SMART amplification combined with cDNA size fractionation in order to obtain large full-length clones

• Main Authors: Poustka Annemarie, Schupp Ingo, Wellenreuther Ruth, Wiemann Stefan
• Format: Article
• Language: English
• Published: BMC 2004-06-01
• Series: BMC Genomics
• Online Access: http://www.biomedcentral.com/1471-2164/5/36

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>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.</p> <p>Conclusions</p> <p>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.</p>