Sensitive Quantification of Somatic Mutations Using Molecular Inversion Probes

Hirani, Rena; Connolly, Ashley R.; Putral, Lisa N.; Dobrovic, Alexander; Trau, Matt

doi:10.1021/ac2019409

Cited by 6 publications

(4 citation statements)

References 40 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This approach was well-suited to maintaining a high level of specificity with the flexibility of incorporating “universal” inosine nucleotides in each hybridization domain. A small amount of nonspecific ligation (3%–7%) was found to be consistent with the promiscuity of DNA ligase in assays containing synthetic DNA. , In practice, however, the specificity of ligation-dependent assays performed on more-complex genomic extracts is expected to approach 0.1% . The specificity is also defined by the uniqueness of the DNA target sequence, which can often be made redundant following bisulfite treatment, which substantially reduces the nucleotide sequence complexity of DNA.…”

Section: Discussionmentioning

confidence: 79%

“…16,40 In practice, however, the specificity of ligation-dependent assays performed on morecomplex genomic extracts is expected to approach 0.1%. 13 The specificity is also defined by the uniqueness of the DNA target sequence, which can often be made redundant following bisulfite treatment, which substantially reduces the nucleotide sequence complexity of DNA.…”

Section: ■ Discussionmentioning

confidence: 99%

“…As a result, ligation-dependent amplification (LDA) assays have emerged as an alternative method for analyzing DNA methylation. − This approach enables many different suspected DNA methylation sites to be analyzed in a single multiplex assay . Two DNA probes designed to “frame” a suspected cytosine methylation site in bisulfite-treated DNA undergo ligation only if they complement the nucleotide at the suspected methylation site.…”

mentioning

confidence: 99%

See 2 more Smart Citations

Accurate Detection of Methylated Cytosine in Complex Methylation Landscapes

2013

Self Cite

View full text Add to dashboard Cite

Monitoring DNA methylation can be a useful biomarker for disease diagnosis and prognosis. However, monitoring the methylation status of a specific cytosine biomarker is often confounded by heterogeneous peripheral DNA methylation. To address this issue, molecular inversion probes were designed with inosine strategically positioned to complement suspected DNA methylation sites. This enabled the methylation status of a specific cytosine to be accurately measured with a high level of specificity, irrespective of adjacent epigenetic modifications.

show abstract

Section: Discussionmentioning

confidence: 79%

Section: ■ Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

Accurate Detection of Methylated Cytosine in Complex Methylation Landscapes

2013

Self Cite

View full text Add to dashboard Cite

show abstract

“…This looped physical conformation differentiates it from a variant of the MIP technology which does not generate a single stranded gap between the binding domains known as padlock probes (PLP). [189][190][191] Padlock probes (PLP) have been developed for several applications including plant pathogen identification. 87,107,192,193 However, PLPs exhibited limited accuracy when detecting different pathogens from closely related species 193 , mostly due to the fact that the specificity of the PLP assay depends on the fidelity of the ligase which has been known to be promiscuous.…”

Section: Discussionmentioning

confidence: 99%

Development of point-of-care and multiplex diagnostic methods for the detection of plant pathogens

