Abstract
Modular primers are strings of three contiguously annealed unligated oligonucleotides (modules) as short as 5- or 6-mers, selected from a presynthesized library. It was previously found that such strings can prime DNA sequencing reactions specifically, thus eliminating the need for the primer synthesis step in DNA sequencing by primer walking. It has remained largely a mystery why modular primers prime uniquely, while a single module, used alone in the same conditions, often shows alternative priming of comparable strength. In a puzzling way, the single module, even in a large excess over the template, no longer primes at the alternative sites, when modules with which it can form a contiguous string are also present. Here we describe experiments indicating that this phenomenon cannot be explained by cooperative annealing of the modules to the template. Instead, the mechanism seems to involve competition between different primers for the available polymerase. In this competition, the polymerase is preferentially engaged by longer primers, whether modular or conventional, at the expense of shorter primers, even though the latter can otherwise prime with similar or occasionally higher efficiency.
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- Blöcker H., Lincoln D. N. The 'shortmer' approach to nucleic acid sequence analysis. I: Computer simulation of sequencing projects to find economical primer sets. Comput Appl Biosci. 1994 Apr;10(2):193–197. doi: 10.1093/bioinformatics/10.2.193. [DOI] [PubMed] [Google Scholar]
- Huber H. E., Tabor S., Richardson C. C. Escherichia coli thioredoxin stabilizes complexes of bacteriophage T7 DNA polymerase and primed templates. J Biol Chem. 1987 Nov 25;262(33):16224–16232. [PubMed] [Google Scholar]
- Kaczorowski T., Szybalski W. Assembly of 18-nucleotide primers by ligation of three hexamers: sequencing of large genomes by primer walking. Anal Biochem. 1994 Aug 15;221(1):127–135. doi: 10.1006/abio.1994.1388. [DOI] [PubMed] [Google Scholar]
- Kieleczawa J., Dunn J. J., Studier F. W. DNA sequencing by primer walking with strings of contiguous hexamers. Science. 1992 Dec 11;258(5089):1787–1791. doi: 10.1126/science.1465615. [DOI] [PubMed] [Google Scholar]
- Kotler L. E., Zevin-Sonkin D., Sobolev I. A., Beskin A. D., Ulanovsky L. E. DNA sequencing: modular primers assembled from a library of hexamers or pentamers. Proc Natl Acad Sci U S A. 1993 May 1;90(9):4241–4245. doi: 10.1073/pnas.90.9.4241. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kotler L., Sobolev I., Ulanovsky L. DNA sequencing: modular primers for automated walking. Biotechniques. 1994 Sep;17(3):554–559. [PubMed] [Google Scholar]
- McCombie W. R., Kieleczawa J. Automated DNA sequencing using 4-color fluorescent detection of reactions primed with hexamer strings. Biotechniques. 1994 Sep;17(3):574–579. [PubMed] [Google Scholar]
- Szybalski W. Proposal for sequencing DNA using ligation of hexamers to generate sequential elongation primers (SPEL-6). Gene. 1990 May 31;90(1):177–178. doi: 10.1016/0378-1119(90)90458-4. [DOI] [PubMed] [Google Scholar]
- Zevin-Sonkin D., Ilan E., Guthmann D., Riss J., Theodor L., Shoham J. Molecular cloning of the bovine thymopoietin gene and its expression in different calf tissues: evidence for a predominant expression in thymocytes. Immunol Lett. 1992 Feb 15;31(3):301–309. doi: 10.1016/0165-2478(92)90131-7. [DOI] [PubMed] [Google Scholar]