Preparing a human membrane and secreted protein-enriched cDNA library using PCR primers derived from a genomic database.
We describe here a strategy for preparing a human membrane and secreted protein (MSP)-enriched cDNA library based on human MSP- and non-MSP-encoding cDNA sequences in the databases. The signal peptide parts of the MSP-encoding cDNA sequences, which currently comprise about half of the estimated total number in humans, were analyzed for common patterns. These patterns form a 'minimal' set of polymerase chain reaction primer candidates of length varying from 9 to 21 nt. The products stemming from each primer candidate were determined and the results allowed us to obtain an 'optimal' mixed-length primer set. Ninety-six percent of the primers in this set were predicted to yield </=10% undesired products, and the desired MSP-cDNA products could be easily separated by gel electrophoresis. The present analysis establishes a methodology for preparing a cDNA library that enables the analysis of individual MSPs. This methodology may also help identify new MSPs. As many cell regulatory processes are mediated by secreted proteins and their membrane-bound receptors, the preparation of a MSP-enriched cDNA library should benefit research on MSPs.
Fan
Y
Y
Department of Life Science, National Tsing Hua University, Hsinchu 300, Taiwan.
Wu
C Y
CY
Chen
C W
CW
Chang
T W
TW
Lim
C
C
D000595:Amino Acid Sequence; D001482:Base Composition; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics*; D018076:DNA, Complementary / Q000235:genetics*; D030561:Databases, Nucleic Acid; D030562:Databases, Protein; D015723:Gene Library; D015894:Genome, Human; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics*; D009841:Oligonucleotides / Q000235:genetics; D016133:Polymerase Chain Reaction; D021382:Protein Sorting Signals / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction
D017931:DNA Primers; D018076:DNA, Complementary; D008565:Membrane Proteins; D009841:Oligonucleotides; D021382:Protein Sorting Signals; D012333:RNA, Messenger