High Efficiency Selection of Full-length cDNA by Improved Biotinylated Cap Trapper

Abstract

We report here an improved protocol for the preparation of full-length cDNA libraries that improves the previously reported method (Carninci, P., Kvam, K., Kitamura, A. et al. 1996, Genomics, 137, 327-336), that allows long cDNAs to be cloned more efficiently. One potential disadvantage of the original biotinylated CAP trapper protocol is the exposure of mRNA to chemical and enzymatic attacks during the biotinylation of the cap structure, before the first-strand cDNA synthesis (and selection of full-length cDNA by biotinylated cap). Here, we show that the biotinylation of the cap structure is very specific and effective even if biotinylation is performed on the mRNA/cDNA hybrid produced by the first-strand cDNA synthesis reaction. Consequently, mRNA remains protected from chemical and enzymatic degradation during the overnight biotinylation step, thus making it possible to select full-length cDNAs of longer average size. We herein report the efficiency and specificity of the new version of the protocol for cap structure biotinylation and capture of full-length cDNA.

Comparative genomics ofPhyscomitrella patensgametophytic transcriptome andArabidopsis thaliana: Implication for land plant evolution

“…The cDNA was synthesized by using trehalose-thermoactivated reverse transcriptase (24), and full-length cDNA was recovered by using the biotinylated CAP trapper method (25,26). The -full-length cDNA vector (27) and the single-strand linker ligation method (28) were used in the construction of the cDNA libraries.…”

Construction of Long-Transcript Enriched cDNA Libraries from Submicrogram Amounts of Total RNAs by a Universal PCR Amplification Method

“…Because they require large amounts of RNA, conventional cDNA construction methods are precluded for such purposes. The problem is especially acute for full-length cDNA libraries, which require 10-50 µg of polyA+ RNA, equivalent to milligram amounts of total RNA (Maruyama and Sugano 1994;Edery et al 1995;Carninci et al 1996Carninci et al , 1997Carninci et al , 2000Suzuki et al 1997Suzuki et al , 2000.PCR amplification can permit the use of smaller amounts of RNA (Belyavsky et al 1989), but presents the technical dilemma that it customarily yields a shorter average insert size (e.g., ∼0.5 kb [Peterson et al 1998] and ∼1.5 kb [Ko et al 2000]), too short to recover many full-length cDNAs. While working on various linker designs and PCR conditions, we serendipitously found a condition that overcomes size limitations.…”

Transgenic Plants as a Tool for Plant Functional Genomics

“…Approximately 240,000 Arabidopsis fl-cDNA clones have been generated (Sakurai et al 2005;Seki et al 2002) using the biotinylated CAP trapper method together with trehalosethermoactivated reverse transcriptase (Carninci et al 1996;Carninci et al 1997;Carninci et al 1998) . Large sets of fl-cDNA clones have also been produced from several plants such as rice (Kikuchi et al 2003), wheat (Ogihara et al 2004), poplar (Nanjo et al 2007), soybean (Umezawa et al 2008), barley (K. Sato et al 2009), cassava (Sakurai et al 2007), sitka spruce (Ralph et al 2008), Physcomitrella patens (Nishiyama et al 2003), and Thellungiella halophila (Taji et al 2008).Well-characterized collections of cDNAs play an essential role in defining the function of genes and proteins in plants.…”