title: Quick and clean cloning: a ligation-independent cloning strategy for selective cloning of specific PCR products from non-specific mixes.
abstract: We have developed an efficient strategy for cloning of PCR products that contain an unknown region flanked by a known sequence. As with ligation-independent cloning, the strategy is based on homology between sequences present in both the vector and the insert. However, in contrast to ligation-independent cloning, the cloning vector has homology with only one of the two primers used for amplification of the insert. The other side of the linearized cloning vector has homology with a sequence present in the insert, but nested and non-overlapping with the gene-specific primer used for amplification. Since only specific products contain this sequence, but none of the non-specific products, only specific products can be cloned. Cloning is performed using a one-step reaction that only requires incubation for 10 minutes at room temperature in the presence of T4 DNA polymerase to generate single-stranded extensions at the ends of the vector and insert. The reaction mix is then directly transformed into E. coli where the annealed vector-insert complex is repaired and ligated. We have tested this method, which we call quick and clean cloning (QC cloning), for cloning of the variable regions of immunoglobulins expressed in non-Hodgkin lymphoma tumor samples. This method can also be applied to identify the flanking sequence of DNA elements such as T-DNA or transposon insertions, or be used for cloning of any PCR product with high specificity.
authors.affiliation: Icon Genetics GmbH, Halle, Germany.
authors.forename: Frank
authors.initials: F
authors.lastname: Thieme
authors.forename: Carola
authors.initials: C
authors.lastname: Engler
authors.forename: Romy
authors.initials: R
authors.lastname: Kandzia
authors.forename: Sylvestre
authors.initials: S
authors.lastname: Marillonnet
mesh_terms: D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000235:genetics; D008228:Lymphoma, Non-Hodgkin / Q000235:genetics; D008957:Models, Genetic; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA; D014764:Viral Proteins / Q000378:metabolism
chemical_list: D007135:Immunoglobulin Variable Region; D014764:Viral Proteins; C069983:gene 43 protein, Enterobacteria phage T4; D004247:DNA; D004259:DNA-Directed DNA Polymerase