Details

Autor(en) / Beteiligte

• PETERSON, L. A
• BROWN, M. R
• CARLISLE, A. J
• KOHN, E. C
• LIOTTA, L. A
• EMMERT-BUCK, M. R
• KRIZMAN, D. B

Titel

An improved method for construction of directionally cloned cDNA libraries from microdissected cells

Ist Teil von

• Cancer research (Chicago, Ill.), 1998-12, Vol.58 (23), p.5326-5328

Ort / Verlag

Philadelphia, PA: American Association for Cancer Research

Erscheinungsjahr

1998

Quelle

Electronic Journals Library

Beschreibungen/Notizen

• Here, we developed an improved method for constructing microdissected cDNA libraries, based on strand-switching properties of reverse transcriptase, followed by PCR amplification with primers to mediate unidirectional insert cloning. Using RNA from microdissected ovarian carcinoma cells, we constructed a cDNA library consisting of 1.3 x 10(6) unidirectional recombinants with an average insert size of 500 bp. Single-pass sequencing of 100 clones with the T7 primer revealed 89 inserts derived from known genes, anonymous expressed sequence tags (ESTs), or novel sequences. Among these clones were known genes and ESTs previously found in cDNA libraries from bulk ovarian tissue RNA, sequences seen for the first time in an ovarian-derived library, and novel sequences not previously seen in any cDNA library. These results demonstrate a methodology for constructing quality cDNA libraries that are cloned in a unidirectional fashion, are complex and diverse, and reflect the tissue of origin.