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High efficiency selection of full-length cDNA by improved biotinylated cap trapper
P Carninci1, A Westover, Y Nishiyama
1Genome Science Laboratory, Tsukuba Life Science Centre, Institute of Physical and Chemical Research (RIKEN), Ibaraki, Japan.
Summary
This study introduces an improved cDNA library preparation protocol. The enhanced method protects messenger RNA (mRNA) during cap biotinylation, enabling more efficient cloning of long full-length complementary DNAs (cDNAs).
Area of Science:
- Molecular Biology
- Genomics
- Biotechnology
Background:
- The biotinylated CAP trapper protocol is a method for preparing complementary DNA (cDNA) libraries.
- A limitation of the original protocol is the exposure of messenger RNA (mRNA) to degradation during cap structure biotinylation before first-strand cDNA synthesis.
Purpose of the Study:
- To report an improved protocol for full-length cDNA library preparation.
- To enhance the efficiency of cloning long cDNAs.
- To protect mRNA from chemical and enzymatic degradation during the biotinylation process.
Main Methods:
- The improved protocol involves biotinylating the mRNA/cDNA hybrid after first-strand cDNA synthesis.
- This strategy protects the mRNA from degradation during the overnight biotinylation step.
- The method allows for the selection of full-length cDNAs with a longer average size.
Main Results:
- Biotinylation of the cap structure on the mRNA/cDNA hybrid is specific and effective.
- Messenger RNA (mRNA) is protected from degradation during the overnight biotinylation step.
- The improved protocol facilitates the selection of full-length cDNAs with a greater average length.
Conclusions:
- The modified protocol offers enhanced protection for mRNA during cDNA library preparation.
- This leads to more efficient cloning of long full-length cDNAs.
- The improved method increases the efficiency and specificity of full-length cDNA capture.