Polymerase chain reaction with nearby primers.

DNA analysis of biological specimens containing degraded nucleic acids such as mortal remains, archaeological artefacts, forensic samples etc. has gained more attention in recent years. DNA extracted from these samples is often inapplicable for conventional polymerase chain reaction (PCR), so for its amplification the nearby primers are commonly used. Here we report the data that clarify the features of PCR with nearby and abutting primers. We have shown that the proximity of primers leads to significant reduction of the reaction time and ensures the successful performance of DNA amplification even in the presence of PCR inhibitors. The PCR with abutting primers is usually characterized by the absence of nonspecific amplification products that causes extreme sensitivity with limit of detection on single copy level. The feasibility of PCR with abutting primers was demonstrated on species identification of 100 years old rotten wood.

The influence of quality of primers on the formation of primer dimers in PCR.

Polymerase chain reaction (PCR) is the most commonly used method for nucleic acids amplification. PCR performance depends on several causes, among which the quality of primers is one of the main determinants affecting specificity, sensitivity and reliability of the reaction. Here, we report on the results of the detailed study devoted to the dimerization of the primers during PCR. The course and specificity of the reaction were studied on the model DNA templates as well as genomic DNA using primers that form amplifiable heterodimeric structures with different thermodynamic stability. It was confirmed that more than two 3'-overlapping nucleotides cause a considerable accumulation of primer dimers. It turned out that the presence of any DNA promotes the formation of dimers even for primers, which do not tend to nonspecific amplification in the absence of DNA. It was shown that dimerization could not be eliminated by commonly used techniques. Even the use of hot-start DNA polymerases does not prevent PD formation if primers with stable 3'-overlapping are employed. Despite several advantages of PCR with abutting primers, their close disposition has no benefits regarding the formation of PD if low-quality primers are utilized.

The influence of CpG (5'-d(CpG)-3' dinucleotides) methylation on ultrasonic DNA fragmentation.

DNA methylation is an important way of gene regulation. The variety of methods for DNA methylation analysis based on chemical modification or enzyme digestion has been proposed. However, DNA is able to undergo transformations under physical power. Here, we report that the cytosine methylation in CpG dinucleotides determines the difference in fragmentation rate of methylated and unmethylated DNA under sonication. We found that at the beginning of sonication, methylated DNAs are degraded faster than unmethylated one, and the difference in fragmentation degree can be evaluated with high reliability by quantitative polymerase chain reaction (qPCR). The optimal parameters that provide the greatest difference in amount of amplifiable DNA targets corresponding to fragmentation degree are the following: moderate amplicon size (about 150-250 bp), medium CpG sparseness (one CpG dinucleotide per ∼12-14 nucleotides of the chain), and short sonication time (less than 5 min). Along with CpG, the CpA and CpT contents of amplified regions should be taken into account for proper DNA fragmentation by ultrasound as well. The obtained data could be used for elaboration of a method for comparative methylation testing, when there is no need to detect methylation of certain CpG dinucleotides. This method will be simple (can be used by any technician familiar with PCR), low cost (no need to use an expensive reagents), and fast (only brief DNA sonication and conventional qPCR are carried out). Communicated by Ramaswamy H. Sarma.