Direct sequencing of double-stranded DNA PCR products via removing the complementary strand with single-stranded DNA of an M13 clone

Abstract To examine the role of the RAD52 recombinational repair pathway in compensating for DNA replication defects in Saccharomyces cerevisiae, we performed a genetic screen to identify mutants that require Rad52p for viability. We isolated 10 mec1 mutations that display synthetic lethality with rad52. These mutations (designated mec1-srf for synthetic lethality with rad-fifty-two) simultaneously cause two types of phenotypes: defects in the checkpoint function of Mec1p and defects in the essential function of Mec1p. Velocity sedimentation in alkaline sucrose gradients revealed that mec1-srf mutants accumulate small single-stranded DNA synthesis intermediates, suggesting that Mec1p is required for the normal progression of DNA synthesis. sml1 suppressor mutations suppress both the accumulation of DNA synthesis intermediates and the requirement for Rad52p in mec1-srf mutants, but they do not suppress the checkpoint defect in mec1-srf mutants. Thus, it appears to be the DNA replication defects in mec1-srf mutants that cause the requirement for Rad52p. By using hydroxyurea to introduce similar DNA replication defects, we found that single-stranded DNA breaks frequently lead to double-stranded DNA breaks that are not rapidly repaired in rad52 mutants. Taken together, these data suggest that the RAD52 recombinational repair pathway is required to prevent or repair double-stranded DNA breaks caused by defective DNA replication in mec1-srf mutants.

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The replication process of single stranded DNA of bacteriophage φX174 II. The non-intermediation of the double stranded DNA as a material precursor

Biochemical and Biophysical Research Communications ◽

10.1016/0006-291x(63)90125-6 ◽

1963 ◽

Vol 11 (5) ◽

pp. 372-377 ◽

Cited By ~ 11

Author(s):

Kenichi Matsubara ◽

Masayuki Takai ◽

Yasuyuki Takagi

Keyword(s):

Replication Process ◽

Single Stranded Dna ◽

Double Stranded Dna

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DNA Binding by the Methanothermobacter thermautotrophicus Cdc6 Protein Is Inhibited by the Minichromosome Maintenance Helicase

Journal of Bacteriology ◽

10.1128/jb.00168-06 ◽

2006 ◽

Vol 188 (12) ◽

pp. 4577-4580 ◽

Cited By ~ 14

Author(s):

Rajesh Kasiviswanathan ◽

Jae-Ho Shin ◽

Zvi Kelman

Keyword(s):

Dna Binding ◽

Binding Activity ◽

Minichromosome Maintenance ◽

Single Stranded Dna ◽

Mutant Proteins ◽

Dna Binding Activity ◽

Double Stranded Dna ◽

Mcm Helicase ◽

Methanothermobacter Thermautotrophicus

ABSTRACT The Cdc6 proteins from the archaeon Methanothermobacter thermautotrophicus were previously shown to bind double-stranded DNA. It is shown here that the proteins also bind single-stranded DNA. Using minichromosome maintenance (MCM) helicase mutant proteins unable to bind DNA, it was found that the interaction of MCM with Cdc6 inhibits the DNA binding activity of Cdc6.

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Detection of erythromycin-resistant determinants by PCR.

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.40.11.2562 ◽

1996 ◽

Vol 40 (11) ◽

pp. 2562-2566 ◽

Cited By ~ 632

Author(s):

J Sutcliffe ◽

T Grebe ◽

A Tait-Kamradt ◽

L Wondrack

Keyword(s):

Direct Sequencing ◽

Pcr Primers ◽

Efflux Pumps ◽

Clinical Isolates ◽

Gene Products ◽

Mechanisms Of Resistance ◽

Erythromycin Resistance ◽

Surveillance Studies ◽

Resistance Determinants ◽

Pcr Products

Erythromycin resistance determinants include Erm methylases, efflux pumps, and inactivating enzymes. To distinguish the different mechanisms of resistance in clinical isolates, PCR primers were designed so that amplification of the partial gene products could be detected in multiplex PCRs. This methodology enables the direct sequencing of amplified PCR products that can be used to compare resistance determinants in clinical strains. Further, this methodology could be useful in surveillance studies of erythromycin-resistant determinants.

