The History of Pyrosequencing(®).

One late afternoon in the beginning of January 1986, bicycling from the lab over the hill to the small village of Fulbourn, the idea for an alternative DNA sequencing technique came to my mind. The basic concept was to follow the activity of DNA polymerase during nucleotide incorporation into a DNA strand by analyzing the pyrophosphate released during the process. Today, the technique is used in multidisciplinary fields in academic, clinical, and industrial settings all over the word.

Collinearity of protease mutations in HIV-1 samples with high-level protease inhibitor class resistance.

Objectives: To determine whether pan-protease inhibitor (PI)-resistant virus populations are composed predominantly of viruses with resistance to all PIs or of diverse virus populations with resistance to different subsets of PIs.

Methods: We performed deep sequencing of plasma virus samples from nine patients with high-level genotypic and/or phenotypic resistance to all licensed PIs. The nine virus samples had a median of 12 PI resistance mutations by direct PCR Sanger sequencing.

Whole-genome sequencing of the efficient industrial fuel-ethanol fermentative Saccharomyces cerevisiae strain CAT-1.

The Saccharomyces cerevisiae strains widely used for industrial fuel-ethanol production have been developed by selection, but their underlying beneficial genetic polymorphisms remain unknown. Here, we report the draft whole-genome sequence of the S. cerevisiae strain CAT-1, which is a dominant fuel-ethanol fermentative strain from the sugarcane industry in Brazil.

Improvement in cell capture throughput using parallel bioactivated microfluidic channels.

Optimization of targeted cell capture with microfluidic devices continues to be a challenge. On the one hand, microfluidics allow working with microliter volumes of liquids, whereas various applications in the real world require detection of target analyte in large volumes, such as capture of rare cell types in several ml of blood. This contrast of volumes (microliter vs.

Comparative analysis of human gut microbiota by barcoded pyrosequencing.

Humans host complex microbial communities believed to contribute to health maintenance and, when in imbalance, to the development of diseases. Determining the microbial composition in patients and healthy controls may thus provide novel therapeutic targets. For this purpose, high-throughput, cost-effective methods for microbiota characterization are needed.

Human papillomavirus (HPV), DNA aberrations and cell cycle progression in anal squamous cell carcinoma patients.

Unlabelled: Human papillomavirus (HPV) infections of the genital tract are sexually transmitted and prevalent worldwide. In this study, the role of HPV in 72 patients with anal squamous cell carcinoma was investigated.

Patients And Methods: Polymerase chain reaction (PCR) in combination with in situ hybridization was used to identify HPV-DNA in the patients' biopsies.

Connector inversion probe technology: a powerful one-primer multiplex DNA amplification system for numerous scientific applications.

We combined components of a previous assay referred to as Molecular Inversion Probe (MIP) with a complete gap filling strategy, creating a versatile powerful one-primer multiplex amplification system. As a proof-of-concept, this novel method, which employs a Connector Inversion Probe (CIPer), was tested as a genetic tool for pathogen diagnosis, typing, and antibiotic resistance screening with two distinct systems: i) a conserved sequence primer system for genotyping Human Papillomavirus (HPV), a cancer-associated viral agent and ii) screening for antibiotic resistance mutations in the bacterial pathogen Neisseria gonorrhoeae. We also discuss future applications and advances of the CIPer technology such as integration with digital amplification and next-generation sequencing methods.

PathogenMip assay: a multiplex pathogen detection assay.

The Molecular Inversion Probe (MIP) assay has been previously applied to a large-scale human SNP detection. Here we describe the PathogenMip Assay, a complete protocol for probe production and applied approaches to pathogen detection. We have demonstrated the utility of this assay with an initial set of 24 probes targeting the most clinically relevant HPV genotypes associated with cervical cancer progression.

The history of pyrosequencing.

One late afternoon in the beginning of January 1986, bicycling from the lab over the hill to the small village of Fullbourn, the idea for an alternative DNA sequencing technique came to my mind. The basic concept was to follow the activity of DNA polymerase during nucleotide incorporation into a DNA strand by analyzing the pyrophosphate released during the process. Today, the technique is used in multidisciplinary fields in academic, clinical, and industrial settings all over the word.

Methodological improvements of pyrosequencing technology.

Pyrosequencing technology is a rather novel DNA sequencing method based on the sequencing-by-synthesis principle. This bioluminometric, real-time DNA sequencing technique employs a cascade of four enzymatic reactions producing sequence peak signals. The method has been proven highly suitable for single nucleotide polymorphism analysis and sequencing of short stretches of DNA.

Sentinel-base DNA genotyping using multiple sequencing primers for high-risk human papillomaviruses.

Despite the various technologies in place for genotyping human papillomaviruses (HPV), clinical use and clinical research demand a method that is fast, more reliable and cost-effective. The technology described here represents a breakthrough development in that direction. By combining the method of multiple sequencing primers with DNA sequencing, we have developed a rapid assay for genotyping HPV that relies on the identification of a single, type-specific 'sentinel' base.

Detection of gyrA mutations associated with ciprofloxacin resistance in Neisseria gonorrhoeae by rapid and reliable pre-programmed short DNA sequencing.

Quinolone resistance is rapidly increasing in Neisseria gonorrhoeae and is posing a significant public health threat that requires constant surveillance. A rapid and reliable mutation detection assay has been developed. The assay is based on pre-programmed short DNA sequencing and is designed to detect point mutations in the gyrA gene that are highly related to ciprofloxacin resistance, i.

Type-specific multiple sequencing primers: a novel strategy for reliable and rapid genotyping of human papillomaviruses by pyrosequencing technology.

