by treatment with the restriction enzyme DpnI, that restricts dam methylated plasmid DNA, but leaves unmethylated PCR-derived DNA intact. Later, PCR entered the scene and variants, known as inverse PCR or whole plasmid synthesis (WHOPS), largely seems to have replaced the Kunkel method. Subsequently, the DNA is transformed into ung+ bacteria, where only the newly synthesized, mutant DNA survives and the primer-introduced mutation is isolated. In the method, dU-containing DNA is used as template in a linear amplification reaction with a mutagenic primer, as well as the Klenow enzyme, dNTPs and a ligase. ![]() This strain lacks dUTPase and uracil deglycosidase and therefore accumulates soluble dUTP and DNA-bound dU nucleotides. One of the early methods for doing site-directed mutagenesis was the Kunkel method that uses template DNA isolated from a ung- dut- E. In addition to PCR, and cloning and fusing genes, site-directed mutagenesis is an indispensable tool for molecular biologists. The useful application of these technologies was further demonstrated by their use in artificial gene synthesis. This led to development of an improved uracil-excision cloning technology and to a new way of doing seamless PCR product fusion that may eventually replace overlapping PCR in many applications. With mutants like PfuV93Q, high-fidelity PCR became compatible with uracil-excision cloning. In 1999, the crystal structure of the DNA polymerase Tgo from the archea Thermococcus gorgonarius was solved, revealing the nature of the uracil-binding pocket, and allowing the design of mutant Tgo- and Pfu-polymerases with reduced stalling at uracil-containing DNA. Hence, the technique relies solely on a pair of properly spaced deoxy adenine- and dT-nucleotides, and, due to the degeneracy of the genetic code, allow seamless translational fusions of virtually any protein coding DNA sequence, making it particular suitable for (but not limited to) applications such as chimeric DNA fusion designs. The overhangs are usually designed to be 7-12 nucleotides long, and therefore can create circular DNA species that are stable enough to allow bacterial transformation without prior ligation. Subsequently, the DNA is treated with uracil DNA glycosidase (UNG) and usually either T4 endonuclease or DNA glycosylase-lyase endo VIII (commercially available as a mix with UNG as the so-called USERâ„¢ enzyme from New England Biolabs), which releases the sequence upstream from the dU's and allows pairing between exposed, compatible ends (and these reactions define the term "uracil-excision" as it is used here). In the technique, compatible single stranded DNA overhangs are created by substituting selected deoxy thymidine (dT) nucleotides with deoxy uridine nucleotides (dU) (Figure 1). ![]() Uracil-excision-based cloning was invented more than 15 years ago, but the technique was left unused, due to incompatibility with high-fidelity PCR.
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