[Detection of cDNA end sequence of porcine fibrinogen like protein2 and it's structure analysis].
The purpose of this study is to detect the end sequence of porcine FGL2 gene cDNA and make a preliminary analysis of it's structure. Porcine DNA library was screened by a cDNA probe labeled with radioactive isotope alpha-32P dCTP. Rapid amplification of cDNA end (RACE): Retroverse transcription product of total RNA extracted from normal porcine tissue was used as the template, gene specific primers were designed and advantage 2 polymerase mix was used in PCR, of which using porcine genomic DNA as the template:forward primer was designed according to the acquired consensus region of human and pig FGL2 3' sequences while reverse primer was designed from human FGL2 3' end downstream sequence; TA cloning. Screening library failed to get any specific positive clone; the specific transcription initiation site and first poly A signal were successfully detected by RACE reaction although it fails again to detect the second poly A signal. The unknown sequence of porcine FGL2 3' end including the second poly A signal was successfully detected by PCR using genomic DNA as the template. RACE reaction can be applied as an effective method to detect the specific transcription initiation and termination sites. Using advantage 2 polymerase mix instead of regular DNA polymerase may significantly improve the sensitivity,accuracy and specificity of PCR reaction. If it met particular difficulties in the regular screening of DNA library, PCR reaction utilizing primers designed from the known consensus sequence and genomic DNA as template may be considered as an appropriate alternative.
Liu
Hao
H
Organ Transplant Unit, First of Filiated Hospital, China Medical Univeristy, Shenyang China,110001. liuhaodoctor@hotmail.com
Ghanekar
Anand
A
Chan
Matthew
M
Liu
Ming Feng
MF
Liu
Yong-Feng
YF
Levy
Gary
G