Pyrosequencing Technology for Short DNA Sequencing and Whole Genome Sequencing

Gharizadeh, Baback; Ghaderi, Mehran; Nyrén, Pål

doi:10.2142/biophys.47.129

Cited by 3 publications

(13 citation statements)

References 33 publications

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“…It employs a series of four enzymes to accurately detect nucleic acid sequences during the synthesis. In Pyrosequencing (Nyren and Skarpnack, 2001) the sequencing primer is hybridized to a single-stranded DNA biotinlabeled template and mixed with the enzymes; DNA polymerase, ATP sulfurylase, luciferase and apyrase and the substrates adenosine 5' phosphosulfate (APS) and luciferin (Gharizadeh et al, 2007).…”

Section: Principles Of Pyrosequencingmentioning

confidence: 99%

“…There is a certain time interval (usually 65 sec) between each nucleotide dispensation to allow complete degradation. For this reason, dNTP addition is performed one at a time (Gharizadeh et al, 2007). Because the added nucleotide is known, the sequence of the template can be determined (Ronaghi, 2001).…”

Section: Principles Of Pyrosequencingmentioning

confidence: 99%

“…The generated light is observed as a peak signal in the pyrogram (corresponding to electropherogram in dideoxy sequencing) proportional to the number of nucleotides incorporated (a triple dGTP incorporation generates a triple higher peak) (Gharizadeh et al, 2007). During this synthesis process, the DNA strand is extended by complementary nucleotides and the DNA sequence is demonstrated by the pyrogram on a screen.…”

Section: Principles Of Pyrosequencingmentioning

confidence: 99%

“…The chain termination sequencing method, also known as Sanger sequencing, was developed by Frederick Sanger and colleagues (Sanger et al, 1977), has been the most widely used sequencing method since its advent in 1977 and still is in use after more than 29 years. Despite all the advantages, there are limitations in this method, which could be complemented with other techniques (Gharizadeh et al, 2007). Recently, pyrosequencing has emerged as a new sequencing methodology (Ronaghi, 2001).…”

Section: Introductionmentioning

confidence: 99%

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Pyrosequencing-An Alternative to Traditional Sanger Sequencing

Fakruddin¹,

Chowdhury²

2012

American Journal of Biochemistry and Biotechnology

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Problem statement: Pyrosequencing has the potential to rapidly and reliably sequence DNA taking advantages over traditional Sanger di-deoxy sequencing approach. Approach: A comprehensive review of the literature on the principles, applications, challenges and prospects of pyrosequencing was performed. Results: Pyrosequencing was a DNA sequencing technology based on the sequencing-by-synthesis principle. It employs a series of four enzymes to accurately detect nucleic acid sequences during the synthesis. Pyrosequencing had the potential advantages of accuracy, flexibility, parallel processing and could be easily automated. The technique dispenses with the need for labeled primers, labeled nucleotides and gel-electrophoresis. Pyrosequencing had opened up new possibilities for performing sequence-based DNA analysis. The method had been proven highly suitable for single nucleotide polymorphism analysis and sequencing of short stretches of DNA. Pyrosequencing had been successful for both confirmatory sequencing and de novo sequencing. By increasing the read length to higher scores and by shortening the sequence reaction time per base calling, pyrosequencing may take over many broad areas of DNA sequencing applications as the trend was directed to analysis of fewer amounts of specimens and large-scale settings, with higher throughput and lower cost. Conclusion/Recommendations: The Competitiveness of pyrosequencing with other sequencing methods can be improved in future.

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Section: Principles Of Pyrosequencingmentioning

confidence: 99%

Section: Principles Of Pyrosequencingmentioning

confidence: 99%

Section: Principles Of Pyrosequencingmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Pyrosequencing-An Alternative to Traditional Sanger Sequencing

Fakruddin¹,

Chowdhury²

2012

American Journal of Biochemistry and Biotechnology

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“…Pyrosequencing can perform faster than Sanger sequencing, the most common sequencing method. Pyrosequencing can give the sequencing signal immediately after reaching the downstream of the primer and analyze 96 samples at the same time 9,10 . There are cascade of steps which have to be done before the pyrosequencing process starts, named as pyrosequencing workflow, which only take 3-4 hours to be completed.…”

Section: Introductionmentioning

confidence: 99%

Optimization of Pyrosequencing Method to Detect IVS1-NT5 ß-Globin Gene Mutation in ß-Thalassemia

Maskoen¹,

Sofiana²,

Reniarti³

2017

IJPTR

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: β-thalassemia incidence in Indonesia is reaching 2.500 cases per year, so that it is still a threat for Indonesian's society due to the high therapeutic cost. Prenatal diagnosis for detecting β-globin gene mutation is important to be developed in order to decrease β-thalassemia incidence. Pyrosequencing, a new DNA sequencing method, owns the ability to detect DNA mutation quickly for prenatal diagnosis importance. A specific β-globin gene mutation for Indonesian population is needed to make the mutation detection process become more effective, which is the mutation in the intervening sequence 1 nucleotide 5 (IVS1-NT5) region. Hence, the objective of this study is to know the optimal pyrosequencing condition to detect the IVS1-NT5 β-globin gene mutation in β-thalassemia. The sample used was the stored DNA material which the mutation had been detected using polymerase chain reactionrestriction fragment length polymorphism (PCR-RFLP) for pyrosequencing optimization. There were two steps of optimization, both in the amplification step using polymerase chain reaction (PCR), and pyrosequencing step. The obtained result in amplification step was 55 o C for primer annealing temperature. In pyrosequencing step, the enzyme and substrate storage condition, cartridge's needle performance, and quality of sequencing primer were observed as factors which influence peak formation in the pyrograms, Nevertheless, due to the absence of the proper peaks on the pyrograms of this study, the pyrosequencing method to detect IVS1-NT5 is not yet optimized.

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Next-Generation Sequencing (NGS) Platforms: An Exciting Era of Genome Sequence Analysis

Krishna

Khan

2019

Microbial Genomics in Sustainable Agroecosystems

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Pyrosequencing Technology for Short DNA Sequencing and Whole Genome Sequencing

Cited by 3 publications

References 33 publications

Pyrosequencing-An Alternative to Traditional Sanger Sequencing

Pyrosequencing-An Alternative to Traditional Sanger Sequencing

Optimization of Pyrosequencing Method to Detect IVS1-NT5 ß-Globin Gene Mutation in ß-Thalassemia

Next-Generation Sequencing (NGS) Platforms: An Exciting Era of Genome Sequence Analysis

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