Baback Gharizadeh

Mutation detection by pyrosequencing : sequencing of exons 5-8 of the p53 tumor suppressor gene

The ability to sequence a large number of DNA samples rapidly and accurately for detection of all possible mutations is a critical goal for the future application of DNA sequencing in routine medical diagnostics. Pyrosequencing() is a non-electrophoretic real-time DNA sequencing method that uses the luciferase-luciferin light release as the detection signal for nucleotide incorporation into target DNA. For pyrosequencing of the human p53 gene, a nested multiplex PCR method for amplification of exons 5-8 was prepared. In order to investigate the use of pyrosequencing in mutation detection, DNA samples from skin-cancer patients were used. Two forms of nucleotide dispensation strategy were used, cyclic and programmed. Bi-directional pyrosequencing was performed and the overlapping sequence data produced were assembled to determine the sequence of the gene. Reliable sequencing data were obtained with both dispensation strategies, but some advantages were obtained using the programmed nucleotide dispensation approach, such as longer and faster reads, and fewer out-of-phase problems. The accuracy of pyrosequencing for detection of p53 mutations and allele distribution was demonstrated.

Minority Human Immunodeficiency Virus Type 1 Variants in Antiretroviral-Naive Persons with Reverse Transcriptase Codon 215 Revertant Mutations

ABSTRACT T215 revertant mutations such as T215C/D/E/S that evolve from the nucleoside reverse transcriptase (RT) inhibitor mutations T215Y/F have been found in about 3% of human immunodeficiency virus type 1 (HIV-1) isolates from newly diagnosed HIV-1-infected persons. We used a newly developed sequencing method—ultradeep pyrosequencing (UDPS; 454 Life Sciences)—to determine the frequency with which T215Y/F or other RT inhibitor resistance mutations could be detected as minority variants in samples from untreated persons that contain T215 revertants (“revertant” samples) compared with samples from untreated persons that lack such revertants (“control” samples). Among the 22 revertant and 29 control samples, UDPS detected a mean of 3.8 and 4.8 additional RT amino acid mutations, respectively. In 6 of 22 (27%) revertant samples and in 4 of 29 control samples (14%; P = 0.4), UDPS detected one or more RT inhibitor resistance mutations. T215Y or T215F was not detected in any of the revertant or control samples; however, 4 of 22 revertant samples had one or more T215 revertants that were detected by UDPS but not by direct PCR sequencing. The failure to detect viruses with T215Y/F in the 22 revertant samples in this study may result from the overwhelming replacement of transmitted T215Y variants by the more fit T215 revertants or from the primary transmission of a T215 revertant in a subset of persons with T215 revertants.

Typing of Human Papillomavirus by Pyrosequencing

The possibility of using a new bioluminometric DNA sequencing technique, called pyrosequencing, for typing of human papillomaviruses (HPV) was investigated. A blinded pyrosequencing test was performed on an HPV test panel of 67 GP5+/GP6+ PCR-derived amplification products. The 67 clinical DNA samples were sequenced up to 25 bases and sequences were searched using BLAST. All of the samples were correctly genotyped by pyrosequencing and the results were unequivocally in accordance with the results obtained from conventional DNA sequencing. Pyrosequencing was found to be a fast and efficient tool for identifying individual HPV types. Furthermore, pyrosequencing has the capability of determining novel HPV types as well as HPV sequence variants harboring mutation(s). The method is robust and well suited for large-scale programs.

Identification of medically important fungi by the Pyrosequencing technology.

The PyrosequencingTM technology was used for identification of different clinically relevant fungi. The tests were performed on amplicons derived from the 18S rRNA gene using polymerase chain reaction (PCR) universal primers for amplification. Sequencing was performed up to 40 bases in a variable region with a designed general sequencing primer and the Pyrosequence data were analyzed by BLAST sequence search in the GenBank database. DNA from a total of 21 fungal specimens consisting of nine strains of clinically relevant fungi and 12 clinical specimens from patients suffering from proven invasive fungal infections were PCR‐amplified and analyzed by gel electrophoresis, PCR‐enzyme‐linked immunosorbent assay (ELISA) and the Pyrosequencing technology. All data obtained by the Pyrosequencing technology were in agreement with the results obtained by PCR‐ELISA using species/genus‐specific oligonucleotides and were as well in accordance with the culture results. The results demonstrate that the Pyrosequencing method is a reproducible and reliable technique for identification of fungal pathogens.

