Repeated Template Switching: Obstacles in cDNA Libraries and Ways to Avoid Them

ABSTRACT

We screened zebra finch cDNA libraries with the plaque-based competitive hybridization technique for genes differentially expressed in the brains of courting and non-courting males. The cDNA libraries were generated by the SMART™ cDNA Library Construction Kit.

Sixty-six percent (35/53) of the isolated clones possessed high homology to sequences present in the cDNA libraries of species ranging from Arabidopsis to humans. The clones had to be false positives since the corresponding sequences are absent in genomic libraries.

The false positive clones were tandem repeats of different lengths of the SMART IV™ oligonucleotide and originated through repeated template switching during reverse transcription. We report here based on in silico data that multiple template switching and subsequent repeated incorporation of primers at 3'end of first strand cDNA is a common event during the generation of cDNA libraries created by the template switching strategy. We demonstrate that the repeat containing clones disturbs hybridization procedures aimed to identify differentially expressed genes. We propose an additional hybridization step with repeat containing clones to avoid the identification of false positives, and recommend the use of switch primers without palindrome sequences to avoid the formation of tandem repeats during cDNA library construction.

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