Negative selection: a method for obtaining low-abundance cDNAs using high-density cDNA clone arrays.
The identification of the entire complement of genes expressed in a cell, tissue, or organism provides a framework for understanding biological properties and establishes a tool set for subsequent functional studies. The large-scale sequencing of randomly selected clones from cDNA libraries has been successfully employed as a method for identifying a large fraction of these expressed genes. However, this approach is limited by the inherent redundancy of cellular transcripts reflecting widely variant levels of gene transcription. As a result, a high percentage of transcript duplications are encountered as the number of sequenced clones accrues. To address this problem, we have developed a negative hybridization selection method that employs the hybridization of complex cDNA probes to high-density arrays of cDNA clones and the subsequent selection of clones with a null or low hybridization signal. This approach was applied to a cDNA library constructed from normal human prostate tissue and resulted in the reduction of highly expressed prostate cDNAs from 6.8 to 0.57% with an overall decline in clone redundancy from 33 to 11%. The selected clones also reflected a more diverse cDNA population, with 89% of the clones representing distinctly different cDNAs compared with 67% of the randomly selected clones. This method compares favorably with cDNA library re-association normalization approaches and offers several distinct advantages, including the flexibility to use previously prepared libraries, and the ability to employ an iterative screening approach for continued accrual of cDNAs representing rare transcripts.
Nelson
P S
PS
Department of Molecular Biotechnology, University of Washington, Seattle 98195, USA. psnels@u.washington.edu
Hawkins
V
V
Schummer
M
M
Bumgarner
R
R
Ng
W L
WL
Ideker
T
T
Ferguson
C
C
Hood
L
L
D018076:DNA, Complementary; D020224:Expressed Sequence Tags; D020869:Gene Expression Profiling / Q000379:methods*; D006801:Humans; D008297:Male; D015336:Molecular Probe Techniques; D020411:Oligonucleotide Array Sequence Analysis; D011467:Prostate / Q000737:chemistry; D012333:RNA, Messenger / Q000032:analysis*
D018076:DNA, Complementary; D012333:RNA, Messenger