Quantitative Investigation of the Modular Primer Effect for DNA and Peptide Nucleic Acid Hexamers

Nilsson, Peter; O’Meara, Deirdre; Edebratt, Fredrik; Persson, Björn; Uhlén, Mathias; Lundeberg, Joakim; Nygren, Per‐Åke

doi:10.1006/abio.1999.4000

Cited by 7 publications

(5 citation statements)

References 27 publications

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“…In the majority of cases we found that the spacer improved the hybridization signal and stringency (data not shown), which is also supported by previous observations (33). Furthermore, we have shown previously that a probe that anneals adjacent to the capture probe improves the hybridization ef®ciency when analyzed in a biosensor format (34,35). Here by comparing hybridization of labeled s/s DNA with and without a modular probe (anneals 5¢ of the allele-speci®c extension probe) we could con®rm a 1.5±5-fold improvement in hybridization signal on the microarray for the MTHFR and p53 targets, respectively (data not shown).…”

Section: Resultssupporting

confidence: 90%

SNP typing by apyrase-mediated allele-specific primer extension on DNA microarrays

O’Meara

2002

Nucleic Acids Research

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This study reports the development of a microarray-based allele-specific extension method for typing of single nucleotide polymorphisms (SNPs). The use of allele-specific primers has been employed previously to identify single base variations but it is acknowledged that certain mismatches are not refractory to extension. Here we have overcome this limitation by introducing apyrase, a nucleotide-degrading enzyme, to the extension reaction. We have shown previously that DNA polymerases exhibit slower reaction kinetics when extending a mismatched primer compared with a matched primer. This kinetic difference is exploited in the apyrase-mediated allele-specific extension (AMASE) assay, allowing incorporation of nucleotides when the reaction kinetics are fast but degrading the nucleotides before extension when the reaction kinetics are slow. Here we show that five homozygous variants (14% of the total number of variants) that were incorrectly scored in the absence of apyrase were correctly typed when apyrase was included in the extension reaction. AMASE was performed in situ on the oligonucleotide microarrays using fluorescent nucleotides to type 10 SNPs and two indels in 17 individuals generating approximately 200 genotypes. Cluster analysis of these data shows three distinct clusters with clear-cut boundaries. We conclude that SNP typing on oligonucleotide microarrays by AMASE is an efficient, rapid and accurate technique for large-scale genotyping.

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Section: Resultssupporting

confidence: 90%

SNP typing by apyrase-mediated allele-specific primer extension on DNA microarrays

O’Meara

2002

Nucleic Acids Research

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“…2). The stacking interaction of these with the adjacent fragments is, therefore, lower (14,15). In order to maximize the possibility for stacking interactions between each of the individual fragments, the molar ratio of capture probe to restriction fragments in the hybridization reaction was kept below 2.5.…”

Section: Resultsmentioning

confidence: 99%

Mass spectrometry of single-stranded restriction fragments captured by an undigested complementary sequence

Chiu

Tang²,

Yip³

et al. 2000

Nucleic Acids Research

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In this report, we describe a simple and accurate method to analyze restriction fragments using matrixassisted laser desorption/ionization time-of-flight mass spectrometry. The two complementary strands of restriction fragments are separated through hybridization to a capture probe, which is a singlestranded undigested fragment. Using the biotinstreptavidin linkage, the hybrid is immobilized on streptavidin-coated magnetic beads. After conditioning the captured restriction fragments, they are eluted from the probe and their molecular weights are determined. The proposed method greatly improves the quality, and reduces the complexity of the mass spectrum by analyzing only one of the complementary strands of restriction fragments.

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“…Future modifications can be envisioned such as designing new probes for capture and adapting the protocol for automated systems. Improvements on probe design could involve varying the capture sequences or the use of nucleotide derivatives (such as PNA) or spacer sequences (10). For such purposes, it would be possible to take advantage of biosensor technology that has been used earlier for real-time, quantitative estimation of hybridization events (10)(11)(12)(13).…”

Section: Discussionmentioning

confidence: 99%

“…Improvements on probe design could involve varying the capture sequences or the use of nucleotide derivatives (such as PNA) or spacer sequences (10). For such purposes, it would be possible to take advantage of biosensor technology that has been used earlier for real-time, quantitative estimation of hybridization events (10)(11)(12)(13). We have previously shown that magnetic separation can successfully be automated using both commercial and in-house instrumentation for similar purposes (7,15,17).…”

Section: Discussionmentioning

confidence: 99%

Cooperative Oligonucleotides in Purification of Cycle Sequencing Products

et al. 2000

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Nucleic acid hybridization is an essential component in many of today's standard molecular biology techniques. In a recent study, we investigated whether nucleic acid capture could be improved by taking advantage of stacking hybridization, which refers to the stabilizing effect that exists between oligonucleotides when they hybridize in a contiguous tandem fashion. Here, we describe a specific approach for purification of sequencing products using cooperative probes that hybridize to single-strand targets where one of the probes has been coupled to a magnetic bead. This approach has been developed for standard sequencing primers and has been applied to shotgun plasmid libraries. The cooperative probes have been designed to anneal within the common vector sequence and to avoid co-purification of nonextended sequencing primers and misprimed sequencing products. The reuse of magnetic beads, together with salt independent elution, makes the approach suitable for high-capacity capillary electrophoresis instruments.

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Quantitative Investigation of the Modular Primer Effect for DNA and Peptide Nucleic Acid Hexamers

Cited by 7 publications

References 27 publications

SNP typing by apyrase-mediated allele-specific primer extension on DNA microarrays

SNP typing by apyrase-mediated allele-specific primer extension on DNA microarrays

Mass spectrometry of single-stranded restriction fragments captured by an undigested complementary sequence

Cooperative Oligonucleotides in Purification of Cycle Sequencing Products

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