Technique / Molecular Biology / PCR / High fidelity long PCR

In the initial report, introducing a single phosphorothioate modification at the very 3' terminus of the oligodeoxynucleotide primer has been shown to effectively protect the oligodeoxynucleotide degradation due to the 3' exonuclease activity. In this study, we reported a novel finding that phosphorothioate modification at the 3' end of primers could not only effectively prevent the primer from degradation, but could also mediate an off-switch extension by Pfu polymerase when primers also carry single or multiple mismatched bases located in the first eight bases of the 3' terminus. This suggests that the combination of 3' phosphorothioate-modified primers with exo+ polymerases such as Pfu constituted an on/off switch, which allows perfectly matched primers to be extended but not mismatched primers. Furthermore, we found that polymerases with different fidelities showed different efficiencies in turning off mismatched-primer mediated extension. So we described here a SYBR green-based real-time quantitative PCR assay for the detection of abundance level of gene expression that did not require fluorescently labeled gene-specific probes or complicated primer combinations. The emergence of real-time quantitative RT-PCR technology is thus suited for a diverse application with a need for high-throughput methods to detect and quantify different gene expressions by way of simplicity, versatility, and accuracy, and thus could complement global microarray-based expression profiling strategies.

Amplifying microbial DNA by the polymerase chain reaction (PCR) from single phytoseiid mites has been difficult, perhaps due to the low titer of bacteria and to interference by the relatively larger amounts of mite genomic DNA. In this paper we evaluate the efficiency of standard and high-fidelity PCR protocols subsequent to amplification of the whole genome by a multiple displacement amplification (MDA) procedure developed by Dean et al. DNA from the phytoseiid Phytoseiulus persimilis (Athias-Henriot) was tested because it lacks a Cytophaga-like organism (CLO) and we could add known amounts of a plasmid containing a cloned 16S rRNA gene fragment from a CLO from Metaseiulus occidentalis (Nesbitt). P. persimilis genomic DNA was mixed with the serially diluted plasmid and amplified using MDA followed by either standard or high-fidelity PCR. MDA followed by high-fidelity PCR was most efficient and successfully amplified an expected 1.5-kb band from as little as 0.01fg of the plasmid, which is equivalent to about 1 copy. MDA followed by high-fidelity PCR also consistently amplified Wolbachia- or CLO-specific products from naturally infected single females or eggs of M. occidentalis, which will allow detailed studies of infection frequency and transmission of several microorganisms associated with this predatory mite.

We introduce the TA cloning antibody method for the high-fidelity PCR product amplified by family B DNA polymerase without purification. This method uses antibodies and Thermus aquaticus (Taq) DNA polymerase. The antibodies can inhibit only the activity of family B DNA polymerase, and Taq can co-work for A-tailing. This method has nearly cloning efficiency to that of the PCR product of Taq.