DNA sequencing is the gold standard method for accurate microbial and viral typing. However, DNA sequencing techniques have been facing limitations in typing of human papillomaviruses when the specimen harbors multiple genotypes and yields nonspecific amplification products, resulting in nonspecific and noninterpretable sequence data. To address these limitations we have developed a type-specific multiple sequencing primer DNA-sequencing method.

DNA gyrase gene in Neisseria gonorrhoeae as indicator for resistance to ciprofloxacin and species verification.

We have identified a unique region of eight amino acids in the quinolone resistance-determining region in the gyrA gene in Neisseria gonorrhoeae as an indicator of resistance to fluoroquinolones. We sequenced that region by the Pyrosequencing technology in 46 N. gonorrhoeae strains and 11 urine samples positive in AMPLICOR N.

7-Deaza-2'-deoxyadenosine-5'-triphosphate as an alternative nucleotide for the pyrosequencing technology.

A new adenosine nucleotide analog suitable for the Pyrosequencing method is presented. The new analog, 7-deaza-2'-deoxyadenosine-5'-triphosphate (c7dATP), has virtually the same low substrate specificity for luciferase as the currently used analog, 2'-deoxyadenosine-5'-O-(1-thiotriphosphate) (dATPalphaS). The inhibitory effect dATPalphaS displays on the nucleotide degrading activity of apyrase was reduced significantly by substituting the c7dATP for the dATPalphaS.

A mathematical model of the Pyrosequencing reaction system.

The Pyrosequencing technology is a newly developed DNA sequencing method that monitors DNA nucleotide incorporation in real-time. A set of coupled enzymatic reactions, together with bioluminescence, detects incorporated nucleotides in the form of light pulses, yielding a characteristic light profile. In this study, a biochemical model of the Pyrosequencing reaction system is suggested and implemented.

Improvements in Pyrosequencing technology by employing Sequenase polymerase.

Pyrosequencing is a DNA sequencing technique based on the bioluminometric detection of inorganic pyrophosphate, which is released when nucleotides are incorporated into a target DNA. Since the technique is based on an enzymatic cascade, the choice of enzymes is a critical factor for efficient performance of the sequencing reaction. In this study we have analyzed the performance of an alternative DNA polymerase, Sequenase, on the sequencing performance of the Pyrosequencing technology.

Toward pyrosequencing on surface-attached genetic material by use of DNA-binding luciferase fusion proteins.

Mutation detection and single-nucleotide polymorphism genotyping require screening of large samples of materials and therefore the importance of high-throughput DNA analysis techniques is significant. Pyrosequencing is a four-enzyme bioluminometric DNA sequencing technology based on the sequencing-by-synthesis principle. Currently, the technique is limited to simultaneous analysis of 96 or 384 samples.

Pyrosequencing trade mark technology at elevated temperature.

To date, the Pyrosequencing trade mark technology has been performed at 28 degrees C due to the low thermostability of the firefly luciferase. In this study, firefly luciferase was stabilized in the presence of glycine betaine, allowing DNA sequencing at 37 degrees C. By increasing the temperature to 37 degrees C, false signals due to primer-dimers and loop-structures were decreased significantly.

Viral and microbial genotyping by a combination of multiplex competitive hybridization and specific extension followed by hybridization to generic tag arrays.

Detection and identification of microbial pathogens are important for disease diagnosis, treatment and prophylaxis measurements. By introducing an innovative technique, we show a robust, reliable and accurate microarray-based method for identification of microbial pathogens. The technique utilizes a unique combination of multiplex competitive hybridization, which enhances hybridization accuracy of oligonucleotides to the specific target, and apyrase-mediated allele-specific extension, which improves specific extension.

Genotypes of CCR2 and CCR5 chemokine receptors in human myasthenia gravis.

The aim of this study was to examine the association of human autoimmune myasthenia gravis (MG) with two DNA polymorphisms of the chemokine receptors CCR5-Delta 32 and CCR2-64I. CCR2 and CCR5 interact primarily with the human CC family ligands CCL2 (formerly called monocyte chemoattractant protein; MCP-1), CCL3 and CCL4 (macrophage inflammatory protein-1 alpha and -1 beta; MIP-1 alpha/beta), and their main function is to recruit leukocytes from circulation into the tissues, thus playing an important role in human inflammatory disorders. A PCR-based genotyping method was used to determine the genetic variation at the CCR5 gene and an automated real-time Pyrosequencing technology was employed for the analysis of G right curved arrow A point mutation at the CCR2 gene.

Multiple group-specific sequencing primers for reliable and rapid DNA sequencing.

Pyrosequencing technology is a bioluminometric DNA sequencing method that employs a cascade of four enzymes to deliver sequence signals. To date this technology has been limited to the sequencing of short stretches of DNA. As an improvement to this technique, we have introduced a bacterial group-specific, multiple sequencing primer approach that circumvents sequencing of less informative semi-conservative regions of the 16S rRNA gene.

Method for clone checking.

A new method for simple and fast clone checking is described. We combined the Pyrosequencing technology with a preprogrammed nucleotide dispensation strategy for fast analysis of DNA constructs. To test this method, the N-terminus region of plasmids constructed for the production of recombinant apyrase was analyzed.

Multiple-primer DNA sequencing method.

A multiple-primer DNA sequencing approach suitable for genotyping, detection and identification of microorganisms and viruses has been developed. In this new method two or more sequencing primers, combined in a pool, are added to a DNA sample of interest. The oligonucleotide that hybridizes to the DNA sample will function as a primer during the subsequent DNA sequencing procedure.