Genomic Maps and Novel Approaches to Sequencing of Repetitive versus Non-repetitive DNA

• Autorzy: Szybalski W.
• Języki publikacji: EN

Abstrakty

EN

As first demonstrated by Avery et al., DNA is a macromolecule which governs most aspects of life [1]. Thus our role as chemists was to determine the structure of this macromolecule, synthesize it, and possibly relate its structure to the genetic function. The first task was to determine the subunit structure of DNA, namely the structure of bases and their organization in relation to the deoxyribose and phosphate backbone. This was done in decades around the 1950-s. Independently, and around the same time, the concept of genes and the gene maps emerged as to relate the linear structure of DNAto its function. Next came the visualization ofDNAby electron microscopy (EM) and its physical mapping using the heteroduplexes between DNA strands of various mutants. This permitted a precise way of measuring the length of DNA, positioning various deletions or other rearrangements and relating these to the genetic and transcriptional maps. The final step was the precise sequencing of DNA, either by the now abandoned chemical based method or by the presently used enzymatic procedure, which led to progressively more genomes being sequenced. Taken together, all this important scientific milestones led to our present day understanding of the chemical structure and function of DNAin relation to the 'puzzle of life'! However, it was soon realized that the precise entire sequence ofDNAcould not be determined for many genomes, especially the eukaryotic ones, because they contain numerous long stretches of highly repetitive sequences, which defy the present computerized overlap procedure required for aligning of fragments and determining the final sequence. Therefore, we had to develop novel strategies to accurately sequence repetitive elements of DNA, as outlined here. These comprise construction and use of special transposons and pBAC/oriV vectors, both equipped with very rare cutting sites. Transposons (Tn) allow determination of 500-1000 nucleotide (nt) sequences on both sides of their insertion, whereas the very rare cutting sites (like I-SceI, PI-SceI or our Achilles heel cleavage sites) allow precise mapping of the positions of the insertions, using pulsed field gel electrophoresis (PFGE) or other physical means, including electron microscopic (EM) mapping. Thus we had to return to some of our earlier methods of physical mapping, which together with transposon-associated priming would allow sequencing of large eukaryotic genomes to be completed. This would be the final triumph of the structural chemistry of the DNA macromolecules which are the essence of genomes and genomics.

Słowa kluczowe

EN

genomics; DNA sequencing; repetitive DNA

• Wydawca: Polskie Towarzystwo Chemiczne
• Czasopismo: Polish Journal of Chemistry
• Rocznik: 2004
• Tom: Vol. 78 / nr 8
• Strony: 1007--1017
• Opis fizyczny: Bibliogr. 21 poz., rys.

Twórcy

autor

Szybalski W.

• McArdle Laboratory, University of Wisconsin, 1400 University Avenue, Madison, WI 53706-1599, USA

Bibliografia

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