Lack of association of human chemokine receptor gene polymorphisms CCR2-64I and CCR5-Delta32 with autoimmune Addison's disease.

The attraction of leukocytes to tissues is essential for inflammation and the initiation of the autoimmune reaction. The process is controlled by chemokines, which are chemotactic cytokines. We investigated whether human chemokine receptor gene polymorphisms, namely CCR5-Delta32 and CCR2-64I, are associated with susceptibility to autoimmune Addison's disease. Genotyping was performed in 56 patients and 127 healthy controls by a new method using pyrosequencing for CCR2-64I and by polymerase chain reaction and detecting gel for CCR5-Delta32. None of the CCR2 or CCR5 alleles was found to be associated, either positively or negatively, with disease risk. Our results indicate that the CCR2-64I and CCR5-Delta32 gene polymorphisms do not play a major role in conferring genetic risk for, and/or protection against, autoimmune Addison's disease.

Identification of medically important fungi by the Pyrosequencing technology.

The Pyrosequencing 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.

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.

Method enabling fast partial sequencing of cDNA clones.

Pyrosequencing is a nonelectrophoretic single-tube DNA sequencing method that takes advantage of cooperativity between four enzymes to monitor DNA synthesis. To investigate the feasibility of the recently developed technique for tag sequencing, 64 colonies of a selected cDNA library from human were sequenced by both pyrosequencing and Sanger DNA sequencing. To determine the needed length for finding a unique DNA sequence, 100 sequence tags from human were retrieved from the database and different lengths from each sequence were randomly analyzed. An homology search based on 20 and 30 nucleotides produced 97 and 98% unique hits, respectively. An homology search based on 100 nucleotides could identify all searched genes. Pyrosequencing was employed to produce sequence data for 30 nucleotides. A similar search using BLAST revealed 16 different genes. Forty-six percent of the sequences shared homology with one gene at different positions. Two of the 64 clones had unique sequences. The search results from pyrosequencing were in 100% agreement with conventional DNA sequencing methods. The possibility of using a fully automated pyrosequencer machine for future high-throughput tag sequencing is discussed.

Method for real-time detection of inorganic pyrophosphatase activity.

A sensitive and simple method for real-time detection of inorganic pyrophosphatase (PPase) (EC 3.6.1.1) activity has been developed. The method is based on PPase-induced activation of the firefly luciferase activity in the presence of inorganic pyrophosphate (PPi). PPi inhibits the luciferase activity, but in the presence of PPase the luciferase activity is restored and the luminescence output increases. The assay yields linear responses between 8 and 500 mU. The detection limit was found to be 8 mU PPase. The method was used to detect the hydrolytic activity of PPases from Saccharomyces cerevisiae, Escherichia coli, and Bacillus stearothermophilus. As substrate for the luciferase, adenosine 5'-phosphosulfate can replace ATP, which is an advantage for detection of PPase activity in crude extracts containing ATP-hydrolyzing activities. The method can be used for kinetic and inhibition studies as well as for detection of PPase activity during different purification procedures.

Quantification of HIV-1 using multiple quantitative polymerase chain reaction standards and bioluminometric detection.

A non-gel-based quantification assay based on competitive PCR and bioluminometric detection has been developed. Samples containing human immunodeficiency virus type 1 (HIV-1) DNA and three quantitative standards at discrete concentrations were coamplified by PCR with primers annealing in the polymerase gene region. The quantitative standards contained the same primer binding sequences and had the same amplicon length as the wild-type DNA, but differed in an internal homopolymeric stretch (A, C, or T) over three base pairs. The PCR products were captured onto a solid support and treated with NaOH to separate the strands. Discrimination between the wild-type DNA and the three quantitative standard amplicons was achieved on the solid support by four parallel extension reactions with 3'-end specific primers. Inorganic pyrophosphate (PPi) released as a result of successful extension was converted to ATP by ATP sulfurylase and the level of ATP was sensed by firefly luciferase, generating a proportional amount of visible light which was detected by a luminometer. Here, we show that the obtained calibration curves, using the signal intensities of the three quantitative standards, enabled determination of the amount of target HIV-1 DNA.

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.

Method enabling pyrosequencing on double-stranded DNA.

Pyrosequencing is a new nonelectrophoretic, single-tube DNA sequencing method that takes advantage of co-operativity between four enzymes to monitor DNA synthesis (M. Ronaghi, M. Uhlén, and P. Nyrén, Science 281, 363-365). Pyrosequencing has so far only been performed on single-stranded DNA. In this paper different enzymatic strategies for template preparation enabling pyrosequencing on double-stranded DNA were studied. High quality data were obtained with several different enzyme combinations: (i) shrimp alkaline phosphatase and exonuclease I, (ii) calf intestine alkaline phosphatase and exonuclease I, (iii) apyrase and inorganic pyrophosphatase together with exonuclease I, and (iv) apyrase and ATP sulfurylase together with exonuclease I. In many cases, when the polymerase chain reaction was efficient exonuclease I could be omitted. In certain cases, additives such as dimethyl sulfoxide, single-stranded DNA-binding protein, and Klenow DNA polymerase improved the sequence quality. Apyrase was the fastest and most efficient of the three different nucleotide degrading enzymes tested. The data quality obtained on double-stranded DNA was comparable with that on single-stranded DNA. Pyrosequencing data for more than 30 bases could be generated on both long and short templates, as well as on templates with high GC content.

