Cloning Of Four FBN1 Gene And Its Auxiliary Effect In Marfan Syndrome Diagnosis

Aims: To develop a DNA-recombination independent cloning method; 2) To clone FBN1 genetic fragments of several types of birds and fishes; and 3). To analyze the evolutionary conservation of the amino acid 1170 in order to determine the possibility of the causal relationship of a rare Rl 170H identified in an atypic Marfan syndrome.Method: Degenerated primers targeting the conserved amino acids flanking the R1170H relevant point mutation were used to clone the DNA fragments of FBN1 of chicken, turkey, carp, rainbow trout, and blackbull head. The amino acid sequences of the cloned DNA fragments of the newly cloned FBN1 were deduced and aligned using the program of Alignx.Results: An inverse nested PCR was developed and successfully applied in the cloning of 4 pieces of new FBN1 genes of turkey, carp, rainbow trout, and blackbull head. These four newly cloned sequences were submitted to NCBI with the accession numbers of DQ314744, DQ314745, DQ314746, and DQ314747 issued. The inverse nested PCR was also used to confirm the FBN1 sequence of chicken. The amino acids at the location of 1170 were histamine (H) for chicken and turkey, glutamine (Q) for the rest three species. The 1170 amino acid is not a highly conserved during evolution.Conclusion: 1). Developed and confirmed the application of using inverse nested PCR for cloning of genes with similar or conserved amino acid sequences within or between gene families during evolution; 2). Cloned 4 new FBN1 fragments from 4 species and submitted to genbank with access numbers issued; 3). The rare mutation of R1170H is not highly conserved during evolution and may not be the cause responsible for the atypic Marfan syndrome recently identified.