Pyrosequencing Sheds Light on DNA Sequencing

DNA sequencing is one of the most important platforms for the study of biological systems today. Sequence determination is most commonly performed using dideoxy chain termination technology. Recently, pyrosequencing has emerged as a new sequencing methodology. This technique is a widely applicable, alternative technology for the detailed characterization of nucleic acids. Pyrosequencing has the potential advantages of accuracy, flexibility, parallel processing, and can be easily automated. Furthermore, the technique dispenses with the need for labeled primers, labeled nucleotides, and gel-electrophoresis. This article considers key features regarding different aspects of pyrosequencing technology, including the general principles, enzyme properties, sequencing modes, instrumentation, and potential applications.

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Molecular Characterization of Multidrug-ResistantStreptococcus pneumoniae Isolates in Korea

Journal of Clinical Microbiology ◽

10.1128/jcm.38.4.1641-1644.2000 ◽

2000 ◽

Vol 38 (4) ◽

pp. 1641-1644 ◽

Cited By ~ 11

Author(s):

Jae-Hoon Song ◽

Ji-won Yang ◽

Joung Hwa Jin ◽

Shin Woo Kim ◽

Choon Kwan Kim ◽

...

Keyword(s):

Amino Acids ◽

Dna Sequencing ◽

Molecular Characterization ◽

Gel Electrophoresis ◽

Multidrug Resistant ◽

Pulsed Field Gel Electrophoresis ◽

Pulsed Field ◽

Fingerprinting Analysis

Pulsed-field gel electrophoresis, ribotyping, and fingerprinting analysis of 22 invasive isolates of multidrug-resistant (MDR) pneumococci from Korea showed that 59 to 82% were genetically related. DNA sequencing of the PBP 2B gene showed relatively uniform alterations in nucleotides (5.4 to 7.8%) and amino acids (3.0 to 4.3%), while Asn-276→Lys, Arg-285→Cys and Ser-305→Phe substitutions were unique to Korean MDR strains, suggesting the spread of a few epidemic clones of resistant pneumococci within Korea.

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Sanger Sequencing as the Holy Grail of Sequencing Technology

10.20944/preprints202011.0163.v1 ◽

2020 ◽

Author(s):

Ayobami Adesiyan ◽

Ayomikun Kade ◽

Kafayat Oladimeji ◽

Kehinde Sowunmi

Keyword(s):

Dna Sequencing ◽

Gel Electrophoresis ◽

Enzymatic Synthesis ◽

Sanger Sequencing ◽

Polyacrylamide Gel Electrophoresis ◽

Chain Termination ◽

Chemical Degradation ◽

Sequencing Technology ◽

Sequencing Method ◽

Dna Template

DNA sequencing methods were first developed more than 20 years ago with the publication of two approaches to sequencing methodology that became known as Sanger sequencing (1), based on enzymatic synthesis from a single-stranded DNA template with chain termination using dideoxynucleotides (ddNTPs) and Maxim-Gilbert sequencing (2),which involved chemical degradation ofend-radio-labeled DNA fragments. Both methods relied on four-lane,high resolution polyacrylamide gel electrophoresis to separate the labeled fragment and allow the base sequence to be read in a staggered ladder-like fashion. Sanger sequencing was technically easier and faster, and thus became the main DNA sequencing method for the vast majority of applications.

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Rapid characterization of HIV-1 sequence diversity using denaturing gradient gel electrophoresis and direct automated DNA sequencing of PCR products.

Genome Research ◽

10.1101/gr.2.4.293 ◽

1993 ◽

Vol 2 (4) ◽

pp. 293-300 ◽

Cited By ~ 11

Author(s):

B Andersson ◽

J H Ying ◽

D E Lewis ◽

R A Gibbs

Keyword(s):

Dna Sequencing ◽

Gel Electrophoresis ◽

Denaturing Gradient Gel Electrophoresis ◽

Sequence Diversity ◽

Automated Dna Sequencing ◽

Pcr Products ◽

Gradient Gel Electrophoresis ◽

Rapid Characterization ◽

Hiv 1

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Application of a colorimetric chain-termination assay for characterization of reverse transcriptase from 3′-azido-2′,3′-deoxythymidine-resistant HIV isolates

Biotechnology and Applied Biochemistry ◽

10.1042/ba20010031 ◽

2002 ◽

Vol 35 (3) ◽

pp. 155 ◽

Cited By ~ 4

Author(s):

Xing-Wu Shao ◽

Sandra Hjalmarsson ◽

Johan Lennerstrand ◽

Bo Svennerholm ◽

Jonas Blomberg ◽

...

