On the mechanism of the modular primer effect.
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.
Beskin
A D
AD
Department of Structural Biology, Weizmann Institute of Science, Rehovot, Israel.
Zevin-Sonkin
D
D
Sobolev
I A
IA
Ulanovsky
L E
LE
D001483:Base Sequence; D001667:Binding, Competitive; D017931:DNA Primers / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004279:DNA, Viral / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D008969:Molecular Sequence Data; D011089:Polydeoxyribonucleotides / Q000378:metabolism; D017422:Sequence Analysis, DNA / Q000379:methods; D013698:Templates, Genetic
D017931:DNA Primers; D004277:DNA, Single-Stranded; D004279:DNA, Viral; D011089:Polydeoxyribonucleotides; C034970:bacteriophage T7 induced DNA polymerase; D004259:DNA-Directed DNA Polymerase