[Cloning of an expressed sequence tag with restriction display polymerase chain reaction].
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.


CONCLUSIONS
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.
Zhang
Bao
B
Institute of Molecular Biology, First Military Medical University, Guangzhou 510515, China.
Ma
Wen-Li
WL
Liu
Li-Yang
LY
Song
Yan-Bin
YB
Zheng
Wen-Ling
WL
D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000032:analysis / Q000235:genetics; D030561:Databases, Nucleic Acid; D020224:Expressed Sequence Tags; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*
D018076:DNA, Complementary