Identification and cloning of differentially expressed genes by long- distance differential display

Roland Jurecic, Ronald G. Nachtman, Suzanne M. Colicos, John W. Belmont

Research output: Contribution to journalArticle

21 Citations (Scopus)

Abstract

Differential mRNA display (DD-PCR) amplifies short cDNAs (average size 100-350 bp), representing mainly the 3' untranslated regions (3' UTR) of transcripts. Sequencing of these cDNAs is predominantly uninformative for prediction of function and selection of clones for further analysis. Differential display of longer amplicons (0.5-2.0 kb) could enable isolation of cDNAs that encompass both 3' UTR and at least part of the 3' end of the coding region. The coding sequence information could facilitate selection of candidate clones for further analysis without the necessity of screening cDNA libraries. By combining DD-PCR protocols with long-distance PCR and using hot-start PCR with rTth DNA polymerase we have successfully amplified and comparatively displayed cDNAs ranging in size from 150 bp to 2 kb. Long- distance DD-PCR (LDD-PCR) has generated highly reproducible primer-specific patterns of cDNA fragments, as well as reproducible duplicate fingerprints, obtained from different RNA and cDNA samples. Sequencing and expression analyses of LDD-PCR clones have shown that LDD-PCR (a) enables nonredundant clone sampling, (b) generates many clones that encompass part of the coding region, and (c) samples both abundant and rare transcripts, ~60% of which are differentially expressed as confirmed by Northern analysis. Coupled with high-throughput cDNA sequencing and multiplex hybridization of cDNA microarrays for confirmation of differential expression, LDD-PCR could prove to be useful for simultaneous scanning of transcripts from multiple cDNA samples and faster selection of differentially expressed transcripts of interest.

Original languageEnglish
Pages (from-to)235-244
Number of pages10
JournalAnalytical Biochemistry
Volume259
Issue number2
DOIs
StatePublished - Jun 1 1998
Externally publishedYes

Fingerprint

Cloning
Organism Cloning
Complementary DNA
Genes
Display devices
Polymerase Chain Reaction
Clone Cells
3' Untranslated Regions
Dermatoglyphics
Gene Expression Profiling
DNA-Directed DNA Polymerase
Oligonucleotide Array Sequence Analysis
Gene Library
Microarrays
Screening
RNA
Throughput
Sampling
Scanning
Messenger RNA

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

Identification and cloning of differentially expressed genes by long- distance differential display. / Jurecic, Roland; Nachtman, Ronald G.; Colicos, Suzanne M.; Belmont, John W.

In: Analytical Biochemistry, Vol. 259, No. 2, 01.06.1998, p. 235-244.

Research output: Contribution to journalArticle

Jurecic, Roland ; Nachtman, Ronald G. ; Colicos, Suzanne M. ; Belmont, John W. / Identification and cloning of differentially expressed genes by long- distance differential display. In: Analytical Biochemistry. 1998 ; Vol. 259, No. 2. pp. 235-244.
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