![]() ![]() In all cases, PCR cannot be used directly to amplify a fragment containing the known and unknown sequence since only the sequence at one end of the fragment to amplify is known. T-DNA), the sequence flanking a transposon insertion, or the sequences of the variable regions of an immunoglobulin. Examples of applications where such problem is encountered include the determination of flanking sequences of stably integrated transgenes (e.g. ![]() One problem in molecular biology consists of identifying unknown sequences that flank a region of known sequence. 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. ![]() 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. coli where the annealed vector-insert complex is repaired and ligated. The reaction mix is then directly transformed into E. 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. Since only specific products contain this sequence, but none of the non-specific products, only specific products can be cloned. 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. 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. As with ligation-independent cloning, the strategy is based on homology between sequences present in both the vector and the insert. We have developed an efficient strategy for cloning of PCR products that contain an unknown region flanked by a known sequence. ![]()
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