Specificity Of Molecular Beacons Research Articles

P07.08 Designing of molecular beacon based polymerase chain reaction method as an unconventional low cost diagnostic assay for sexually transmitted diseases

Introduction About 300 million new infections of gonorrhoea, Chlamydia, or trichomoniasis occur each year. Young adults, population with health inequities and people in resource poor settings bear the significant proportion of sexually transmitted infections (STI). Therefore, rapid, inexpensive and acceptable tests are needed to address STIs in developing countries. PCR based tests are recommended for diagnosis of several STIs, but such tests require sophisticated infrastructure, need to be imported and hence cannot be used in resource limited settings. Affordability can be countered by the development of in house diagnostic assays which are practical and easy to use, and produced within country to further lower the cost. Methods Endocervical swabs were collected from patients visiting gynaecology department of hospitals in Delhi. In-house PCR based assay was developed for C. trachomatis , N. gonorrhoeae and T. vaginalis and modified to visual assay using molecular beacon for end-point detection. Results The molecular beacon based PCR assay was developed for C. trachomatis , N. gonorrhoeae and T. vaginalis and evaluated against different commercial kits viz; Roche AMPLICOR NG/CT kit and qPCR kit of Abbott and fast-track diagnostics. Specificity of molecular beacon was confirmed by competition experiments. Diagnostic tests were more than 95% specific and 99.0% sensitive for all the three pathogens and negative and positive predicted values were around 98.5% and 97.5%, respectively at 95% CI for these three pathogens. We also observed that dry swab samples gave concordant results with that of wet swabs. Assay reagents were stable for more than 6 months at room temperature. We also report, high rates of co-infection for C. trachomatis + N. gonorrhoeae , C. trachomatis + T. vaginalis , N. gonorrhoeae + T. vaginalis amongst women patients in India. Conclusions Development of a rapid, sensitive, specific and PCR based visual diagnostic assay, suitable for developing countries will provide a better disease management.

Molecular Beacon Based DNA Computing Model for General Satisfiability Problem

Molecular Beacon is a hairpin-shaped fluorescent probe,which can hybridize with great specificity target sequence that is complement to its loop sequence.The specificity of Molecular Beacon is as high as single base mismatch detection.Peptide Nucleic Acid(PNA)is an artificial synthesized analogue of nature occurring DNA,in which the DNA sugar-phosphate backbone has been replaced by a pseudo-peptide.Thus the hybridization of PNA to complement DNA strand is more specific and stable than that of DNA to DNA.PNA can stop polymerase extension reaction as well.In this paper,Molecular Beacons were employed to encode variables in satisfiability problem and complement PNA strands were added and allowed to hybridize with Molecular Beacon.The hybridized PNA strands on Molecular Beacon stopped polymerase extension reaction,causing Molecular Beacon corresponding to non-solution were digested by means of restriction endonuclease EcoRI.The remaining Molecular Beacons encoding solutions were read out via heating.The appealing characteristics of proposed method in this paper are:Reliable,no observation and record of midst solution,easy solution detection.

Effects of the 2′,4′-BNA modification on the sequence specificity of molecular beacons

Molecular beacons (MBs) are stem-loop hairpin oligonucleotide probes with an internally quenched fluorophore. These probes recognize their targets with higher specificity than conventional linear probes. To further enhance sequence-specificity of MBs, we have designed and synthesized MBs having 2',4'-BNA modification. Thermodynamic analysis revealed that the MBs with the 2',4'-BNA-modified stem region have high sequence-specificity. In addition, the 2',4'-BNA modification in the loop region was also found to be efficient for discrimination of one base mismatch.

Collection of trace amounts of DNA/mRNA molecules using genomagnetic nanocapturers.

The collection and then the separation of rare DNA/mRNA targets with single-base mismatches in a complex matrix is critically important in human disease diagnostics, gene expression studies, and gene profiling. The major result of this work is the development and application of a novel genomagnetic nanocapturer (GMNC) for the collection, separation, and detection of trace amounts of DNA/RNA molecules with one single-base difference. The GMNC is constructed by bioconjugating molecular beacon DNA probes onto magnetic nanoparticle surfaces. We have successfully applied the GMNC in artificial buffer solution samples and in cancer cell samples, both containing different proteins and random DNA sequences. Our method has three distinctly useful features: highly efficient collection of trace amount of DNA/mRNA samples down to femtomolar (10(-15) M) concentrations; excellent ability to differentiate single-base-mismatched DNA/mRNA samples by combining the exceptional specificity of molecular beacons and the separation power of magnetic nanoparticles; and real-time monitoring and confirmation of the collected gene products. The newly developed genomagnetic nanocapturers will be highly useful for the collection of trace amounts of DNA/mRNA targets in a variety of sample sources in forensic, medical, and biotechnological fields.