The application of the polymerase chain reaction to cloning members of the protein tyrosine kinase family.
Degenerate oligodeoxyribonucleotide (oligo) primers derived from amino acid (aa) sequence motifs held in common between all members of the protein tyrosine kinase (PTK) family were used to prime the amplification of PTK-related sequences from a variety of murine cDNA sources, including the haemopoietic cell lines, FDC-P1 and WEHI-3B D+, peritoneal macrophages and whole brain. Several parameters, such as the length (short, i.e., less than 20 nucleotides (nt) vs. long, i.e., greater than 30 nt) and degeneracy (i.e., moderately degenerate vs. highly degenerate) of the oligo primers and the temperature of the extension phase of the reaction, were examined. The data from these analyses suggest that the most effective type of primer in this application of the polymerase chain reaction is a short, moderately degenerate oligo such as that which might be derived from the small patches of aa sequence homology that are frequently found to be held in common among members of protein families. In addition to a number of previously described PTK sequences, a novel mammalian PTK-related sequence was uncovered.
Wilks
A F
AF
Melbourne Tumour Biology Branch, Ludwig Institute for Cancer Research, Parkville, Victoria, Australia.
Kurban
R R
RR
Hovens
C M
CM
Ralph
S J
SJ
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002999:Clone Cells; D003001:Cloning, Molecular; D015723:Gene Library; D015151:Immunoblotting; D051379:Mice; D008969:Molecular Sequence Data; D005810:Multigene Family; D021141:Nucleic Acid Amplification Techniques; D015345:Oligonucleotide Probes; D011061:Poly A / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D011505:Protein-Tyrosine Kinases / Q000235:genetics*; D012313:RNA / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics
D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D011061:Poly A; D012313:RNA; D011505:Protein-Tyrosine Kinases