Efficiency and limitations of the hn-cDNA library approach for the isolation of human transcribed genes from hybrid cells.
The use of splice donor site consensus sequences as primers in cDNA synthesis (to make a cDNA library from heterogeneous RNA or unprocessed transcript--an hn-cDNA library) and the screening of such an hn-cDNA library with human repeat DNA probe in order to isolate human genes from somatic cell hybrids have been demonstrated. Here, we optimize and evaluate the efficiency and limitations of the approach. Computer analysis of genomic sequences of 22 randomly selected human genes indicated that hexamers CTTACC, CTCACC, and CCTACC were most efficient at beginning first-strand cDNA synthesis at donor splice sites of hnRNA and suggested that the procedure is efficient for priming cDNA synthesis of at least one exon from most every gene. Primer extension experiments established conditions in which the primers would initiate synthesis of cDNA starting from a perfectly matched position on the RNA template at more than 60-fold higher yield than any other product. By isolation of a clone containing exon III of the human DNA repair gene ERCC1, we indicate that the approach is capable of cloning exons from weakly expressed genes. Sequencing of clones revealed a structure of hn-cDNA clones consistent with the expectations of the cloning strategy and indicated the potential of the clones in detecting polymorphisms. Finally, we demonstrate that the expression of these hn-cDNA sequences in cells can be detected efficiently at the hnRNA level by reverse transcriptase-polymerase chain reaction (RT/PCR).
Department of Molecular Genetics, University of Texas M.D. Anderson Cancer Center, Houston.
P
P
Liu
M B
MB
Perryman
W
W
Liao
M J
MJ
Siciliano
D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry* / Q000302:isolation & purification; D015342:DNA Probes; D015894:Genome, Human; D015698:Genomic Library; D006801:Humans; D006822:Hybrid Cells / Q000737:chemistry*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000737:chemistry; D012194:RNA-Directed DNA Polymerase; D011817:Rabbits; D012984:Software; D014158:Transcription, Genetic
D015342:DNA Probes; D012333:RNA, Messenger; D004247:DNA; D012194:RNA-Directed DNA Polymerase