title: SMART amplification combined with cDNA size fractionation in order to obtain large full-length clones.
abstract: 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.
authors.affiliation: Department of Molecular Genome Analysis, German Cancer Research Center (DKFZ), Heidelberg, Germany. r.wellenreuther@dkfz.de
authors.forename: Ruth
authors.initials: R
authors.lastname: Wellenreuther
authors.forename: Ingo
authors.initials: I
authors.lastname: Schupp
authors.forename: Annemarie
authors.initials: A
authors.lastname: Poustka
authors.forename: Stefan
authors.initials: S
authors.lastname: Wiemann
mesh_terms: D045744:Cell Line, Tumor; D003001:Cloning, Molecular / Q000379:methods; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004273:DNA, Neoplasm / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004587:Electrophoresis, Agar Gel / Q000379:methods; D016889:Endometrial Neoplasms / Q000235:genetics / Q000473:pathology; D005260:Female; D015723:Gene Library; D006801:Humans; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D012333:RNA, Messenger / Q000235:genetics; D012334:RNA, Neoplasm / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
chemical_list: D018076:DNA, Complementary; D004273:DNA, Neoplasm; D012333:RNA, Messenger; D012334:RNA, Neoplasm