Assembly and cloning of coding sequences for neurotrophic factors directly from genomic DNA using polymerase chain reaction and uracil DNA glycosylase.
The polymerase chain reaction (PCR) was used to amplify individual exons of the gene (CNTF) coding for human ciliary neurotrophic factor (CNTF) directly from genomic DNA. Inclusion of deoxyuracil in place of thymine in the PCR primers permits removal of dU residues in the primer after amplification using uracil DNA glycosylase, generating single-stranded 3' overhangs. Thus, the individual exons were assembled to generate the full-length CNTF sequence. A similar strategy was also used to generate a chimeric gene (BDNF) encoding brain-derived neurotrophic factor (BDNF) with the pre-pro sequence of nerve growth factor (NGF). The method described allows direct amplification of coding sequence from genomic DNA and ordered assembly of amplified exons to generate a clone containing the complete coding sequence of the gene without the need for splicing; a clone which is equivalent to a cDNA clone.
Booth
P M
PM
Department of Cellular Biochemistry, Life Technologies, Inc., BRL, Gaithersburg, MD 20884-9980.
Buchman
G W
GW
Rashtchian
A
A
D000595:Amino Acid Sequence; D020934:Ciliary Neurotrophic Factor; D003001:Cloning, Molecular / Q000379:methods*; D005091:Exons / Q000235:genetics*; D005784:Gene Amplification / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D009414:Nerve Growth Factors / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D009419:Nerve Tissue Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D021141:Nucleic Acid Amplification Techniques; D016133:Polymerase Chain Reaction / Q000379:methods; D011993:Recombinant Fusion Proteins / Q000737:chemistry
D020934:Ciliary Neurotrophic Factor; D009414:Nerve Growth Factors; D009419:Nerve Tissue Proteins; D011993:Recombinant Fusion Proteins