Cloning and Analysis of Intron 3 of Human Telomerase Reverse Transcriptase (hTERT) Gene.
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.
Said
A. Saleh
AS
Institute of Genetics, School of Life Science, State Key Laboratory of Genetic Engineering, Fudan University, Shanghai 200433, China. dmren@sina.com
Sun
Jian-Long
JL
Chen
Zhao
Z
Huang
Jiang-Sheng
JS
Zhan
Bo-Sheng
BS
Ren
Da-Ming
DM