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BMC Genomics. 2004 Jun 15;5(1):36.

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

Author information

1
Department of Molecular Genome Analysis, German Cancer Research Center (DKFZ), Heidelberg, Germany. r.wellenreuther@dkfz.de

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.

PMID:
15198809
PMCID:
PMC436056
DOI:
10.1186/1471-2164-5-36
[Indexed for MEDLINE]
Free PMC Article

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