Recombinant enrichment by exploitation of restriction sites with interrupted palindromes: design, synthesis and incorporation of zero-background linkers in cloning and expression vectors.
A universal approach for improving the efficiency of cloning through a selective enrichment of recombinants has been developed. This was achieved by using novel polylinkers or multiple cloning sites (MCS) termed zero-background linkers (ZBL). These MCS have short-cutter site(s) incorporated in the wobble portion of the recognition sequence of the long-cutter(s) in such a fashion that cloning at the short-cutter site disrupts the continuity of the long-cutter-specific sequence. Consequently, digestion of the ligation mixture with the long-cutter, prior to transformation, essentially eliminates the insert negative clones from transformants. The usefulness of such background removal has been illustrated through the design and synthesis of a model ZBL molecule and then its incorporation into various popular cloning and expression vectors. We demonstrate that replacement of the residing MCS of a vector with ZBL leads to enhanced cloning efficiency, as evidenced by the marked increase in the ratio of recombinants to non-recombinants. Such vector improvement is nearly universal as zero backgrounding is an add-on feature and can be adapted to practically any vector system, without having to alter the essential features of the parent vectors.
Majumder
K
K
International Center for Genetic Engineering and Biotechnology, New Delhi, India.
Choudhury
S
S
Bhatnagar
R K
RK
D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004262:DNA Restriction Enzymes / Q000378:metabolism*; D015246:Deoxyribonuclease EcoRI / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis*; D015183:Restriction Mapping; D013379:Substrate Specificity; D014170:Transformation, Genetic
D011994:Recombinant Proteins; D004247:DNA; D004262:DNA Restriction Enzymes; D015246:Deoxyribonuclease EcoRI; C047564:CCANNNNNNTGG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific