Detection, simultaneous display and direct sequencing of multiple nuclear hormone receptor genes using bilaterally targeted RNA fingerprinting.
We have developed a PCR-based method to detect, display and directly sequence multiple members of the nuclear hormone receptor (NHR) gene family. Our approach employs the basic concepts of RNA fingerprinting (Welsh et al. (1992) Nucleic Acids Res. 20, 4965-4970; Stone, B. and Wharton, W. (1994) Nucleic Acids Res. 22, 2612-2618) and differential display PCR (Liang, P. and Pardee, A.B. (1992) Science 257, 967-971), with modifications. In contrast to the previous methods, two conserved regions within the gene family were targeted to derive primers for PCR amplification. One of the conserved sites was used to deduce primers for cDNA synthesis. We believe that this strategy led to increased specificity. The use of degenerate primers with low redundancy in both reverse transcription and PCR steps also contributed to enhanced signal-to-noise ratio. The ability to directly sequence the amplified fragments constitutes a vast improvement over the previous methods. This method permitted the successful identification and simultaneous display of six different NHR genes, which included the previously unreported rat homolog of COUP-TFI and a recently described orphan receptor. We believe that this approach provides a convenient and rapid screening method for detecting and characterizing members of a gene family.
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Unit on Gene Mapping and Expression, National Institute of Mental Health, National Institutes of Health, Bethesda, MD 20892, USA.
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D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000196:embryology / Q000378:metabolism; D005260:Female; D005784:Gene Amplification; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011247:Pregnancy; D012333:RNA, Messenger / Q000302:isolation & purification; D051381:Rats; D017207:Rats, Sprague-Dawley; D011987:Receptors, Steroid / Q000737:chemistry / Q000235:genetics*; D014158:Transcription, Genetic
D009687:Nuclear Proteins; D012333:RNA, Messenger; D011987:Receptors, Steroid