Single-nucleotide polymorphism analysis by pyrosequencing.

There is a growing demand for high-throughput methods for analysis of single-nucleotide polymorphic (SNP) positions. Here, we have evaluated a novel sequencing approach, pyrosequencing, for such purposes. Pyrosequencing is a sequencing-by-synthesis method in which a cascade of enzymatic reactions yields detectable light, which is proportional to incorporated nucleotides. One feature of typing SNPs with pyrosequencing is that each allelic variant will give a unique sequence compared to the two other variants. These variants can easily be distinguished by a pattern recognition software. The software displays the allelic alternatives and allows for direct comparison with the pyrosequencing raw data. For optimal determination of SNPs, various protocols of nucleotide dispensing order were investigated. Here, we demonstrate that typing of SNPs can efficiently be performed by pyrosequencing using an automated system for parallel analysis of 96 samples in approximately 5 min, suitable for large-scale screening and typing of SNPs.

Direct analysis of single-nucleotide polymorphism on double-stranded DNA by pyrosequencing.

Pyrosequencing, a new method for DNA sequencing, is gaining widespread use for many different types of DNA analysis. The method takes advantage of four coupled enzymes in a single tube assay to monitor DNA synthesis in real time using a luminometric detection system. Here, we demonstrate the use of pyrosequencing for direct analysis of single-nucleotide polymorphism on double-stranded PCR product. Pyrosequencing data on the human glutathione peroxidase gene (GPX1) from several individuals were analysed and three different allelic variants were determined and confirmed. The possibility of further simplifying the sequencing and template-preparation steps is discussed.

Analysis of the p53 tumor suppressor gene by pyrosequencing.

Tumor suppressor genes are implicated in cell cycle progression. Inactivation of these genes predominantly occurs through mutations and/or allelic loss that involves both alleles. With inactivation by multiple mutations in a single gene, cloning of the amplified gene is necessary to determine whether the mutations reside on one or both alleles. Using pyrosequencing, a recently developed approach based on sequencing-by-synthesis, we studied genetic variability in the p53 tumor suppressor gene and could quantify the ratio between the mutated and wild-type amplified fragments. Furthermore, this sequencing technique also allows allelic determination of adjacent mutations with no cloning of amplified fragments.

Real-time detection and quantification of adenosine triphosphate sulfurylase activity by a bioluminometric approach.

A real-time, sensitive, and simple assay for detection and quantification of adenosine triphosphate sulfurylase (ATP:sulfate adenylytransferase, EC 2.7.7.4) activity has been developed. The method is based on detection of ATP generated in the ATP sulfurylase reaction between APS and PPi by the firefly luciferase system. For the Saccharomyces cerevisiae ATP sulfurylase, the concentrations of APS and PPi at the half-maximal rate were found to be about 0.5 and 7 microM, respectively. The assay is sensitive and yields linear response between 0.1 microU and 50 mU. The method can be used for monitoring and quantification of recombinant ATP sulfurylase activity in Escherichia coli lysate, as well as for detection of the activity during different purification procedures.

Real-time bioluminometric method for detection of nucleoside diphosphate kinase activity.

A real-time, simple and sensitive method for detection of nucleoside diphosphate (NDP) kinase activity has been developed. The assay is based on detection of ATP, generated in the NDP kinase reaction between a nucleoside triphosphate and adenosine diphosphate (ADP), by the firefly luciferase system. In the presence of 0.3 mM dGTP, the Km for ADP was found to be approximately 30 microM for the NDP kinase from Baker's yeast. In the presence of 250 microM ADP, the Km for dATP alpha S, dTTP alpha S, dGTP, dTTP, dCTP and GTP was found to be approximately 0.01, 0.03, 0.05, 0.25, 0.75 and 0.2 mM, respectively. The assay is sensitive and yields linear responses between 0.05-50 mU. The detection limit was found to be 0.05 mU of NDP kinase. The method was used to detect NDP kinase contamination in commercial enzyme preparations.

Production, purification, and luminometric analysis of recombinant Saccharomyces cerevisiae MET3 adenosine triphosphate sulfurylase expressed in Escherichia coli.

ATP sulfurylase cDNA from MET3 on chromosome X of Saccharomyces cerevisiae was amplified and cloned, and recombinant ATP sulfurylase was expressed in Escherichia coli. The synthesis of ATP sulfurylase was directed by an expression system that employs the regulatory genes of the luminous bacterium Vibrio fischeri. A soluble, biologically active form was purified to electrophoretic homogeneity from lysates of recombinant E. coli by ammonium sulfate fractionation, ion-exchange chromatography, and gel filtration. The specific activity of the purified enzyme was estimated to 140 U/mg. The apparent molecular mass of the recombinant enzyme was determined by gel filtration to be 470 kDa, which indicates that the active enzyme is an octamer of identical subunits (the molecular mass of a single subunit is 59.3 kDa). The ATP sulfurylase activity was monitored in real time by a very sensitive bioluminometric method.

