Single-Tube Genotyping without Oligonucleotide Probes

Germer, Søren; Higuchi, Russell

doi:10.1101/gr.9.1.72

Cited by 166 publications

(14 citation statements)

References 31 publications

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“…During the past decade, DNA microarray technology combined with on-chip diagnostic approaches, such as isothermal "padlock" probes, 7 DNA polymerase selectivity, 8 the TaqMan method 9 and the sequence-specific invasive cleavage assay, 10,11 has become a promising technology for the analysis of billions of genetic differences within a single-pot reaction. This has had a great influence on next generation personalized drug discovery and the emerging field of bioinformatics.…”

Section: Introductionmentioning

confidence: 99%

Surface invasive cleavage assay on a maskless light-directed diamond DNA microarray for genome-wide human SNP mapping

Nie

Yang

et al. 2015

Analyst

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The surface invasive cleavage assay, because of its innate accuracy and ability for self-signal amplification, provides a potential route for the mapping of hundreds of thousands of human SNP sites. However, its performance on a high density DNA array has not yet been established, due to the unusual "hairpin" probe design on the microarray and the lack of chemical stability of commercially available substrates. Here we present an applicable method to implement a nanocrystalline diamond thin film as an alternative substrate for fabricating an addressable DNA array using maskless light-directed photochemistry, producing the most chemically stable and biocompatible system for genetic analysis and enzymatic reactions. The surface invasive cleavage reaction, followed by degenerated primer ligation and post-rolling circle amplification is consecutively performed on the addressable diamond DNA array, accurately mapping SNP sites from PCR-amplified human genomic target DNA. Furthermore, a specially-designed DNA array containing dual probes in the same pixel is fabricated by following a reverse light-directed DNA synthesis protocol. This essentially enables us to decipher thousands of SNP alleles in a single-pot reaction by the simple addition of enzyme, target and reaction buffers.

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Section: Introductionmentioning

confidence: 99%

Surface invasive cleavage assay on a maskless light-directed diamond DNA microarray for genome-wide human SNP mapping

Nie

Yang

et al. 2015

Analyst

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“…Ct (cycles needed to surpass a threshold) delay of about four cycles is adequate to allow typing. 29 In general, the quantity of the vent (exo-) DNA polymerase affects the allele-specific PCR efficiency. For the codon 259 (AGA) detection in several breast cancer samples, Cdt values for the G:A/T/G mismatched systems were 10 cycles higher than those from G:C matched system when 1 unit of Vent R (exo-) DNA polymerase was used.…”

Section: Discussionmentioning

confidence: 99%

“…Allele specificity can be affected by the 3‘-exonuclease activity of DNA polymerase. − The amplification of the mismatched template is usually delayed compared with the matched ones. Ct (cycles needed to surpass a threshold) delay of about four cycles is adequate to allow typing . In general, the quantity of the vent (exo-) DNA polymerase affects the allele-specific PCR efficiency.…”

Section: Discussionmentioning

confidence: 99%

Universal Molecular Beacon-Based Tracer System for Real-Time Polymerase Chain Reaction

Yong

Guan

et al. 2006

Anal. Chem.

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DNA diagnostic has been moving from expensive, low-throughput, multistep methods to inexpensive, higher throughput, closed-tube, and automated methods. Fluorescence is the favored signaling technology for such assays. In this method, we describe a universal molecular beacon (U-MB) as the fluorescent tracer in the real-time PCR technique. A 5'-universal template primer (5'-UT primer) has been designed with a tail in complementary to the loop and 5'-side arm sequence of U-MB at the 5'-end of forward target specific primer. As PCR cycles increase, a new DNA fragment with a 5'-UT primer tail is synthesized, which is used as the template for next PCR cycle. As the reverse primer extends to the 5'-UT primer tail, the U-MB hybridized is displaced and the fluorescence from the fluorophore of the U-MB is quenched, indicating that the allele-specific PCR is in progress. This tracing system combined with an allele-specific reverse primer and vent (exo-) DNA polymerase, a polymerase that lacks 3'- to 5'-exonuclease activity, was used for the detection of point mutations of base G in codon 259 (AGA) of exon 7 of p53 gene on a panel of breast cancer individuals.

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“…Most of the existing techniques used to profile genetic variation with a specific disease or trait are based on either primer extension or direct hybridization. The primerextension methods include DNA sequencing, 19 allele-specific primer extension 20 and polymerase chain reaction, 21 which use the ability of the polymerase to incorporate specific deoxyribonucleosides complementary to a template DNA for analysis of genetic variation. The direct hybridization approach, such as single-strand conformation polymorphism, 22 heteroduplex analysis, 23 and denaturing gradientgel electrophoresis, 24 allows the unknown target sequence to be identified based on its ability to suffer sequence-specific binding with a designed probe.…”

Section: Introductionmentioning

confidence: 99%

A biocompatible open-surface droplet manipulation platform for detection of multi-nucleotide polymorphism

Huang

Fang

et al. 2014

Lab Chip

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We present a novel and simple method to manipulate droplets applicable to an open-surface microfluidic platform. The platform comprised a control module for pneumatic droplets and a superhydrophobic polydimethylsiloxane (PDMS) membrane. With pneumatic suction to cause deflection of the flexible PDMS-based superhydrophobic membrane, the sample and reagent droplets on the membrane become transported and mixed. A facile one-step laser micromachining technique serves to fabricate a superhydrophobic surface; a contact angle of 150° and a hysteresis angle of 4° were achieved without chemical modification. Relative to previous open-surface microfluidic systems, this platform is capable of simultaneous and precise delivery of droplets in two-dimensional (2D) manipulation. Droplets were manipulated with suction, which avoided interference from an external driving energy (e.g. heat, light, electricity) to affect the bio-sample inside the droplets. Two common bio-samples, namely protein and DNA, verified the performance of the platform. Based on the experimental results, operations on protein can be implemented without adsorption on the surface of the platform. Another striking result is the visual screening for multi-nucleotide polymorphism with hybridization-mediated growth of gold-nanoparticle (AuNP) probes. The detection results are observable with the naked eye, without the aid of advanced instruments. The entire procedure only takes 5 min from the addition of the sample and reagent to obtaining the results, which is much quicker than the traditional method. The total sample volume consumed in each operation is only 10 μL, which is significantly less than what is required in a large system. According to this approach, the proposed platform is suitable for biological and chemical applications.

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Single-Tube Genotyping without Oligonucleotide Probes

Cited by 166 publications

References 31 publications

Surface invasive cleavage assay on a maskless light-directed diamond DNA microarray for genome-wide human SNP mapping

Surface invasive cleavage assay on a maskless light-directed diamond DNA microarray for genome-wide human SNP mapping

Universal Molecular Beacon-Based Tracer System for Real-Time Polymerase Chain Reaction

A biocompatible open-surface droplet manipulation platform for detection of multi-nucleotide polymorphism

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