Search results for "Pcr cloning"

2017

Despite rapid progress, many problems and limitations persist and limit the applicability of gene-editing techniques. Making use of meganucleases, TALENs, or CRISPR/Cas9-based tools requires an initial step of pre-screening to determine the efficiency and specificity of the designed tools. This step remains time consuming and material consuming. Here we propose a simple, cheap, reliable, time-saving, and highly sensitive method to evaluate a given gene-editing tool based on its capacity to induce chromosomal translocations when combined with a reference engineered nuclease. In the proposed technique, designated engineered nuclease-induced translocations (ENIT), a plasmid coding for the DNA-…

Isolation of Abiotrophia adiacens from a brain abscess which developed in a patient after neurosurgery.

1999

ABSTRACT We report the case of a patient who developed a large brain abscess after neurosurgery. Cerebrospinal fluid from the abscess drainage yielded Abiotrophia adiacens -specific PCR products and microorganisms that were identified by conventional microbiological methods and by 16S ribosomal DNA analysis as Abiotrophia adiacens , which was formerly classified as a member of nutritionally variant streptococci.

2020

Droplet microfluidics is a technology that enables the production and manipulation of small volumes. In biosciences, the most popular application of this technology is Droplet Digital™ PCR (ddPCR™), where parallel nanoliter-scale PCR assays are used to provide a high sensitivity and specificity for DNA detection. However, the recovery of PCR products for downstream applications such as sequencing can be challenging due to the droplets' stability. Here we compared five methods for disrupting the droplets to recover DNA. We found that rapid freezing in liquid nitrogen results in a clear phase separation and recovery of up to 70% of the DNA content. Liquid nitrogen freezing can thus offer a s…

Application of Nanogen microarray technology for forensic SNP analysis

2006

Abstract The NanoChip® Molecular Biology Workstation using electronic microarrays is an approach for rapid and high throughput analysis of SNPs. This instrument is fully automated and uses a microchip for electronic addressing of capture probes to specific array sites followed by electronic hybridisation of the single stranded PCR products, and passive hybridisation of fluorescently labelled reporter probes. Discrimination is achieved by applying thermal stringency to denature the mismatched reporters. 48 SNP assays have been designed using the ‘capture down’ assay which applies a thermal ‘touch down’ strategy to obtain the best reporter probe discrimination.

Multiplex Ligation-dependent Probe Amplification (MLPA)

2011

The Multiplex Ligation-dependent Probe Amplification (MLPA) is a PCR-based method. The procedure relies on sequence-specific probe hybridization of genomic DNA, followed by multiplex-PCR amplification of the hybridized probe and a semiquantitative analysis of the resulting PCR products. MLPA allows the analysis of around 40 loci in the same reaction, and is a sensitive and relatively fast technique. Only a small amount of DNA is required and results are available within 2 days.The critical factors when performing MLPA analyses from formalin-fixed paraffin-embedded (FFPE) tissues are DNA integrity and purity; for this reason, a suitable DNA extraction method must be chosen.The MLPA protocol …

Quantification of PCR products by phosphate measurement

2008

Various techniques for quantification of PCR are available. Most frequently, the densitometric intensities of ethidium bromide-stained PCR products separated in gels are compared after normalizing to the levels of housekeeping gene products such as beta-actin. More precise, but extremely time consuming, is the technique of competitive PCR. Newer methods, such as tracking amplification in real-time, have high start-up and maintenance costs (e.g., TaqMan, Applied Biosystems; LightCycler, Roche; I-Cycler, Bio-Rad). Here, I describe an alternative, simple technique to quantify PCR products by determining the entire phosphate released during PCR. The method can be performed using common laborato…