Construction and characterization of a cDNA library from human liver tissue with chronic hepatitis B / 浙江大学学报(英文版)(B辑:生物医学和生物技术)
Journal of Zhejiang University. Science. B
;
(12): 288-294, 2005.
Artigo
em Inglês
| WPRIM
| ID: wpr-249219
ABSTRACT
<p><b>OBJECTIVE</b>To construct a cDNA library from human liver tissue with chronic hepatitis B and check its quality for investigating the expression level of liver tissue infected by hepatitis B virus. This will then be used to find the relevant genes and interesting proteins associated with the development of hepatitis B.</p><p><b>METHODS</b>The total RNA from liver tissue with chronic hepatitis B was extracted and the mRNA was purified using TRIZOL method. Switching mechanism at 5' end of the RNA transcript (SMART) technique and CDS III/3' primer were used for first-strand cDNA synthesis. Long distance polymerase chain reaction (LD PCR) was then used to synthesize the double-strand cDNA that was then digested by Sfi I and fractionated by CHROMA SPIN-400 column. The longer than 0.4 kb cDNAs were collected and ligated to lambdaTriplEx2 vector. Then lambda phage packaging reaction and library amplification were performed. The qualities of both unamplified and amplified cDNA libraries were strictly checked by conventional titer determination. Fourteen plaques were randomly picked and tested using PCR with universal primers derived from the sequence flanking the vector.</p><p><b>RESULTS</b>The titers of unamplifed and amplified libraries were 1.94 x 10(6) pfu/ml and 1.49 x 10(9) pfu/ml respectively. The percentages of recombinants from both libraries were 98.15% in unamplified library and 98.76% in amplified library. The lengths of the inserts were 1.23 kb in average, 1-2 kb in 64.29%, and 0.5-1.0 kb in 35.71%.</p><p><b>CONCLUSION</b>A high quality cDNA library from human liver tissue with chronic hepatitis B was successfully constructed.</p>
Texto completo:
DisponíveL
Índice:
WPRIM (Pacífico Ocidental)
Assunto principal:
Patologia
/
RNA
/
Biblioteca Gênica
/
Reação em Cadeia da Polimerase
/
DNA Complementar
/
Hepatite B Crônica
/
Genética
/
Fígado
/
Metabolismo
Limite:
Humanos
Idioma:
Inglês
Revista:
Journal of Zhejiang University. Science. B
Ano de publicação:
2005
Tipo de documento:
Artigo
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