Identification of chromosome-specific sequence-tagged sites by Alu-PCR.
Recently, methods have been developed for the isolation of expressed sequences from particular human chromosomes. Using Alu consensus sequences as primers, cDNA synthesis has been initiated from interspecies hybrid cell lines that contain single human chromosomes. Alu consensus sequences have also been utilized to amplify human genomic sequences via polymerase chain reaction (PCR). Here, we describe the use of Alu-PCR to isolate expressed sequences from human chromosomes selectively. Heteronuclear (hn) RNA is transcribed into cDNA by using poly-(dT)15 primer sequences. Subsequently, human specific cDNA sequences are amplified by Alu-PCR and cloned into pBluescript. To verify the chromosomal assignment, cloned PCR products are sequenced, converted into STS markers, and tested on a different somatic hybrid that contains human chromosome 22. The method provides a fast, reliable way to identify expressed sequence tagged sites from selected human chromosomes.
Klein
V
V
Department of Human Genetics, University of Saarland, Homburg/Saar, Germany.
Piontek
K
K
Brass
N
N
Subke
F
F
Zang
K D
KD
Meese
E
E
D000818:Animals; D015152:Blotting, Northern; D015139:Blotting, Southern; D002892:Chromosomes, Human, Pair 22; D003001:Cloning, Molecular; D004247:DNA; D006801:Humans; D006822:Hybrid Cells; D016133:Polymerase Chain Reaction; D012332:RNA, Heterogeneous Nuclear / Q000302:isolation & purification; D012091:Repetitive Sequences, Nucleic Acid; D012377:Rodentia; D016324:Sequence Tagged Sites
D012332:RNA, Heterogeneous Nuclear; D004247:DNA