However, such a vector has been realized only partially. Ideally, a complete vector system that covers both choices should be available. coli cells carrying empty vectors cannot survive in this system because the expression of CcdB protein is lethal 15.īased on their needs, researchers should therefore choose either a T-overhang vector or a blunt-end vector including the ccdB gene 14, 16, 17. To overcome the low efficiency, ZERO-background-cloning using the ccdB gene, an active cytotoxic factor in Escherichia coli, was developed as a positive-selection system 14. In contrast to TA cloning using a single nucleotide T-overhang, blunt-end cloning of PCR products is less efficient because the terminal ends of the DNA fragment are not sticky 12, 13. However, XcmI-dependent T-vector preparation is hampered by a high background colony rate this is attributable to re-ligation of small excised DNA fragments into the digested empty vector 9, 10. Preparation of T-vectors by XcmI cleavage is a relatively easy way to produce T-overhangs, because XcmI is a commonly used restriction enzyme. To use these T-vectors, the two XcmI sites that are designed to produce the 3′-end T-overhang are introduced into a specific vector 9, 10, 11. Another T-vector preparation-method uses XcmI (a well-known type IIS restriction enzyme that recognizes an asymmetric nucleotide sequence) to digest outside of this recognition sequence and generate a T-overhang for TA cloning 8. The availability of commercially available ready-to-use T-vectors has obviated the need for very specific and complicated procedures however, while the vectors are generally very convenient, they are expensive. Unfortunately, this strategy is not widely adopted by researchers, as the reagents are not frequently used. To prepare the T-overhang vector from general cloning vectors, dideoxythimidine triphosphate (ddT) is incorporated at the 3′-terminus of a linearized blunt end vector by using terminal deoxynucleotidyl-transferase 7. Vectors that have a T-overhang at the 3′-end were developed as so-called ‘T-vectors’ for cloning of dA-tailed PCR products amplified by Taq DNA polymerase 7, 8. Due to the diverse types of PCR product that are generated, choice of cloning vector is a critical step that can ultimately determine cloning efficiency 6. In contrast, various high-fidelity thermostable DNA polymerases that possess 3′ to 5′ exonuclease activity for proofreading, such as Pfu DNA polymerase 3, KOD DNA polymerase 4 and Phusion DNA polymerase 5, produce the blunt-end DNA fragments. Of these, Taq DNA polymerase is the most widely used this enzyme attaches a deoxyadenosine triphosphate (dA) to the 3′-end of amplified DNA 2. Various types of thermostable DNA polymerases are commercially available for PCR amplification. Improvements to the basic technique to facilitate highly efficient molecular cloning of PCR products into a vector are required to promote research projects. Polymerase chain reaction (PCR) is an indispensable tool for amplification of genomic DNA and transcripts to analyze their functions 1.
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