Construction of Long-Transcript Enriched cDNA Libraries from Submicrogram Amounts of Total RNAs by a Universal PCR Amplification Method | Zendy

Yulan Piao | Zendy; Naomi T. Ko | Zendy; Meng K. Lim | Zendy; Minoru S.H. Ko | Zendy

Open Access

Construction of Long-Transcript Enriched cDNA Libraries from Submicrogram Amounts of Total RNAs by a Universal PCR Amplification Method

Author(s) -

Yulan Piao,

Naomi T. Ko,

Meng K. Lim,

Minoru S.H. Ko

Publication year - 2001

Publication title -

genome research

Language(s) - English

Resource type - Journals

SCImago Journal Rank - 9.556

H-Index - 297

eISSN - 1549-5469

pISSN - 1088-9051

DOI - 10.1101/gr.185501

Subject(s) - biology , complementary dna , primer (cosmetics) , insert (composites) , cdna library , microbiology and biotechnology , rna , rapid amplification of cdna ends , messenger rna , genomic library , genetics , computational biology , dna , gene , base sequence , molecular cloning , mechanical engineering , chemistry , organic chemistry , engineering

Here we report a novel design of linker primer that allows one to differentially amplify long tracts (average 3.0 kb with size ranges of 1-7 kb) or short DNAs (average 1.5 kb with size ranges of 0.5-3 kb) from a complex mixture. The method allows one to generate cDNA libraries enriched for long transcripts without size selection of insert DNAs. One representative library from newborn kidney includes 70% of clones bearing ATG start codons. A comparable library has been generated from 20 mouse blastocysts, containing only approximately 40 ng of total RNA. This universal PCR amplification scheme can provide a route to isolate very large cDNAs, even if they are expressed at very low levels.

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