Improved Efficiency and Robustness in qPCR and Multiplex End-Point PCR by Twisted Intercalating Nucleic Acid Modified Primers

We introduce quantitative polymerase chain reaction (qPCR) primers and multiplex end-point PCR primers modified by the addition of a single ortho-Twisted Intercalating Nucleic Acid (o-TINA) molecule at the 5′-end. In qPCR, the 5′-o-TINA modified primers allow for a qPCR efficiency of 100% at significantly stressed reaction conditions, increasing the robustness of qPCR assays compared to unmodified primers. In samples spiked with genomic DNA, 5′-o-TINA modified primers improve the robustness by increased sensitivity and specificity compared to unmodified DNA primers. In unspiked samples, replacement of unmodified DNA primers with 5′-o-TINA modified primers permits an increased qPCR stringency. Compared to unmodified DNA primers, this allows for a qPCR efficiency of 100% at lowered primer concentrations and at increased annealing temperatures with unaltered cross-reactivity for primers with single nucleobase mismatches. In a previously published octaplex end-point PCR targeting diarrheagenic Escherichia coli, application of 5′-o-TINA modified primers allows for a further reduction (>45% or approximately one hour) in overall PCR program length, while sustaining the amplification and analytical sensitivity for all targets in crude bacterial lysates. For all crude bacterial lysates, 5′-o-TINA modified primers permit a substantial increase in PCR stringency in terms of lower primer concentrations and higher annealing temperatures for all eight targets. Additionally, crude bacterial lysates spiked with human genomic DNA show lesser formation of non-target amplicons implying increased robustness. Thus, 5′-o-TINA modified primers are advantageous in PCR assays, where one or more primer pairs are required to perform at stressed reaction conditions

Protein-Protein Interactions of Proteins from the ESAT-6 Family of Mycobacterium tuberculosis

In the present study, we demonstrate that, in analogy with the genes encoding ESAT-6 and CFP-10, the genes rv0287 and rv0288 from the ESAT-6 gene family are cotranscribed. Using Western-Western blotting and protein-print overlay methodologies, we demonstrate that ESAT-6 and CFP-10, as well as the protein pair Rv0288/Rv0287, interact pairwise in a highly specific way. Most notably, the ESAT-6 proteins interact directly with Rv3873, a possible cell envelope component of the ESAT-6 secretion pathway

CFP10 discriminates between nonacetylated and acetylated ESAT-6 of Mycobacterium tuberculosis by differential interaction

ESAT-6 (the 6 kDa early secreted antigenic target) protein species in short-term culture filtrate of Mycobacterium tuberculosis were separated in a 4-5 narrow range p/ gradient two-dimensional gel electrophoresis (2-DE). Eight ESAT-6 protein species were analyzed in detail by peptide mass fingerprinting matrix-assisted laser desorption/ionization-mass spectrometry as well as by electrospray ionization-tandem mass spectrometry. An N-terminal Thr acetylation was identified in four species and a C-terminal truncation was identified in two species. In 2-DE blot overlay assays, the recombinant 10 kDa culture filtrate protein (CFP10) discriminated N-terminal acetylated and nonacetylated ESAT-6 by differential interaction, whereas removal of the C-terminal 11 residues of ESAT-6 had no effects thereon. This example shows that the access to the protein species level can be a prerequisite to understand regulation of protein-protein interaction

Comparison of primers in samples spiked with human genomic DNA (gDNA).

<p>(a-c) unmodified DNA primers and (d-f) 5′-<i>o</i>-TINA modified primers. Each ○ on the efficiency curves represents one threshold cycle (Cq) determination on an amplification curve with a corresponding melting curve, reported as the first derivative. Cq determinations highlighted in red would normally have been excluded based on the amplification curve and melting curve evaluation. (a, d) Unspiked samples. (b, e) All samples and negative controls spiked with 10 ng gDNA. (c, f) All samples and negative controls spiked with 100 ng gDNA. A uniform primer concentration of 200 nM was used in all samples and negative controls. The annealing temperatures for unmodified and <i>o-</i>TINA primers were 60.0°C and 66.0°C, respectively.</p

PPE Protein (Rv3873) from DNA Segment RD1 of Mycobacterium tuberculosis: Strong Recognition of Both Specific T-Cell Epitopes and Epitopes Conserved within the PPE Family

Proteins encoded by DNA segment RD1 of Mycobacterium tuberculosis have recently been demonstrated to play important roles in bacterial virulence, vaccine development, and diagnostic reagent design. Previously, we characterized two immunodominant T-cell antigens, the early secreted antigen target (ESAT-6) and the 10-kDa culture filtrate protein (CFP10), which are encoded by the esx-lhp operon in this region. In the present study we characterized a third putative open reading frame in this region, rv3873, which encodes a PPE protein. We found that the rv3873 gene is expressed in M. tuberculosis H37Rv and that the native protein, Rv3873, is predominantly associated with the mycobacterial cell or wall. When tested as a His-tagged recombinant protein, Rv3873 stimulated high levels of gamma interferon secretion in peripheral blood mononuclear cells isolated from tuberculosis (TB) patients, as well as from healthy tuberculin purified protein derivative-positive donors. In contrast to other RD1-encoded antigens, Rv3873 was also found to be recognized by a significant proportion of Mycobacterium bovis BCG-vaccinated donors. Epitope mapping performed with overlapping peptides revealed a broad pattern of T-cell recognition comprising both TB-specific epitopes and epitopes also recognized by BCG-vaccinated donors. The immunodominant epitope (residues 118 to 135) for both TB patients and BCG-vaccinated individuals was found to be highly conserved among a large number of PPE family members

Efficiency curves for unmodified and 5′-<i>o</i>-TINA modified primers.

<p>All experiments were conducted at an annealing temperature (T<i>a</i>) of 66.0°C and primer concentrations (C<i>primers</i>) of unmodified and <i>o</i>-TINA modified primers were compared on the same plate. The melting curves corresponding to the amplification curves used for efficiency curve determination are included for the lowest C<i>primers</i> that allows an efficiency of 100% for unmodified and 5′-<i>o</i>-TINA modified primers.</p

<i>Ortho</i>-TINA (<i>o</i>-TINA) modification attached to the 5′-end of the primer sequence.

<p><i>Ortho</i>-TINA (<i>o</i>-TINA) modification attached to the 5′-end of the primer sequence.</p

Comparison of unmodified and 5′-<i>o</i>-TINA modified primer concentrations on a temperature gradient.

<p>(a) Unmodified DNA primer concentrations from 50 nM (⧫) to 800 nM (▾). (b) 5′-<i>o</i>-TINA modified primer concentrations from 50 nM (<b>◊</b>) to 800 nM (∇). Each threshold cycle (Cq) determination is presented as mean +/− standard deviation (SD) established by triplicate measurements with 1000 copies per well of target. Inter-plate normalization was based on triplicate measurements using 800 nM of unmodified primers and an annealing temperature of 61.0°C (mean Cq for normalization was 25.11 with a SD of 0.1 on each plate).</p