Immobilization of acrylamide-modified oligonucleotides by co-polymerization

Rehman, Fida; Audeh, M; Abrams, Ezra S.; Hammond, P W; Kenney, M. Cristina; Boles, T. Christian

doi:10.1093/nar/27.2.649

Cited by 155 publications

(137 citation statements)

References 35 publications

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“…Because the acrylamide matrix restricts the diffusion of amplification products, double-stranded DNA accumulates around the chromosome, forming two overlapping polonies, each amplified from a different region on the same molecule of DNA. A key feature of this protocol is the use of modified primers in the PCR reaction that covalently attach one strand of the amplified DNA to the acrylamide matrix (19,20). This feature allows us to remove the unattached strand from all polonies by heating and washing the slide, leaving single-stranded templates for subsequent SBE reactions (21)(22)(23).…”

Section: Resultsmentioning

confidence: 99%

Digital genotyping and haplotyping with polymerase colonies

Mitra

Butty

Shendure

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

139

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Polymerase colony (polony) technology amplifies multiple individual DNA molecules within a thin acrylamide gel attached to a microscope slide. Each DNA molecule included in the reaction produces an immobilized colony of double-stranded DNA. We genotype these polonies by performing single base extensions with dye-labeled nucleotides, and we demonstrate the accurate quantitation of two allelic variants. We also show that polony technology can determine the phase, or haplotype, of two singlenucleotide polymorphisms (SNPs) by coamplifying distally located targets on a single chromosomal fragment. We correctly determine the genotype and phase of three different pairs of SNPs. In one case, the distance between the two SNPs is 45 kb, the largest distance achieved to date without separating the chromosomes by cloning or somatic cell fusion. The results indicate that polony genotyping and haplotyping may play an important role in understanding the structure of genetic variation.T he study of genetic variation in the human population has the potential to greatly improve human health, both by predicting susceptibility to disease and guiding choice of therapy. The most common genetic variations in the human population are single-nucleotide polymorphisms (SNPs). By studying candidate genes and performing genome-wide linkage disequilibrium studies, scientists are trying to uncover the ''causative SNP,'' the SNP that alters gene function and thereby increases the risk of disease. It has been shown that deriving haplotypes increases the efficiency of linkage disequilibrium studies (1). Surprisingly, recent studies (2, 3) suggest that haplotypes will also be critical for candidate gene studies. These studies found that for some diseases, there is not one single SNP that is responsible for altering gene function, but instead, multiple SNPs interact to alter the function or expression of a protein (4). These alterations occur only when specific combinations of SNPs are present on the same chromosome, so one must determine the haplotype to find a correlation to the observed phenotype. In these cases, the focus has shifted from a causative SNP to a causative haplotype.The most common approach for determining the haplotype, or phase, of a set of SNPs is computational inference from unphased data (3,(5)(6)(7)(8). Although this methodology has greatly increased the power of both linkage and candidate gene studies, a recent study estimates the error rate to be between 19 and 48%, depending on the algorithm used (6). This error rate presents challenges for the use of this method as a research tool and makes it an unlikely candidate for use in the clinical setting. Recent findings suggest a way to improve the accuracy of haplotype inference. Daly et al. (9) and others (10, 11) have shown that SNPs tend to be inherited in larger haplotype blocks than previously thought, and that there are relatively few variants of each block. This observation has sparked a public effort to characterize all common haplotypes in the human population (12). ...

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Section: Resultsmentioning

confidence: 99%

Digital genotyping and haplotyping with polymerase colonies

Mitra

Butty

Shendure

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

139

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“…Any DNA without an acrydite group will be washed away. 33,34 Isothermal titration calorimetry (ITC). ITC was performed using a VP-ITC Microcalorimeter instrument (MicroCal).…”

Section: Dynamic Light Scattering (Dls)mentioning

confidence: 99%

Molecularly Imprinted Polymers with DNA Aptamer Fragments as Macromonomers

Zhang

Liu

2016

ACS Appl. Mater. Interfaces

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Molecularly imprinted polymers (MIPs) are produced in the presence of a template molecule. After removing the template, the cavity can selectively rebind the template. MIPs are attractive functional materials with a low cost and high stability, but traditional MIPs often suffer from low binding affinity. This study employs DNA aptamer fragments as macromonomers to improve MIPs. The DNA aptamer for adenosine was first split into two halves, fluorescently labeled, and co-polymerized into MIPs. With a fluorescence quenching assay, the importance of imprinting was confirmed. Further studies were carried out using isothermal titration calorimetry (ITC).Compared to the mixture of the free aptamer fragments, their MIPs doubled the binding affinity.Each free aptamer fragment alone cannot bind adenosine, while MIPs containing each fragment are effective binders. We further shortened one of the aptamer fragments, and the DNA length was pushed to as short as six nucleotides, yielding MIPs with a dissociation constant of 27 µM adenosine. This study provides a new method for preparing functional MIP materials by combining high affinity biopolymer fragments with low cost synthetic monomers, allowing higher binding affinity and providing a method for signaling binding based on DNA chemistry.

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“…An acrydite DNA has a similar reactivity as free acrylamide monomers, allowing a high incorporation efficiency [43]. While the field of DNA-functionalized hydrogels began in 1996 [44], most of the developments have occurred in the past ten years [45][46][47][48].…”

Section: Dna Attachmentmentioning

confidence: 99%