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...

BACKGROUND Despite considerable advances, DNA sequencing has remained somewhat time-consuming and expensive, requiring three separate steps to generate sequencing products from a template: amplification of the target sequence; purification of the amplified product; and a sequencing reaction. Our aim was to develop a method to routinely combine PCR amplification and cycle sequencing into one sin...

We demonstrate that routine PCR product analytical agarose gels can also serve as preparative gels for quick DNA template purification before sequencing. The band of interest is excised, placed into a Gel Nebulizer inside a Micropure separator and rapidly purified in a single centrifugation step. Gel-purified PCR product, suitable for manual and automated sequencing, is delivered within 10 min.

Fluorescence-based capillary DNA sequencing has facilitated the early completion of several complex sequencing projects. While capillary systems offer great benefits in terms of ease of use and automation, we find that they are sufficiently different from slab gel separation methodologies, demanding re-examination of the protocols used to generate and use DNA sequencing templates. We have recen...

The highly thermostable DNA polymerase from Thermus aquaticus (Taq) is ideal for both manual and automated DNA sequencing because it is fast, highly processive, has little or no 3'-exonuclease activity, and is active over a broad range of temperatures. Sequencing protocols are presented that produce readable extension products greater than 1000 bases having uniform band intensities. A combinati...

BACKGROUND Testing for tumor specific mutations on routine formalin-fixed paraffin-embedded (FFPE) tissues may predict response to treatment in Medical Oncology and has already entered diagnostics, with KRAS mutation assessment as a paradigm. The highly sensitive real time PCR (Q-PCR) methods developed for this purpose are usually standardized under optimal template conditions. In routine diagn...

dna methylation is an important biological process involving in human disease such as cancer insomia and diabetes. bisulfite sequencing (bs-seq) with next-generation technology is an accurate method for measuring dna methylation. bs-seq data analysis is a considerable way to recognize methylated cytosines and several tools have been developed to analysis bs-seq such as bs-seeker, b-solana, brat...

Routine blood analysis indicated the presence of Mycoplasma-like bodies in a guigna (Leopardus guigna). Evidence infection with Candidatus Mycoplasma haemominutum was found samples using PCR and DNA sequencing 16S rRNA gene Mycoplasma. spp. are documented cats but their role transmission to populations requires investigation.

Using some recent parasitology case reports involving zoonoses, I will cover aspects such as sample preparation, DNA extraction, primer choice, types of PCR and sequencing methods. also briefly discuss the use GenBank importance phylogenetic trees for diagnostic purposes. The NGS other technologies be reviewed.