Lau¹

View full text Add to dashboard Cite

New diagnostic technologies for the detection of plant pathogens with high multiplexing ability and point-of-care (POC) capability are an essential tool in the fight to reduce the large agricultural production losses caused by plant diseases. The main desirable characteristics for such diagnostic assays are high specificity, sensitivity, reproducibility, quickness, cost efficiency and highthroughput multiplex detection capability. The aim of this project is to develop new methods combining nucleic acid amplification, multiplexing and point-of-care application for the detection of plant pathogens.Isothermal amplification methods, such as recombinase polymerase amplification (RPA) which operates at low constant temperature are especially suited for POC applications. However, the development of a rapid and simplified detection method for the isothermal amplification products in resource-poor settings is still challenging. A new method to visualize the success of the amplification step, and therefore the presence of pathogen DNA in a sample, was developed based on bridging flocculation. This method requires minimal equipment and can be assessed by the naked eye allowing its use in POC settings. The method was initially applied to the rapid and sensitive detection of several important plant pathogens and subsequently extended to animal and human pathogens to demonstrate the wide applications of the approach. A nanoparticle based electrochemical biosensor was also developed for rapid and sensitive detection of amplified plant pathogen DNA on screen-printed carbon electrodes. Gold nanoparticles were employed as a probe for electrochemical assessment by differential pulse voltammetry (DPV). This method was 10,000 times more sensitive than conventional polymerase chain reaction (PCR)/gel electrophoresis and could readily identify P. syringae infected plant samples even before the disease symptoms were visible.To allow multiplex detection of plant pathogen, a novel method that can screen for thousands of plant pathogens with high specificity and sensitivity using molecular inversion probes (MIPs) was been developed. As proof of concept, a MIP targeting a unique DNA sequence present in the F.oxysporum f.sp. conglutinans (Foc) genome was designed. The specificity, sensitivity and detection limit of the assay were assessed and used to detect the presence of pathogen in infected Arabidopsis thaliana samples. This methodology successfully detected as little as 2.5 ng of pathogen DNA and it was highly specific, being able to accurately differentiate Fusarium oxysporum f.sp. conglutinans from other fungal pathogens such as Botrytis cinerea and even pathogens of the same species such as Fusarium oxysporum f.sp. lycopersici. Finally, a diagnostic platform for POC plant pathogen detection using a combination of surface-enhanced Raman ii scattering (SERS) and RPA was developed for rapid multiplex detection. The RPA-SERS method was faster, more sensitive than polymerase chain reaction and could detect as little as 2 copies of...

show abstract

Future of Biosensors: A Personal View

Scheller

Yarman

Bachmann

et al. 2013

Advances in Biochemical Engineering/Biotechnology

View full text Add to dashboard Cite

Biosensors representing the technological counterpart of living senses have found routine application in amperometric enzyme electrodes for decentralized blood glucose measurement, interaction analysis by surface plasmon resonance in drug development, and to some extent DNA chips for expression analysis and enzyme polymorphisms. These technologies have already reached a highly advanced level and need minor improvement at most. The dream of the "100-dollar" personal genome may come true in the next few years provided that the technological hurdles of nanopore technology or of polymerase-based single molecule sequencing can be overcome. Tailor-made recognition elements for biosensors including membrane-bound enzymes and receptors will be prepared by cell-free protein synthesis. As alternatives for biological recognition elements, molecularly imprinted polymers (MIPs) have been created. They have the potential to substitute antibodies in biosensors and biochips for the measurement of low-molecular-weight substances, proteins, viruses, and living cells. They are more stable than proteins and can be produced in large amounts by chemical synthesis. Integration of nanomaterials, especially of graphene, could lead to new miniaturized biosensors with high sensitivity and ultrafast response. In the future individual therapy will include genetic profiling of isoenzymes and polymorphic forms of drug-metabolizing enzymes especially of the cytochrome P450 family. For defining the pharmacokinetics including the clearance of a given genotype enzyme electrodes will be a useful tool. For decentralized online patient control or the integration into everyday "consumables" such as drinking water, foods, hygienic articles, clothing, or for control of air conditioners in buildings and cars and swimming pools, a new generation of "autonomous" biosensors will emerge.

show abstract

Sensitive Quantification of Somatic Mutations Using Molecular Inversion Probes

Cited by 6 publications

References 40 publications

Accurate Detection of Methylated Cytosine in Complex Methylation Landscapes

Accurate Detection of Methylated Cytosine in Complex Methylation Landscapes

Development of point-of-care and multiplex diagnostic methods for the detection of plant pathogens

Future of Biosensors: A Personal View

Contact Info

Product

Resources

About