Multiplex PCR: use of heat-stable Thermus thermophilus RecA protein to minimize non-specific PCR products

Shigemori, Yasushi; Mikawa, Tsutomu; Shibata, Takehiko; Oishi, Michio

doi:10.1093/nar/gni111

Cited by 50 publications

(41 citation statements)

References 29 publications

(16 reference statements)

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“…Protein Expression and Purification-Unlabeled and uniformly 2 H/ 15 N-or 15 N-labeled T. thermophilus HB8 RecR and RecA (ttRecA) proteins were prepared as previously described (23,24). RecO (ttRecO), RecF (ttRecF), and SSB (ttSSB) proteins were prepared as described below.…”

Section: Methodsmentioning

confidence: 99%

Identification of the RecR Toprim Domain as the Binding Site for both RecF and RecO

Honda

Inoue

Yoshimasu

et al. 2006

Journal of Biological Chemistry

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The RecR protein forms complexes with RecF or RecO that direct the specific loading of RecA onto gapped DNA. However, the binding sites of RecF and RecO on RecR have yet to be identified. In this study, a Thermus thermophilus RecR dimer model was constructed by NMR analysis and homology modeling. NMR titration analysis suggested that the hairpin region of the helix-hairpin-helix motif in the cavity of the RecR dimer is a binding site for double-stranded DNA (dsDNA) and that the acidic cluster region of the Toprim domain is a RecO binding site. Mutations of Glu-84, Asp-88, and Glu-144 residues comprising that acidic cluster were generated. The E144A and E84A mutations decreased the binding affinity for RecO, but the D88A did not. Interestingly, the binding ability to RecF was abolished by E144A, suggesting that the region surrounding the RecR Glu-144 residue could be a binding site not only for RecO but also for RecF. Furthermore, RecR and RecF formed a 4:2 heterohexamer in solution that was unaffected by adding RecO, indicating a preference by RecR for RecF over RecO. The RecFR complex is considered to be involved in the recognition of the dsDNA-ssDNA junction, whereas RecO binds single-stranded DNA (ssDNA) and ssDNA-binding protein. Thus, the RecR Toprim domain may contribute to the RecO interaction with RecFR complexes at the dsDNA-ssDNA junction site during recombinational DNA repair mediated by the RecFOR.Maintaining genomic integrity by repairing damaged DNA is crucial for all organisms. DNA damage can arise during normal DNA metabolism such as the introduction of mismatches during replication and the deamination of bases, or it can be caused by exposure to exogenous factors such as ultraviolet radiation, ␥-radiation, and chemical mutagens. Such DNA damage is mostly repaired by base excision repair, nucleotide excision repair, and mismatch repair pathways, based on information provided by the complementary strand in a damaged duplex. However, double-stranded DNA (dsDNA) 2 break and base lesions in single-stranded DNA (ssDNA) gap regions having no complementary strands can also arise, mainly during replication. To cope with this category of DNA damage, organisms have developed recombinational repair pathways that minimize the loss of genetic information by using homologous DNA as a template for repair.RecFOR and/or RecBCD pathways are required to initiate homologous DNA recombination in bacteria (1). The Escherichia coli RecFOR pathway is mainly used for ssDNA gap repair, whereas the RecBCD pathway is responsible for dsDNA break repair. However, the RecFOR pathway can also repair dsDNA breaks, as has been demonstrated in recBC sbcB mutants, which are deficient in the RecBCD pathway (2, 3). In the RecFOR pathway, dsDNA breaks are unwound by the RecQ helicase and processed by the RecJ 5Ј-to 3Ј-exonuclease, and the resulting 3Ј-tailed ssDNA is coated with the ssDNA-binding protein (SSB). The RecF, RecO, and RecR proteins then mediate the loading of RecA protein onto the SSB-coated ssDNA, specifically at junctio...

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Section: Methodsmentioning

confidence: 99%

Identification of the RecR Toprim Domain as the Binding Site for both RecF and RecO

Honda

Inoue

Yoshimasu

et al. 2006

Journal of Biological Chemistry

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“…The antibody-mediated hot start method is significantly more convenient than the hot start method using wax; however, hot start is not always successful, especially when long DNA and high-GC-content DNA are used as the templates. A thermostable RecA protein that is involved in DNA recombination reduces nonspecific amplification in PCR (11,12). In addition, a technique was reported in which the mismatchrecognizing protein MutS from a thermophilic bacterium was added to the PCR mixture for accurate DNA amplification (13).…”

mentioning

confidence: 99%

Application of a Euryarchaeota-Specific Helicase from Thermococcus kodakarensis for Noise Reduction in PCR

