Cloning And Bioinformatics Analysis Of Novel Gene Associated With Gastric Carcinoma

Objective: To screen high frequency Loss of Heterozygosity sites associated with gastric carcinoma exist on chromosome 8p21-22, and clone the novel gene which replaced of EST(DA931869), primaly analyze the structure and function of the unknown gene, which provided basis for further identify novel genes associated with the development of gastric carcinoma.Methods: 42 cases of gastric cancer surgery samples were collected as cancer groups, corresponding normal gastric mucosa away from the cancer point as the normal control groups. All normal and cancer samples were pathologically diagnosed. The total RNA of samples was extracted out, with reverse transcription polymerase chain reaction, to screen the expression of EST (DA931869). Then in silico clone the EST which expressed differently between gastric carcinoma and normal gastric mucosa through the tool of BLAST soft ware,joint and extend the cDNA with the software of DNASTAR. It was predicted the ORF, Chromosome Location for the cDNA, and predicted protein homology, protein's secondary structure and functions for the protein which coding by the cDNA. The full lenth of unknown gene which replaced by EST (DA931869) was identified by chromosome walking.Results: The RT-PCR results showed that EST (DA931869) expressed significantly different between gastric carcinoma and normal gastric mucosa by RT-PCR approach (p<0.05). The positive expression rate of EST was 83.33%(35/42)in normal gastric mucosa, and 40.48%(17/42)in gastric carcinoma, reduced or absent expressed compared with in normal.In silico clonging results: The EST was blasted with dbESTdatabase, extended the homology. Firstly we extended 87bp to3'end, and then extended respectively 443bp, 343bp, 421bp and 473bp to5'end. The full lenth of cDNA was 2326bp through extending five times. It's located on chromosome 8p21, accrossed 770Kbp(24,080-24,850Kbp),the genome sequence (NT-167187.1)included the cDNA. The cDNA was blasted with nr database, the result showed that the identity of the unknown gene and neurofilament, medium polypeptide were 55%.The lenth of ORF which consisted of 117aa was 354bp which predicted By online software ORF FINDER.The predicted values of starting codon was 0.78. Bioinformatics tools found that the TATA box at 367bp, the poly(A) signal at 1932bp, and existed a CpG island within 202-587bp, G+C% was 59%.Protein mocular weight was 13016.04 Daltons, PI was 11.70, which analyzed by DNASTAR software. The protein might be basic protein. The secondary structure of protein was predicted thatαhelix1-9,13-18,40-49,βsheet111-117, respectively. Structure domain predicted results showed that 32-43aa was MORN domain. The protein was blasted with protein database of NCBI, the result showed that the protein was similar with Sec16B and SCOP.The results of Chromosome Walking showed that, we have got the 641bp to 3'end of the cDNA through walking two times. Blasting with genome sequences, the results of sequencing were correctly.Conclusion:1. We have obtained the full lenth of cDNA sequence 2326bp represented by the EST(DA931869) through in silico cloning, which consisted of 3 extrons and 2 introns. the lenth of ORF was 354bp. It's precisely located at chromosome 8p21, contains PolyA tails.2. Protein which consisted of 117aa contains MORN domain(32-43aa). The protein was Hydrophilia protein.3. we have got the 641bp to 3'end of the cDNA which represented by the EST(DA931869) through Chromosome Walking techniques.