Translocation of Rodlike Polymers through Membrane Channels

A theory of channel-facilitated transport of long rodlike macromolecules through thin membranes under the influence of a driving force of arbitrary strength is developed. Analytic expressions are derived for the translocation probability and the Laplace transform of the probability density of time that a macromolecule spends in the channel. We also derive expressions for the (conditional) probability densities of time spent in the channel by translocating and nontranslocating (returning back) macromolecules. These results are used to study how the distribution of the macromolecule lifetime in the channel depends on a polymer chain length and the driving force. It is shown that depending on the values of the parameters, the lifetime probability density may have one or two peaks. Our theory is a generalization of the theory developed by Lubensky and Nelson, who were inspired by recent experiments on driven translocation of single-stranded RNA and DNA molecules through single channels in narrow membranes.     

Polymerase chain reaction: a Markov process approach

A probabilistic approach to the kinetics of the polymerase chain reaction (PCR) is developed. The approach treats the primer extension step of PCR as a microscopic Markov process in which the molecules of deoxy-nucleoside triphosphate (dNTP) are bound to the 3' end of the primer strand one at a time. The binding probability rates are prescribed by combinatorial rules in accord with the microscopic chemical kinetics. As an example, a simple model based on this approach is proposed and analysed, and an exact solution for the probability distribution of lengths of synthesized DNA strands is found by analytical means. Using this solution, it is demonstrated that the model is able to reproduce the main features of PCR, such as extreme sensitivity to the variation of control parameters and the existence of an amplification plateau. A multidimensional optimization technique is used to find numerically the optimum values of control parameters which maximize the yield of the target sequence for a given PCR run while minimizing the overall run time.     

DNA primase-DNA polymerase alpha from simian cells: sequence specificity of initiation sites on simian virus 40 DNA.

Unique single-stranded regions of simian virus 40 DNA, phage M13 virion DNA, and several homopolymers were used as templates for the synthesis of (p)ppRNA-DNA chains by CV-1 cell DNA primase-DNA polymerase alpha. Intact RNA primers, specifically labeled with an RNA capping enzyme, were typically 6 to 8 ribonucleotides long, although their lengths ranged from 1 to 9 bases. The fraction of intact RNA primers 1 to 4 ribonucleotides long was 14 to 73%, depending on the template used. RNA primer length varied among primers initiated at the same nucleotide, as well as with primers initiated at different sites. Thus, the size of an RNA primer depended on template sequence. Initiation sites were identified by mapping 5' ends of nascent RNA-DNA chains on the template sequence, identifying the 5'-terminal ribonucleotide, and partially sequencing one RNA primer. A total of 56 initiation events were identified on simian virus 40 DNA, an average of 1 every 16 bases. Some sites were preferred over others. A consensus sequence for initiation sites consisted of either 3'-dCTTT or 3'-dCCC centered within 7 to 25 pyrimidine-rich residues; the 5' ends of RNA primers were complementary to the dT or dC. High ATP/GTP ratios promoted initiation of RNA primer synthesis at 3'-dCTTT sites, whereas low ATP/GTP ratios promoted initiation at 3'-dCCC sites. Similarly, polydeoxythymidylic acid and polydeoxycytidylic acid were the only effective homopolymer templates. Thus, both template sequence and ribonucleoside triphosphate concentrations determine which initiation sites are used by DNA primase-DNA polymerase alpha. Remarkably, initiation sites selected in vitro were strikingly different from initiation sites selected during simian virus 40 DNA replication in vivo.