Cloning and Analysis of
Intron 3 of Human Telomerase Reverse Transcriptase (hTERT) Gene
SAID A.Saleh, SUN Jian-Long, CHEN Zhao, HUANG Jiang-Sheng,
ZHAN Bo-Sheng, REN Da-Ming*
( Institute of Genetics, School of Life Science, State Key Laboratory of
Genetic Engineering, Fudan University, Shanghai 200433, China )
Abstract Total RNA
was extracted from telomerase-positive cell line SPC-A, and reverse transcribed
to cDNA. The long template PCR was performed using this cDNA as template and
the hTERT cDNA specific oligonucleotides as primers. A long fragment
of about 2.2 kb was produced as well as a short fragment of about 150 bp. The
long fragment was purified from the gel, cloned into T-easy vector and
sequenced from two directions. The sequencing result and homologous comparison
indicated that the fragment contained the intron 3 of hTERT gene.
Further assay showed that the precursor of this fragment is premature mRNA
(pre-mRNA) of hTERT gene and the copy number varied among different
cell lines, as verified in this study. These results suggest the feasibility of
cloning the intron of eucaryotic gene by the combination of reverse
transcription and long template PCR system. And the different splicing
efficiency of the intron 3 from the hTERT pre-mRNA was also implied from this
assay.
Key words hTERT； RT-PCR； intron
*Corresponding author： Tel, 86-21-65642506； Fax, 86-21-65648376； e-mail, [email protected]