The purpose of this study was to develop a real time polymerase chain reaction (PCR) assay for the detection of the JAK2 V617F mutation that could be used in diagnostic laboratories. Sanger sequencing and a newly developed locked nucleic-acid, real-time PCR assay were used to detect the JAK2 V617F mutation. There was 100% agreement between the sequencing and PCR analysis. Both assays were able ...

A broad-range bacterial PCR target to conserved regions of the 23S rDNA was applied to 306 blood culture samples from 295 infants (up to one year of age) admitted to a neonatal intensive care unit. Classic blood culture results were compared to DNA sequencing analysis of the PCR amplification products. Culture results were in agreement to DNA sequencing in 90.5% (277) of 306 samples tested, inc...

Integration of viral vectors into a host genome is associated with insertional mutagenesis and subjects in clinical gene therapy trials must be monitored for this adverse event. Several PCR based methods such as ligase-mediated (LM) PCR, linear-amplification-mediated (LAM) PCR and non-restrictive (nr) LAM PCR were developed to identify sites of vector integration. Coupling the power of next-gen...

Germline mutations in the TP53 gene are associated with Li-Fraumeni and Li-Fraumeni-Like Syndromes, characterized by increased predisposition to early-onset cancers. In Brazil, the prevalence of the TP53-p.R337H germline mutation is exceedingly high in the general population and in cancer-affected patients, probably as result of a founder effect. Several genotyping methods are used for the mole...

Whole transcriptome RNA-sequencing is a powerful tool, but is costly and yields complex data sets that limit its utility in molecular diagnostic testing. A targeted quantitative RNA-sequencing method that is reproducible and reduces the number of sequencing reads required to measure transcripts over the full range of expression would be better suited to diagnostic testing. Toward this goal, we ...

Direct sequencing of polymerase chain reaction (PCR) products is a very powerful technique, especially for clinical investigations where quantities of biological material are often very limited and results are rapidly needed. However, numerous researchers still prefer to sequence cloned PCR fragments, a very time-consuming strategy, as most published protocols for direct sequencing of PCR produ...

BACKGROUND Multiplexed detection of low-level mutations presents a technical challenge for many technologies, including cancer gene panels used for targeted-resequencing. Analysis of mutations below approximately 2%-5% abundance in tumors with heterogeneity, samples with stromal contamination, or biofluids is problematic owing to increased noise from sequencing errors. Technologies that reduce ...

Next-generation sequencing (NGS) technologies have enabled genome re-sequencing for exploring genome-wide polymorphisms among individuals, as well as targeted re-sequencing for the rapid and simultaneous detection of polymorphisms in genes associated with various biological functions. Therefore, a simple and robust method for targeted re-sequencing should facilitate genotyping in a wide range o...

Next-generation sequencing (NGS) has revolutionized the genetic landscape. It is a lengthy, labor-intensive process that yields results never before achieved. As a result, it is imperative that the quality of the DNA sample be evaluated from the start, as most NGS sample preparation protocols require PCR amplification to generate DNA libraries prior to sequencing. The likelihood of artifact gen...

Studies on the value of culture-independent molecular identification of bacteria in cardiac valves are mostly restricted to comparing agreement of identification to what is obtained by culture to the number of identified bacteria in culture-negative cases. However, evaluation of the usefulness of direct molecular identification should also address weaknesses, their relevance in the given settin...