We describe doublex sequencing of human genomic PCR products using two differently labeled primers in a single reaction and analysis on two automated DNA sequencing devices. Feasibility of the methodology is demonstrated by isothermal and cycle sequencing for two different PCR products and by cycle sequencing on both strands of a single product. It was applied to analyze mutations in patient DN...

The polymerase chain reaction (PCR) and DNA sequence determination (4,5) represent two of the most routine and reliable approaches to determine genic sequence variation. Several automated and manual sequencing kits are commercially available, and they provide adequate and satisfactory results. Compressions caused by formation of secondary structure, however, remain particularly problematic when...

Tumor specimens are often preserved as formalin-fixed paraffin-embedded (FFPE) tissue blocks, the most common clinical source for DNA sequencing. Herein, we evaluated the effect of pre-sequencing parameters to guide proper sample selection for targeted gene sequencing. Data from 113 FFPE lung tumor specimens were collected, and targeted gene sequencing was performed. Libraries were constructed ...

Oropharyngeal Cancer Patients Sarah Clark Biochemistry, Biology, Chemistry Background: Circulating tumor DNA (ctDNA) can be distinguished from other cell-free DNA in the body due to its unique mutations. Next generation sequencing and digital PCR have allowed detection of ctDNA to become a more commonly used tool, and because it can be detected directly out of blood, is called the liquid biopsy...

We propose a genome sequencing strategy, which is neither divide-and-conquer (clone by clone) nor the shotgun approach. Random PCR-based and PCR relay sequencing constitute the basis of this novel strategy. Most of the genome is sequenced by the former process that requires only a set of non-specific primers and a template DNA. Random PCR-based sequencing reduces redundancy in sequencing by exp...

In a validation trial of a DNA microarray test for chlamydiae we repeatedly observed false-positive PCR amplicons from truly negative samples and non-template controls. Various PCR tests, microarray hybridization and DNA sequencing, revealed that residual Escherichia coli DNA from thermostable DNA polymerases was the cause of this cross-reaction. A subsequent survey showed that only five out of...

A method is described to simultaneously amplify and sequence DNA using a new class of nucleotides containing boron. During the polymerase chain reaction, boron-modified nucleotides, i.e. 2'-deoxynucleoside 5'-alpha-[P-borano]-triphosphates, are incorporated into the product DNA. The boranophosphate linkages are resistant to nucleases and thus the positions of the boranophosphates can be reveale...

Background The prevalence of infertility is about 10-15% among the couples overally. Several factors can affect fertility ability of men and women. Chlamydia is a non-motile gram negative obligatory interacellular pathogenic organism. It can cause infections in females as cervicitis, urethritis, endometritis, pelvic inflammatory disease also prostatitis and epidydidimitis in male as well. The a...

Nextgen sequencing technologies have enabled noninvasive prenatal testing of fetal aneuploidies by shotgun sequencing of maternal plasma DNA during pregnancy (1). A statistically significant change (e.g., using z-score statistics) in the number of reads mapped to a chromosome of interest (such as chromosome 21) is used to identify fetal aneuploidy. The accuracy of the test is largely dependent ...

The Scottish Meningococcus and Pneumococcus Reference Laboratory provides a national service for the laboratory confirmation of meningococcal and pneumococcal disease in Scotland. The main tests used for the laboratory confirmation of meningococcal disease are culture, the polymerase chain reaction (PCR), antibody testing, and more recently DNA sequencing. This paper describes the automation of...