Sequencing clone-based

The primary structure of AspAT from Thermophilic bacillus was determined from cDNA sequence.24 Sequence information of an N-terminal portion of the native enzyme and 19 tryptic peptide fragments, recovered from HPLC, was obtained from gas phase sequencer analyses. Based on such sequence information, four oligonucleotide probes were prepared. cDNA encoding AspAT was cloned by screening restriction enzyme fragments from genomic cDNA of Thermophilic bacillus species YM-2. Amino acid sequence of Thermophilic bacillus AspAT deduced from cDNA was confirmed by the sequences made available by gas phase sequencer analysis. 

The other tool necessary for a successful cloning project is a probe with specificity adequate for the library of interest. The specificity required will vary greatly depending on the complexity of the library. Most often, cloning projects begin with the use of synthetic oligonucleotides whose sequences are based on available protein sequence data. In fact this technique has been employed in... 

The clone-based strategy is currently used to sequence the human ge-... 

The polymerase gene has been cloned, and the complete nucleotide sequence (2694 base, 898 codons) has been determined (35). The 898 codon ORF is terminated by UGAUAG. Codon usage analysis shows that T4 gene 43 has a preference for T or A in the third codon position (72% T + A), presumably reflecting the relative abundance (67%) of T A in T4 DNA. 

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