Pyrosequencing for discovery and analysis of DNA sequence variations

Since the invention of pyrosequencing, more than 500 articles have been published describing different applications of this technology, most notably for DNA structure variation and microbial detection. Technological advances have been made to enhance the robustness and accuracy of this technique as well as to reduce the cost and increase the throughput. This review intends to cover recent advances in this technology and discuss its application for low and high-throughput DNA variation studies.

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. This new approach is suitable for challenging templates, improving sequence data quality, avoiding sequencing of non-specific amplification products, lessening sequencing time, and moreover, this strategy should open the way for many new applications in the future. The group-specific, multiple sequencing primers can be applied in the Sanger dideoxy sequencing method as well. In addition, we have improved the chemistry of the Pyrosequencing system enabling sequencing of longer stretches of DNA, which allows numerous new applications.

Methylotrophic yeast Pichia pastoris as a host for production of ATP-diphosphohydrolase (apyrase) from potato tubers (Solanum tuberosum).

ATP-diphosphohydrolase (apyrase) catalyzes the hydrolysis of phosphoanhydride bonds of nucleoside tri- and di-phosphates in the presence of divalent cations. This enzyme has broad substrate specificity for nucleotides, which makes it an ideal enzyme for different biotechnical applications, such as DNA sequencing and platelet-aggregation inhibition. The only commercially available apyrase is isolated from potato tubers. To avoid batch-to-batch variations in activity and quality, we decided to produce a recombinant enzyme. The methylotrophic yeast Pichia pastoris was chosen as an eukaryotic expression host. The coding sequence of potato apyrase, without the signal peptide, was cloned into the YpDC541 vector to create a fusion with the alpha-mating secretion signal of Saccharomyces cerevisiae. The gene was placed under the control of the methanol-inducible alcohol oxidase promoter. The YpDC541-apyrase construct was integrated into P. pastoris strain SMD1168. Methanol induction resulted in secretion of apyrase to a level of 1mg/L. The biologically active recombinant apyrase was purified by hydrophobic interaction and ion exchange chromatography. According to SDS-PAGE and Western blot analysis, the purified enzyme showed to be hyperglycosylated. By enzymatic removal of N-glycans, a single band corresponding to a molecular mass of 48kDa was detected. The recombinant apyrase was found to function well when it was used in combination with the Pyrosequencing technology.

Single-nucleotide polymorphisms in the B7H3 gene are not associated with human autoimmune myasthenia gravis

Single-nucleotide polymorphisms in B7H3 gene are not associated with human autoimmune myasthenia gravis

Quinolone-resistant Neisseria gonorrhoeae – response

Sir: In the February 2005 issue of the International Journal of STD & AIDS, Lindbäck et al. describe a novel method for molecular detection of quinoloneresistant Neisseria gonorrhoeae (QRNG). Culture/ urine pairs were analysed by both phenotypic analyses and molecular characterization of gonococcal quinolone resistance-determining regions (QRDRs), which are located within the gyrA and parC genes. Although the authors’ experimental technique is sound, in their Discussion section they claim that ‘The Pyrosequencing method sequenced and analysed accurately all 11 urine samples, and is so far the only method for molecular susceptibility testing in N. gonorrhoeae’. In contrast to this assertion, previously published methods employed molecular approaches to analyse gonococcal QRDRs. Li et al. first described a rapid method for detecting gyrA mutations using the Roche LightCycler. This method utilizes realtime PCR and FRET-based fluorescence to detect point mutations in gyrA by melt curve analysis. In 2002 and 2003, Ng et al. and Booth et al. also described molecular detection methods for QRDR mutations. Using a microarray format, they showed that point mutation patterns in gonococcal QRDRs could be detected from DNA extracts, including previously undescribed mutations. In 2004, our group published a rapid, real-time molecular method for detection of gonococcal QRDR mutations, using the ABI 7900HT platform and Taqman probe technology. In our paper, we suggested that nucleic acid amplification test (NAAT) samples, which are urine-based, could be feasibly used for this analysis. Two months after our method was published, Zhou et al. described another biochip detection system for QRDR mutation detection in N. gonorrhoeae. The pyrosequencing technology is novel, but it is not the only published molecular approach to fluoroquinolone susceptibility testing in N. gonorrhoeae.