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      Large scale cDNA sequencing for analysis of quantitative and qualitative aspects of gene expression.

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      Journal: Nature genetics
      Keywords: Base Sequence, Cloning, Molecular, DNA, Complementary, genetics, Gene Expression, Gene Frequency, Gene Library, Genome, Human, Humans, Liver, chemistry, cytology, Molecular Sequence Data, Proteins, RNA, Messenger, analysis, Sequence Alignment, Sequence Analysis, DNA, Tumor Cells, Cultured

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          Abstract

          Large scale sequencing of cDNAs provides a complementary approach to structural analysis of the human genome by generating expressed sequence tags (ESTs). We have initiated the large-scale sequencing of a 3'-directed cDNA library from the human liver cell line HepG2, that is a non-biased representation of the mRNA population. 982 random cDNA clones were sequenced yielding more than 270 kilobases. A significant portion of the identified genes encoded secretable proteins and components for protein-synthesis. The abundance of cDNA species varied from 2.2% to less than 0.004%. Fifty two percent of the mRNA were abundant species consisting of 173 genes and the rest were non-abundant, consisting of about 6,600 genes.

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          Improved tools for biological sequence comparison.

          W R Pearson, D J Lipman (1988)
          We have developed three computer programs for comparisons of protein and DNA sequences. They can be used to search sequence data bases, evaluate similarity scores, and identify periodic structures based on local sequence similarity. The FASTA program is a more sensitive derivative of the FASTP program, which can be used to search protein or DNA sequence data bases and can compare a protein sequence to a DNA sequence data base by translating the DNA data base as it is searched. FASTA includes an additional step in the calculation of the initial pairwise similarity score that allows multiple regions of similarity to be joined to increase the score of related sequences. The RDF2 program can be used to evaluate the significance of similarity scores using a shuffling method that preserves local sequence composition. The LFASTA program can display all the regions of local similarity between two sequences with scores greater than a threshold, using the same scoring parameters and a similar alignment algorithm; these local similarities can be displayed as a "graphic matrix" plot or as individual alignments. In addition, these programs have been generalized to allow comparison of DNA or protein sequences based on a variety of alternative scoring matrices.
          Article 0 comments Cited 855 times – based on 0 reviews     Review now
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            Complementary DNA sequencing: expressed sequence tags and human genome project

            Rachelle M. M. Adams, Brendan Kelley, J Gocayne … (1991)
            Automated partial DNA sequencing was conducted on more than 600 randomly selected human brain complementary DNA (cDNA) clones to generate expressed sequence tags (ESTs). ESTs have applications in the discovery of new human genes, mapping of the human genome, and identification of coding regions in genomic sequences. Of the sequences generated, 337 represent new genes, including 48 with significant similarity to genes from other organisms, such as a yeast RNA polymerase II subunit; Drosophila kinesin, Notch, and Enhancer of split; and a murine tyrosine kinase receptor. Forty-six ESTs were mapped to chromosomes after amplification by the polymerase chain reaction. This fast approach to cDNA characterization will facilitate the tagging of most human genes in a few years at a fraction of the cost of complete genomic sequencing, provide new genetic markers, and serve as a resource in diverse biological research fields.
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              Construction of improved M13 vectors using oligodeoxynucleotide-directed mutagenesis.

              T Kempe, J Messing, J M Norrander (1983)
              The restriction endonuclease cleavage sites for SphI and KpnI have been added to the lac cloning region of the phage vectors M13mp10 and M13mp11, using oligodeoxynucleotide-directed in vitro mutagenesis. Complementary deoxy 16-, 21- or 18-mers with the desired base changes were annealed to the M13mp DNA strand and extended with the Klenow fragment of DNA polymerase I. In adding these sites we have shown that this technique can be used as a general method for inserting sequences of DNA as well as introducing deletions and base pair changes.
              Article 0 comments Cited 152 times – based on 0 reviews     Review now
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                Author and article information

                Journal
                PubMed ID:: 1345164
                DOI:: 10.1038/ng1192-173

                ScienceOpen disciplines: Chemistry
                Keywords: Base Sequence,Cloning, Molecular,DNA, Complementary,genetics,Gene Expression,Gene Frequency,Gene Library,Genome, Human,Humans,Liver,chemistry,cytology,Molecular Sequence Data,Proteins,RNA, Messenger,analysis,Sequence Alignment,Sequence Analysis, DNA,Tumor Cells, Cultured
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                ScienceOpen disciplines: Chemistry
                Keywords: Base Sequence, Cloning, Molecular, DNA, Complementary, genetics, Gene Expression, Gene Frequency, Gene Library, Genome, Human, Humans, Liver, chemistry, cytology, Molecular Sequence Data, Proteins, RNA, Messenger, analysis, Sequence Alignment, Sequence Analysis, DNA, Tumor Cells, Cultured

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