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[Cloning of an expressed sequence tag with restriction display polymerase chain reaction]
Bao Zhang1, Wen-Li Ma, Li-Yang Liu
1Institute of Molecular Biology, First Military Medical University, Guangzhou 510515, China.
Objective:
To isolate gene fragments from SH-SY5Y cells by way of restriction display polymerase chain reaction (RD-PCR).
Methods:
Total mRNA was extracted from SH-SY5Y cells followed by synthesis of the single-strand cDNA with Oligo (dT18) as the anchored primer, and the second strand was synthesized by nick translation. The double strands were cleft with restriction enzyme Sau3A I and the fragments ligated with a universal adapter to be amplified with the universal primers and selected primers. The products were then ligated into the pMD18-T vector and sequenced.
Results:
One of the sequenced clones was retrieved in the National Center for Biotechnology Information (NCBI) databases with Blast program. The results showed that the sequence possessed great similarity to one fragment of the 17th chromosome in the genome. Sequence analysis with GenScan software indicated that the EST might be one section of an unknown gene.
Conclusion:
RD-PCR provides simple and efficient approach for isolating EST from cells, and cDNA clone sequencing combined with bioinformatics analysis may be helpful in identifying new genes.