Since the seminal discoveries of the basic principles underlying molecular cloning, a number of cloning strategies have been developed to improve on the ease and speed at which DNA fragments can be recombined. Read about the most common strategies used in molecular cloning or get your desired gene in the vector you want the easy way with GenEZ™ ORF clones. Start with a search for your gene.
Traditional cloning, also called PCR cloning, requires the use of the polymerase chain reaction (PCR) to amplify the template sequence of interest (usually the gene of interest) and add restriction sites to the ends of the sequence. Restriction enzymes are used to cut both the template of interest and the target vector, and DNA ligase is used to join the sticky ends of the template and vector together. Traditional cloning allows for ﬂexible DNA sequence manipulation, which facilitates the building of nearly any desired construct. However, the checkpoints and optimization procedures required for traditional cloning can be cumbersome, and the reagents required can be expensive.
TA cloning is one of the simplest forms of cloning. In this method, vectors containing 5' thymine overhangs are used to accept PCR products in which additional 3' adenosine overhangs have been added on by the nature of TAQ polymerase ampliﬁcation. TA cloning has the advantage of ease and speed, since no restriction digestion step is required. In addition, TA cloning kits contain reaction buffers that contain the pre-mixed vector, ligase, and buffer, cutting ligation reaction time to as few as 5 minutes. The disadvantage of TA cloning technology is that the cloning is not directional, meaning the gene of interest may be inserted into the target vector in either the sense or antisense orientation. Normally, half of the subsequent transformants will contain the gene in the sense direction and half will contain the gene in the antisense direction. However, cells transformed with toxic genes may all display the genes in the antisense direction, since cells containing the sense directed genes will not survive. In addition, survivor cells containing toxic genes oriented in the sense direction may be mutated to encode a less toxic protein.
Seamless cloning technologies eliminate the requirement for restriction enzymes. This can be advantageous when an insert contains a number of restriction sites within its sequence, making it diﬃcult to identify restriction enzymes that will not cut the gene of interest during the cloning procedure. Seamless cloning takes advantage of homologous recombination and there are numerous variations on the technique. In general, the procedure consists of adding ﬂanking sequences approximately 15 bp in length to both the insert and vector via PCR. Exonucleases are used to chew back the insert and vector sequences and the DNA is joined using recombinase enzymes or DNA ligase. Seamless cloning has been simpliﬁed by the development of kits that already contain the target vector and a proprietary mix of enzymes required for the recombination reaction. For instance, GenScript’s GenBuilder™ Kit can clone inserts up to 10 kb in 30 minutes, and can also be used for high-throughput cloning projects.