[Construction and characterization of a cDNA library from human liver tissue of cirrhosis].
To construct a cDNA library from human liver tissue of cirrhosis.



The total RNA from human liver tissue of cirrhosis was extracted using Trizol method, and the mRNA was purified using mRNA purification kit. SMART technique and CDSIII/3' primer were used for first-strand cDNA synthesis. Long distance PCR was then used to synthesize the double-strand cDNA that was then digested by proteinase K and Sfi I, and was fractionated by CHOMA SPIN-400 column. The cDNA fragments longer than 0.4 kb were collected and ligated to lambdaTripl Ex2 vector. Then lambda-phage packaging reaction and library amplification were performed. The qualities of both unamplified and amplified cDNA libraries was strictly checked by conventional titer determination. Eleven plaques were randomly picked and tested using PCR with universal primers derived from the sequence flanking the vector.



The titers of unamplifed and amplified libraries were 1.03 x 10(6) pfu/ml and 1.36 x 10(9) pfu/ml respectively. The percentages of recombinants from both libraries were 97.24 % in unamplified library and 99.02 % in amplified library. The lengths of the inserts were 1.02 kb in average (36.36 % 1 approximately equals 2 kb and 63.64 % 0.5 approximately equals 1.0 kb).



A high quality cDNA library from human liver tissue of cirrhosis was constructed successfully, which can be used for screening and cloning new special genes associated with the occurrence of cirrhosis.
Chen
Xiao-hong
XH
Institute of Infectious Diseases, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310003, China.
Chen
Zhi
Z
Chen
Feng
F
Zhu
Hai-hong
HH
Zhou
Hong-juan
HJ
Yao
Hang-ping
HP
D018076:DNA, Complementary / Q000096:biosynthesis*; D015723:Gene Library; D019694:Hepatitis B, Chronic / Q000150:complications / Q000235:genetics* / Q000821:virology; D006801:Humans; D008103:Liver Cirrhosis / Q000235:genetics* / Q000821:virology*; D016133:Polymerase Chain Reaction; D011995:Recombination, Genetic; D014158:Transcription, Genetic / Q000235:genetics
D018076:DNA, Complementary