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Archaeal Orc1 protein interacts with T-rich single-stranded DNA

BMC Research Notes ◽

10.1186/s13104-021-05690-w ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Katarzyna Wegrzyn ◽

Igor Konieczny

Keyword(s):

Dna Replication ◽

Replication Initiation ◽

Single Stranded Dna ◽

Dna Replication Initiation ◽

Double Stranded Dna ◽

Nucleoprotein Complexes ◽

Eukaryotic Organisms ◽

Replication Initiation Proteins ◽

Hydrolysis Of ◽

Replication Activity

Abstract Objective The ability to form nucleoprotein complexes is a fundamental activity of DNA replication initiation proteins. They bind within or nearby the region of replication origin what results in melting of a double-stranded DNA (dsDNA) and formation of single-stranded DNA (ssDNA) region where the replication machinery can assemble. For prokaryotic initiators it was shown that they interact with the formed ssDNA and that this interaction is required for the replication activity. The ability to interact with ssDNA was also shown for Saccharomyces cerevisiae replication initiation protein complex ORC. For Archaea, which combine features of both prokaryotic and eukaryotic organisms, there was no evidence whether DNA replication initiators can interact with ssDNA. We address this issue in this study. Results Using purified Orc1 protein from Aeropyrum pernix (ApOrc1) we analyzed its ability to interact with ssDNA containing sequence of an AT-rich region of the A. pernix origin Ori1 as well as with homopolymers of thymidine (polyT) and adenosine (polyA). The Bio-layer interferometry, surface plasmon resonance and microscale thermophoresis showed that the ApOrc1 can interact with ssDNA and it binds preferentially to T-rich ssDNA. The hydrolysis of ATP is not required for this interaction.

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Human Rhinovirus Molecular Epidemiology and Genotyping in Iranian Military Trainees with Acute Respiratory Symptoms

Journal of Archives in Military Medicine ◽

10.5812/jamm.111224 ◽

2021 ◽

Vol 8 (4) ◽

Author(s):

Fahimeh Safarnezhad Tameshkel ◽

Ali Salimi Jeda ◽

Ahmad Tavakoli ◽

Mohammad Hadi Karbalaie Niya ◽

Morteza Izadi ◽

...

Keyword(s):

Molecular Epidemiology ◽

Clinical Symptoms ◽

Respiratory Infections ◽

Direct Sequencing ◽

Human Rhinovirus ◽

P Value ◽

Rt Pcr ◽

Respiratory Problems ◽

Pcr Products ◽

Tract Infections

Background: Human rhinovirus (HRV) is still the most prevalent viral infection in humans and a significant cause of acute respiratory tract infections (ARTIs) in many communities, including military personnel undergoing basic training. Objectives: In this research, we assessed the molecular epidemiology, genotyping, and phylogenetic classification of HRVs in Iranian military trainees with respiratory infections (RI). Methods: For HRV identification and genotyping, respiratory specimens were obtained, and RT-PCR was conducted for genotyping and phylogenetic analysis of HRV utilizing primers for the 5-UTR region. Results: Among 400 Iranian military trainees (average age of 21 ± 4 years, the range of 18 - 57 years) with respiratory infections, HRV was detected in 29 patients (7%) using RT-PCR. The direct sequencing of PCR products from 10 specimens showed that the incidence of type A (n = 5, 50%) was higher than that of type B (n = 4, 40%) and type C (n = 1, 10%). There were no significant associations between HRV and respiratory and clinical symptoms, blood group, and indoor or outdoor conditions (P-value > 0.05). Conclusions: This research was the first to record HRV as a significant cause of respiratory problems among military trainees in Iran, with a frequency of 7%. The most prevalent genotype was HRV-A, which may be applicable in epidemiological and clinical studies, as well as vaccination plans.

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A colorimetric method for the sequence-specific recognition of double-stranded DNA on the surface of a silver-coated glass slide

Canadian Journal of Chemistry ◽

10.1139/cjc-2017-0544 ◽

2018 ◽

Vol 96 (5) ◽

pp. 466-470

Author(s):

Xiaoting Guo ◽

Jing Wang ◽

Zhifang Zhu ◽

Manjun Zhang ◽

Haigang Li ◽

...