Analyses of secondary structures in DNA by pyrosequencing.

A common problem in conventional DNA sequencing is the occurrence of DNA sequence compressions during gel electrophoresis, leading to misreading of the sequence. These compressions are usually due to secondary structures in the DNA fragment. In this study, we present a non-gel-based DNA sequencing technique that facilitates analysis of such DNA regions. A part of the polymorphic pertussis toxin promoter region in five different Bordetella species was successfully resolved by the new technique. The obtained sequence data revealed four related palindromic sequences. The ability of different DNA polymerases to read through such secondary structures is also described.

PCR-introduced loop structure as primer in DNA sequencing.

The need for a primer hybridization step before sequencing has been eliminated using a stem-loop structure generated by PCR. The loop structure is obtained by careful design of the PCR primer or by cloning the target DNA into a dedicated vector (pRIT 28HP). After solid-phase capture of the PCR product, the loop is formed by elution of the non-bound strand. Here, we show that both the immobilized and the eluted strand can be analyzed using conventional Sanger DNA sequencing and the novel pyrosequencing method as described previously. By using a stem-loop structure as a primer for DNA sequencing, the risk for mispriming is minimized.

Bioluminometric method for real-time detection of reverse transcriptase activity.

A simple and sensitive technique for detection of reverse transcriptase (RT) activity in real time has been developed. The technique is based on continuous detection of the inorganic pyrophosphate formed in the RT-catalyzed reaction by a luminometric method. The technique has been used for continuous monitoring of RT-catalyzed DNA synthesis on both homo- and heteropolymeric templates. The assay is sensitive and yields linear responses between 1.5-960 mU of avian myeloblastosis virus RT (AMV-RT). The assay was used for detection of the inhibitory effect of dideoxythymidine (ddTTP) on the AMV-RT activity and also for real-time detection of single-base incorporation events catalyzed by AMV-RT. The possibility of using the new technique for other applications is discussed.

Detection of single-base changes using a bioluminometric primer extension assay.

A rapid bioluminometric technique for real-time detection of known single-base changes is presented. The concept relies on the measurement of the difference in primer extension efficiency by a DNA polymerase of a matched over a mismatched 3' terminal. The rate of the DNA polymerase-catalyzed primer extension is measured by an enzymatic luminometric inorganic pyrophosphate (PPi) detection assay (ELIDA) (P. Nyrén (1987) Anal. Biochem. 167, 235-238). The PPi formed in the polymerization reaction is converted to ATP by ATP sulfurylase and the ATP production is continuously monitored by the firefly luciferase. In the single-base detection assay, immobilized single-stranded DNA fragments are used as template. Two detection primers differing with one base at the 3' end are designed, one precisely complementary to the nonmutated DNA sequence and the other precisely complementary to the mutated DNA sequence. The primers are hybridized with the 3'-termini over the base of interest and the primer extension rates are, after incubation with DNA polymerase and deoxynucleotides, measured with the ELIDA. We show that the relative mismatch extension efficiency is strongly decreased by substituting the alpha-thiotriphosphate analog for the next correct natural deoxynucleotide after the 3'-mismatch termini. The possibility of using the technique for studies of mismatch extension kinetics for two polymerases lacking exonucleolytic activity is shown.

A 3-hydroxy-3-methylglutaryl-CoA lyase gene in the photosynthetic bacterium Rhodospirillum rubrum.

A 1.2 kb long DNA segment from Rhodospirillum rubrum has been sequenced (EMBL/GenBank accession number: U41280). This DNA segment includes the first sequenced gene for a putative 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) lyase, termed hmgL, from a photosynthetic organism. The sequenced segment also contains a ribosome-binding site and two clusters of possible-35 and -10 promotor sequences preceding the hmgL gene. Translation of the gene would yield a 303 amino-acid-long protein with a calculated molecular weight of 31.1 kDa. This protein shows 55-60% identity and approx. 75% similarity, including conservative substitutions, with the three eukaryotic and the single prokaryotic HMG-CoA lyases which previously have been sequenced. The R. rubrum enzyme showed stronger homology to the chicken HMG-CoA lyase than to the other bacterial protein. Significant sequence similarity was also found with homocitrate synthases from nitrogen-fixing prokaryotes. In contrast to the other sequenced prokaryotic HMG-CoA lyase (from Pseudomonas mevalonii), the R. rubrum hmgL does not seem to appear in an operon together with a HMG-CoA reductase. The hmgL gene was transcribed in photosynthetically grown cells as judged by amplification of cDNAs synthesised from DNA-free total RNA. In addition, HMG-CoA lyase activity was found in R. rubrum cells grown anaerobically in the light with leucine as the carbon source.