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Updated: Aug 23, 2026
Detection of Copy Number Alterations Using Single Cell Sequencing
Published on: February 17, 2017
SMART amplification combined with cDNA size fractionation in order to obtain large full-length clones
Ruth Wellenreuther1, Ingo Schupp, Annemarie Poustka
1Department of Molecular Genome Analysis, German Cancer Research Center (DKFZ), Heidelberg, Germany. r.wellenreuther@dkfz.de
Background:
cDNA libraries are widely used to identify genes and splice variants, and as a physical resource for full-length clones. Conventionally-generated cDNA libraries contain a high percentage of 5'-truncated clones. Current library construction methods that enrich for full-length mRNA are laborious, and involve several enzymatic steps performed on mRNA, which renders them sensitive to RNA degradation. The SMART technique for full-length enrichment is robust but results in limited cDNA insert size of the library.
Results:
We describe a method to construct SMART full-length enriched cDNA libraries with large insert sizes. Sub-libraries were generated from size-fractionated cDNA with an average insert size of up to seven kb. The percentage of full-length clones was calculated for different size ranges from BLAST results of over 12,000 5'ESTs.
Conclusions:
The presented technique is suitable to generate full-length enriched cDNA libraries with large average insert sizes in a straightforward and robust way. The representation of full-coding clones is high also for large cDNAs (70%, 4-10 kb), when high-quality starting mRNA is used.