Keyword(s):

Reverse Transcriptase ◽

Chain Termination

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Isolation and Characterization of Plasminogen Activator from Pig Leucocytes

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649147 ◽

1974 ◽

Vol 31 (01) ◽

pp. 072-085 ◽

Cited By ~ 5

Author(s):

M Kopitar ◽

M Stegnar ◽

B Accetto ◽

D Lebez

Keyword(s):

Molecular Weight ◽

Plasminogen Activator ◽

Gel Electrophoresis ◽

Gel Filtration ◽

Ph Optimum ◽

Isolation And Characterization ◽

Ph Range ◽

Inhibition Studies ◽

Final Purification

SummaryPlasminogen activator was isolated from disrupted pig leucocytes by the aid of DEAE chromatography, gel filtration on Sephadex G-100 and final purification on CM cellulose, or by preparative gel electrophoresis.Isolated plasminogen activator corresponds No. 3 band of the starting sample of leucocyte cells (that is composed from 10 gel electrophoretic bands).pH optimum was found to be in pH range 8.0–8.5 and the highest pH stability is between pH range 5.0–8.0.Inhibition studies of isolated plasminogen activator were performed with EACA, AMCHA, PAMBA and Trasylol, using Anson and Astrup method. By Astrup method 100% inhibition was found with EACA and Trasylol and 30% with AMCHA. PAMBA gave 60% inhibition already at concentration 10–3 M/ml. Molecular weight of plasminogen activator was determined by gel filtration on Sephadex G-100. The value obtained from 4 different samples was found to be 28000–30500.

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Prothrombin Metz : Purification and Characterization of a Variant of Human Prothrombin

10.1055/s-0038-1665670 ◽

1979 ◽

Author(s):

M Ribieto ◽

J Elion ◽

D Labie ◽

F Josso

Keyword(s):

Molecular Weight ◽

Gel Electrophoresis ◽

Polyacrylamide Gel Electrophoresis ◽

Factor Xa ◽

Polyacrylamide Gel ◽

Cleavage Pattern ◽

Purification And Characterization ◽

Double Immunodiffusion ◽

The Family

For the purification of the abnormal prothrombin (Pt Metz), advantage has been taken of the existence in the family of three siblings who, being double heterozygotes for Pt Metz and a hypoprothrombinemia, have no normal Pt. Purification procedures included barium citrate adsorption and chromatography on DEAE Sephadex as for normal Pt. As opposed to some other variants (Pt Barcelona and Madrid), Pt Metz elutes as a single symetrical peak. By SDS polyacrylamide gel electrophoresis, this material is homogeneous and appears to have the same molecular weight as normal Pt. Comigration of normal and abnormal Pt in the absence of SDS, shows a double band suggesting an abnormal charge for the variant. Pt Metz exhibits an identity reaction with the control by double immunodiffusion. Upon activation by factor Xa, Pt Metz can generate amydolytic activity on Bz-Phe-Val-Arg-pNa (S2160), but only a very low clotting activity. Clear abnormalities are observed in the cleavage pattern of Pt Metz when monitored by SDS gel electrophoresis. The main feature are the accumulation of prethrombin l (Pl) and the appearance of abnormal intermediates migrating faster than Pl.

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Intramolecular Proton Transfer in the Isomerization of Hydroxyacetone: A Detailed Characterization Based on Reaction Force Analysis and the Bond Fragility Spectrum

10.26434/chemrxiv.12049062 ◽

2020 ◽

Author(s):

Wallace Derricotte ◽

Huiet Joseph

Keyword(s):

Chemical Potential ◽

Reaction Force ◽

Intramolecular Proton Transfer ◽

Intermediate Energy ◽

Force Analysis ◽

Activation Energy Barrier ◽

Detailed Characterization ◽

Proton Transfers ◽

Reaction Force Constant

The mechanism of isomerization of hydroxyacetone to 2-hydroxypropanal is studied within the framework of reaction force analysis at the M06-2X/6-311++G(d,p) level of theory. Three unique pathways are considered: (i) a step-wise mechanism that proceeds through formation of the Z-isomer of their shared enediol intermediate, (ii) a step-wise mechanism that forms the E-isomer of the enediol, and (iii) a concerted pathway that bypasses the enediol intermediate. Energy calculations show that the concerted pathway has the lowest activation energy barrier at 45.7 kcal mol<sup>-1</sup>. The reaction force, chemical potential, and reaction electronic flux are calculated for each reaction to characterize electronic changes throughout the mechanism. The reaction force constant is calculated in order to investigate the synchronous/asynchronous nature of the concerted intramolecular proton transfers involved. Additional characterization of synchronicity is provided by calculating the bond fragility spectrum for each mechanism.

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