![]() ![]() Standard techniques that rely on digestion of insert and vector with restriction enzymes are not well suited for cloning fragments containing unknown sequences since presence of restriction sites in the unknown region may prevent cloning of such sequences. There are many approaches available for cloning of PCR products. Rather, the amplified products have to be cloned, and recombinant plasmids individually sequenced. However, if several specific products are expected to be amplified in the same reaction (for example a DNA sample may contain several transgenes and therefore several different flanking sequences, or an RNA sample extracted from a B-cell population will contain a large number of different immunoglobulin variable regions), direct sequencing will not be useful. Identification of the unknown sequence can then be done simply by sequencing the amplified product with a nested gene-specific primer. Therefore, one or two additional PCR amplifications performed using nested primers binding in the known region are usually necessary to increase the ratio of specific to non-specific products. Since for all of these protocols the adaptor sequence is not exclusively attached to the desired sequence, many non-specific products are also amplified in a first PCR. Basically, most of these protocols rely on attaching an adapter sequence to the end of the unknown sequence and using PCR for amplification of a fragment containing both known and unknown flanking sequences using a first primer binding to the adaptor sequence and a second primer binding to the known sequence. Such protocols cover a wide range of approaches, including inverse PCR, Tail PCR and adaptor PCR, , for DNA targets, and 5′ RACE for RNA targets. However, over the years, many protocols have been developed to bypass this problem and allow the identification of unknown flanking sequences. 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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