An improved method for construction of directionally cloned cDNA libraries from microdissected cells.
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.
Peterson
L A
LA
Laboratory of Pathology, National Cancer Institute, Bethesda, Maryland 20892, USA.
Brown
M R
MR
Carlisle
A J
AJ
Kohn
E C
EC
Liotta
L A
LA
Emmert-Buck
M R
MR
Krizman
D B
DB
D000199:Actins / Q000235:genetics; D000368:Aged; D003001:Cloning, Molecular; D018283:Cystadenocarcinoma, Papillary / Q000235:genetics*; D004273:DNA, Neoplasm / Q000032:analysis / Q000235:genetics*; D005260:Female; D006801:Humans; D010051:Ovarian Neoplasms / Q000235:genetics*; D012334:RNA, Neoplasm / Q000032:analysis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity
D000199:Actins; D004273:DNA, Neoplasm; D012334:RNA, Neoplasm