SMART amplification combined with cDNA size fractionation in order to obtain large full-length clones.
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.
Wellenreuther
Ruth
R
Department of Molecular Genome Analysis, German Cancer Research Center (DKFZ), Heidelberg, Germany. r.wellenreuther@dkfz.de
Schupp
Ingo
I
Poustka
Annemarie
A
Wiemann
Stefan
S
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*
D018076:DNA, Complementary; D004273:DNA, Neoplasm; D012333:RNA, Messenger; D012334:RNA, Neoplasm