Keyword(s):

Colorimetric Method ◽

Colour Change ◽

Glass Slide ◽

Coated Glass ◽

Specific Recognition ◽

Double Stranded Dna ◽

Pcr Products ◽

Polymerase Chain ◽

Good Sequence ◽

Coated Glass Slide

In this study, a colorimetric method for sequence-specific recognition of double-stranded DNA (dsDNA) was established on the surface of a silver-coated glass slide. Oligo-1 was assembled on the surface of a silver-coated glass slide through an Ag–S bond, and Oligo-2 as reporter was used to bind with streptavidin-horseradish peroxidase (SA–HRP). They could bind with target dsDNA that was composed of Oligo-3 and Oligo-4 on the surface of a silver-coated glass slide through triplex formation. The bound HRP could be moved into the solution by DNase I and catalyze the oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB). Therefore, the concentration of target dsDNA could be determined with the colour change of TMB. Under the optimum conditions, the absorbance was proportional to the concentration of target dsDNA over the range of 100 pmol/L to 2.0 nmol/L, with a detection limit of 13 pmol/L. In addition, this method showed good sequence selectivity, enabling it to be further developed for the detection of other polymerase chain reaction (PCR) products.

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How to copy and paste DNA microarrays

Scientific Reports ◽

10.1038/s41598-019-50371-1 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 1

Author(s):

Stefan D. Krämer ◽

Johannes Wöhrle ◽

Philipp A. Meyer ◽

Gerald A. Urban ◽

Günter Roth

Keyword(s):

Real Time ◽

Dna Microarray ◽

Dna Microarrays ◽

Label Free ◽

Binding Assays ◽

Single Stranded Dna ◽

Whole Process ◽

Double Stranded Dna ◽

Cost Efficient ◽

Copy And Paste

Abstract Analogous to a photocopier, we developed a DNA microarray copy technique and were able to copy patterned original DNA microarrays. With this process the appearance of the copied DNA microarray can also be altered compared to the original by producing copies of different resolutions. As a homage to the very first photocopy made by Chester Charlson and Otto Kornei, we performed a lookalike DNA microarray copy exactly 80 years later. Those copies were also used for label-free real-time kinetic binding assays of apo-dCas9 to double stranded DNA and of thrombin to single stranded DNA. Since each DNA microarray copy was made with only 5 µl of spPCR mix, the whole process is cost-efficient. Hence, our DNA microarray copier has a great potential for becoming a standard lab tool.

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Effects of recJ, recQ, and recFOR Mutations on Recombination in Nuclease-Deficient recB recD Double Mutants of Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.187.4.1350-1356.2005 ◽

2005 ◽

Vol 187 (4) ◽

pp. 1350-1356 ◽

Cited By ~ 28

Author(s):

Ivana Ivančić-Baće ◽

Erika Salaj-Šmic ◽

Krunoslav Brčić-Kostić

Keyword(s):

Escherichia Coli ◽

Double Mutant ◽

Reca Protein ◽

Recq Helicase ◽

Single Stranded Dna ◽

Double Stranded Dna ◽

Recf Pathway ◽

Recombination Pathway ◽

Recbcd Enzyme ◽

Dna Ends

ABSTRACT The two main recombination pathways in Escherichia coli (RecBCD and RecF) have different recombination machineries that act independently in the initiation of recombination. Three essential enzymatic activities are required for early recombinational processing of double-stranded DNA ends and breaks: a helicase, a 5′→3′ exonuclease, and loading of RecA protein onto single-stranded DNA tails. The RecBCD enzyme performs all of these activities, whereas the recombination machinery of the RecF pathway consists of RecQ (helicase), RecJ (5′→3′ exonuclease), and RecFOR (RecA-single-stranded DNA filament formation). The recombination pathway operating in recB (nuclease-deficient) mutants is a hybrid because it includes elements of both the RecBCD and RecF recombination machineries. In this study, genetic analysis of recombination in a recB (nuclease-deficient) recD double mutant was performed. We show that conjugational recombination and DNA repair after UV and gamma irradiation in this mutant are highly dependent on recJ, partially dependent on recFOR, and independent of recQ. These results suggest that the recombination pathway operating in a nuclease-deficient recB recD double mutant is also a hybrid. We propose that the helicase and RecA loading activities belong to the RecBCD recombination machinery, while the RecJ-mediated 5′→3′ exonuclease is an element of the RecF recombination machinery.

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