Fujiwara

Kawato

Kato

et al. 2016

Appl Environ Microbiol

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DNA/RNA helicases, which are enzymes for eliminating hydrogen bonds between bases of DNA/DNA, DNA/RNA, and RNA/ RNA using the energy of ATP hydrolysis, contribute to various biological activities. In the present study, the Euryarchaeota-specific helicase EshA (TK0566) from the hyperthermophilic archaeon Thermococcus kodakarensis (Tk-EshA) was obtained as a recombinant form, and its enzymatic properties were examined. Tk-EshA exhibited maximal ATPase activity in the presence of RNA at 80°C. Unwinding activity was evaluated with various double-stranded DNAs (forked, 5= overhung, 3= overhung, and blunt end) at 50°C. Tk-EshA unwound forked and 3= overhung DNAs. These activities were expected to unwind the structured template and to peel off misannealed primers when Tk-EshA was added to a PCR mixture. To examine the effect of Tk-EshA on PCR, various target DNAs were selected, and DNA synthesis was investigated. When 16S rRNA genes were used as a template, several misamplified products (noise DNAs) were detected in the absence of Tk-EshA. In contrast, noise DNAs were eliminated in the presence of Tk-EshA. Noise reduction by Tk-EshA was also confirmed when Taq DNA polymerase (a family A DNA polymerase, PolI type) and KOD DNA polymerase (a family B DNA polymerase, ␣ type) were used for PCR. Misamplified bands were also eliminated during toxA gene amplification from Pseudomonas aeruginosa DNA, which possesses a high GC content (69%). Tk-EshA addition was more effective than increasing the annealing temperature to reduce misamplified DNAs during toxA amplification. Tk-EshA is a useful tool to reduce noise DNAs for accurate PCR. IMPORTANCEPCR is a technique that is useful for genetic diagnosis, genetic engineering, and detection of pathogenic microorganisms. However, troubles with nonspecific DNA amplification often occur from primer misannealing. In order to achieve a specific DNA amplification by eliminating noise DNAs derived from primer misannealing, a thermostable Euryarchaeota-specific helicase (Tk-EshA) was included in the PCR mixture. The addition of Tk-EshA has reduced noise DNAs in PCR.

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“…While some researchers have suggested the addition of certain reagents (Shigemori et al 2005) or the use of software tools (Nicodeme and Steyaert 1997) in order to increase the compatibility of primers in a reaction, others had seemingly little trouble amplifying hundreds of targets without prior optimizations (Eichinger et al 2005). To evaluate the general applicability of our approach, we conducted further experiments with both modern DNA and permafrost-derived Figure 2.…”

Section: Dmps With Modern Dna and Other Primer Mixesmentioning

confidence: 99%

Direct multiplex sequencing (DMPS)—a novel method for targeted high-throughput sequencing of ancient and highly degraded DNA

Stiller¹,

Knapp²,

Stenzel³

et al. 2009

Genome Res.

103

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Although the emergence of high-throughput sequencing technologies has enabled whole-genome sequencing from extinct organisms, little progress has been made in accelerating targeted sequencing from highly degraded DNA. Here, we present a novel and highly sensitive method for targeted sequencing of ancient and degraded DNA, which couples multiplex PCR directly with sample barcoding and high-throughput sequencing. Using this approach, we obtained a 96% complete mitochondrial genome data set from 31 cave bear (Ursus spelaeus) samples using only two 454 Life Sciences (Roche) GS FLX runs. In contrast to previous studies relying only on short sequence fragments, the overlapping portion of our data comprises almost 10 kb of replicated mitochondrial genome sequence, allowing for the unambiguous differentiation of three major cave bear clades. Our method opens up the opportunity to simultaneously generate many kilobases of overlapping sequence data from large sets of difficult samples, such as museum specimens, medical collections, or forensic samples. Embedded in our approach, we present a new protocol for the construction of barcoded sequencing libraries, which is compatible with all current high-throughput technologies and can be performed entirely in plate setup.

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Multiplex PCR: use of heat-stable Thermus thermophilus RecA protein to minimize non-specific PCR products

Cited by 50 publications

References 29 publications

Identification of the RecR Toprim Domain as the Binding Site for both RecF and RecO

Identification of the RecR Toprim Domain as the Binding Site for both RecF and RecO

Application of a Euryarchaeota-Specific Helicase from Thermococcus kodakarensis for Noise Reduction in PCR

Direct multiplex sequencing (DMPS)—a novel method for targeted high-throughput sequencing of ancient and highly degraded DNA

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