The gene-specific 5′ primer must start with ATG, and the 3′ primer must start at the end of the position of the common stop codon. Note that in the Gateway® scheme the gene of interest will be amplified first with a gene-specific primer set (containing partial att sequences) and then reamplified with a universal primer set to create the entire attB sequence.Īll primers must have a similar melting temperature and must be anchored in start or stop positions of the open reading frame. Only the open reading frame (ORF) will be amplified from ATG to the stop codon, using gene-specific primers.
Schematic view of primer design and PCR amplification. For convenience, cost, and lower error rates, it is ideal to employ just one PCR step to build a recombination-competent fragment, which is usually possible with the In-Fusion® cloning scheme where only 15 base pair (bp) are needed for successful homologous recombination (In-Fusion® scheme Fig. Applying a second universal set of primers also aids in lowering the cost of the PCR scheme by avoiding lengthier gene specific primers (Gateway® scheme Fig. The rest of the recombination site can then be added efficiently by a secondary PCR employing a universal primer set.
In this case, the gene-specific primers can be designed to contain a 5’ tail sequence including only part of the recombination site. If the required flanking recombination sequences are long (minimum of 35 base pair), as is often the case in the Gateway® scheme, some of the single stranded DNA in the primer synthesis may contain errors. The PCR primers used to amplify a gene are designed to contain a 3’ gene-specific portion along with a common 5’ tail sequence for adding flanking recombination sites. The similarities and differences between the systems will be described in further detail below. The differences between the systems can be observed in the sequence and length of the recombination sequences, enzymes required for the homologous recombination event to occur, and the need for an intermediate construction of an entry clone. In all cloning schemes described here, they all share the similar molecular mechanism of recombinational cloning. Although the Cre-Lox system is no longer commercially sold as a kit, the protocol is intended for those still currently using or have reagents for this system. Lastly, we provide a supplemental protocol describing the construction of expression clones utilizing the Cre-Lox system in Alternate Protocol 1. Basic Protocol 1 describes the amplification of target genes and addition of the required recombination sites by PCR, Basic Protocols 2 and 3 describe the generation of entry (master) clones, and Basic Protocol 4 illustrates the construction of expression clones through the Gateway® system. The methods described in this unit are designed for use with two commercially available recombinational cloning systems, Gateway® (Life Technologies) and In-Fusion® (Clontech). Recombination-based universal cloning technology enables efficient parallel transfer of your favorite gene (YFG) from an entry clone into various different expression systems for protein production and functional analysis. In addition, multiple entry clones of varied sequences can be simultaneously transferred into a single expression vector to make a specific kind of library.Īdvantages of universal cloning technology. Another advantage of recombinational cloning is the construction of an entry clone, an intermediate clone that functions as a holding or storage clone that allows the flexibility of transferring a single insert into multiple expression vectors ( Fig.
These nucleotide-specific sequences are referred to as the recombination sequences. Recombinational cloning uses site-specific recombination, where both the insert and vector contain the required nucleotide sequences recognized by the cloning enzymes necessary for the recombinational event to occur. In contrast, recombinational cloning, or the transfer of DNA from one vector to another based on sequence homology, allows high-throughput cloning of genes into protein expression vectors under largely universal conditions without the use of restriction digests or ligation reactions. Traditional cloning methods based on restriction enzyme digestion and ligation are not practical due to the individual requirements and specifications for each construct. Large-scale experiments requiring protein expression from thousands of genes require an efficient method for cloning the genes into protein expression vectors.
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