Use of deoxyinosine-containing primers vs degenerate primers for polymerase chain reaction based on ambiguous sequence information

Rossolini, Gian María; Cresti, Stefania; Ingianni, Angela; Cattani, Paola; Riccio, Maria Letizia; Satta, G.

doi:10.1006/mcpr.1994.1013

Cited by 32 publications

(20 citation statements)

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“…Integrated DNA Technologies) provides an option to either ‘machine-mix’ (default) or ‘hand-mix’ the proportions of nucleotides in mixed-base substitutions to achieve specific ratios for each base14, and the machine-mixed option was selected for this study. Judging from the results obtained, the default ‘machine-mix’ option does not skew the results in bisulfite multiplex sequencing as the methylation pattern of assays using the same primers with deoxyinosine modification were similar.…”

Section: Discussionmentioning

confidence: 99%

“…Four modifications: deoxyinosine-, 5-nitroindole-, mixed- (where Y contains a ‘mixture’ of cytosines and thymines at the cytosine site, and R contains a mixture of guanines and adenines at the guanine site) and primers with an abasic site were selected based on their reported stabilities in previous studies131415. The effects of selected modified bases using a number of different conditions were examined, with a focus on the use of different primer substitutions, position of the modified base within the primer, the impact of having a modification in only one primer verses having a modification in both the forward and reverse primers, as well as the optimal conditions under which to perform bisulfite multiplex PCR when using some of these modified bases.…”

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confidence: 99%

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Evaluation of Different Oligonucleotide Base Substitutions at CpG Binding sites in Multiplex Bisulfite-PCR sequencing

Candiloro

et al. 2017

Sci Rep

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Multiplex bisulfite-PCR sequencing is a convenient and scalable method for the quantitative determination of the methylation state of target DNA regions. A challenge of this application is the presence of CpGs in the same region where primers are being placed. A common solution to the presence of CpGs within a primer-binding region is to substitute a base degeneracy at the cytosine position. However, the efficacy of different substitutions and the extent to which bias towards methylated or unmethylated templates may occur has never been evaluated in bisulfite multiplex sequencing applications. In response, we examined the performance of four different primer substitutions at the cytosine position of CpG’s contained within the PCR primers. In this study, deoxyinosine-, 5-nitroindole-, mixed-base primers and primers with an abasic site were evaluated across a series of methylated controls. Primers that contained mixed- or deoxyinosine- base modifications performed most robustly. Mixed-base primers were further selected to determine the conditions that induce bias towards methylated templates. This identified an optimized set of conditions where the methylated state of bisulfite DNA templates can be accurately assessed using mixed-base primers, and expands the scope of bisulfite resequencing assays when working with challenging templates.

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

Evaluation of Different Oligonucleotide Base Substitutions at CpG Binding sites in Multiplex Bisulfite-PCR sequencing

Candiloro

et al. 2017

Sci Rep

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“…Some of the identified regions have already been targeted by other primers previously reported, such as CHR2, a primer targeting the 2C-3A junction of EV (Kottaridi et al, 2007), or the primers EUC12-a, -b and -c designed to partly sequence the 3D-coding region of HEV-B (Oprisan et al, 2002). After back-translation, the linear increase of the primer degeneracy level can be detrimental to amplification sensitivity due to the geometric increase in combinatory possibilities (Rossolini et al, 1994); for that reason, deoxyinosine was introduced in the primers at the nucleotidic positions displaying 4-fold degeneration.…”

Section: Discussionmentioning

confidence: 99%

Characterization of the genome of human enteroviruses: Design of generic primers for amplification and sequencing of different regions of the viral genome

Bessaud

Jégouic

Joffret

et al. 2008

Journal of Virological Methods

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This article reports the development of a simple method allowing amplification and partial sequencing of the P1, P2 and P3 genomic regions of field human enterovirus strains isolated in cell cultures, by performing PCR on cDNAs generated through a single RT reaction. A set of generic primers were designed and tested on a panel of 90 field and prototype viruses belonging to the five species of human enteroviruses. This assay was shown to amplify efficiently the targeted regions of all the 90 genomes tested. The generated amplicons were sequenced successfully without the need for gel purification.This assay could be a precious tool for laboratories interested in molecular epidemiology and evolution studies implicating a great number of human enterovirus strains isolated from human or environmental samples.

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“…Degenerate PCR primers were designed guided by Hanks’ consensus sequence domains VI, VIII and IX with inosine used in high‐degeneracy codons (Hanks and Quinn, 1991; Wilkie and Simon, 1991; Rossolini et al , 1994). Using this strategy, six primers were assembled, which were designated YF, YR, SF, SR, SFn and SRn (Table 1).…”

Section: Methodsmentioning

confidence: 99%

StoPK‐1, a serine/threonine protein kinase from the glycopeptide antibiotic producer Streptomyces toyocaensis NRRL 15009, affects oxidative stress response

Neu

MacMillan

Nodwell

et al. 2002

Molecular Microbiology

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StoPK-1, a serine/threonine protein kinase from the glycopeptide antibiotic producer Streptomyces toyocaensis NRRL 15009, affects oxidative stress response in signalling pathways sensitive to oxidative stress and/or glucose metabolism. These results broaden the roles of Ser/Thr protein kinases in bacteria and underscore the diversity of signal transduction mechanisms available to respond to various stimuli.

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Use of deoxyinosine-containing primers vs degenerate primers for polymerase chain reaction based on ambiguous sequence information

Cited by 32 publications

References 0 publications

Evaluation of Different Oligonucleotide Base Substitutions at CpG Binding sites in Multiplex Bisulfite-PCR sequencing

Evaluation of Different Oligonucleotide Base Substitutions at CpG Binding sites in Multiplex Bisulfite-PCR sequencing

Characterization of the genome of human enteroviruses: Design of generic primers for amplification and sequencing of different regions of the viral genome

StoPK‐1, a serine/threonine protein kinase from the glycopeptide antibiotic producer Streptomyces toyocaensis NRRL 15009, affects oxidative stress response

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