Quick and Clean Cloning: A Ligation-Independent Cloning Strategy for Selective Cloning of Specific PCR Products from Non-Specific Mixes

Frank Thieme,Romy Kandzia,Carola Engler,Sylvestre Marillonnet,Sudha Agarwal

doi:10.1371/journal.pone.0020556

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.

Highlights

One problem in molecular biology consists of identifying unknown sequences that flank a region of known sequence
In contrast to other ligationindependent strategies, the cloning vector has homology with only one of the two primers used for amplification, the primer designed to bind to the adaptor sequence
Non-specific products Empty vector called sequence and ligation independent cloning (SLIC), that is performed with T4 DNA polymerase on DNA fragments with overlapping ends consisting of any native sequence could be used to assemble up to 9 PCR-amplified fragments into a vector

Summary

Introduction

One problem in molecular biology consists of identifying unknown sequences that flank a region of known sequence. Over the years, many protocols have been developed to bypass this problem and allow the identification of unknown flanking sequences Such protocols cover a wide range of approaches, including inverse PCR [1], Tail PCR [2] and adaptor PCR [3,4,5] for DNA targets, and 59 RACE for RNA targets [6,7]. 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. The amplified products have to be cloned, and recombinant plasmids individually sequenced

Methods

Results

Discussion

Conclusion