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Construction and partial characterization of a human liver cDNA library
Biomedica Biochimica Acta
|January 1, 1985
Summary
Researchers created a human liver complementary DNA (cDNA) library for gene discovery. The library showed instability after multiple bacterial cultures, but key gene sequences were successfully isolated.
Area of Science:
- Molecular Biology
- Genetics
Background:
- Messenger RNA (mRNA) isolation from adult human liver is crucial for gene expression studies.
- Complementary DNA (cDNA) libraries serve as valuable resources for identifying and analyzing genes.
Purpose of the Study:
- To construct a stable human liver cDNA library for gene discovery.
- To assess the stability of the constructed cDNA library during bacterial culture.
- To isolate specific human genes, including mitochondrial ribosomal RNA (rRNA) and alpha-1 antitrypsin.
Main Methods:
- Total mRNA was isolated from adult human liver and reverse transcribed into cDNA.
- Double-stranded cDNA was cloned into the pAT153 plasmid using GC tailing.
- Recombinant clones were screened using a heterologous rat albumin cDNA and specific oligonucleotides.
Main Results:
- Approximately 2000 recombinant clones were initially isolated.
- The cDNA library composition significantly changed after three consecutive overnight bacterial cultures, indicating instability.
- Clones containing human mitochondrial rRNA and alpha-1 antitrypsin gene sequences were successfully identified and isolated.
Conclusions:
- The study successfully constructed a human liver cDNA library.
- Bacterial culture conditions can impact the stability and composition of cDNA libraries.
- The developed library is suitable for isolating specific human gene sequences of interest.