A light-up fluorescence platform based DNA: RNA hybrid G-quadruplet for detecting single nucleotide variant of ctDNA and miRNA-21.

The nucleic acid assay is an area of great concern in the diagnosis and treatment of breast cancer. Here, we developed a DNA: RNA hybrid G-quadruplet (HQ) detection platform based on strand displacement amplification (SDA) and Baby Spinach RNA aptamer for single nucleotide variant (SNV) of circulating tumor DNA (ctDNA) and miRNA-21. This was the first in vitro construction of HQ for the biosensor. It found that HQ had much stronger ability to switch on fluorescence of DFHBI-1T than Baby Spinach RNA alone. Taking advantage of the platform and the FspI enzyme with high specificity, the biosensor achieved ultra-sensitive detection of SNV of the ctDNA (PIK3CA H1047R gene) and miRNA-21. The light-up biosensor had high anti-interference ability in complex actual samples. Hence, the label-free biosensor provided a sensitive and accurate method for early diagnosis of breast cancer. Moreover, it opened a new application model for RNA aptamers.

Rapid and Ultrasensitive Approach for the Simultaneous Detection of Multilocus Mutations to Distinguish Rifampicin-Resistant Mycobacterium tuberculosis.

The untested empirical medications exacerbated the development of multidrug-resistant Mycobacterium tuberculosis (MDR-TB). Here, we develop a rapid and specific method based on loop-mediated isothermal amplification and duplex-specific nuclease for distinguishing rifampicin-resistant M. tuberculosis. Three probes were designed for the codons 516, 526, and 531 on the RNA polymerase ß-subunit (rpoB) gene. These three sites accounted for more than 90% of the total mutations of the ropB gene in the rifampicin-resistant strain. The approach can perform simultaneous and sensitive detection of three mutant sites with the actual detection limit as 10 aM of DNA and 62.5 cfu·mL-1 of bacteria in 67 min under isothermal conditions. Moreover, the positive mode of the approach for MDR-TB can not only deal with the randomness and diversity of mutations but also provide an easier way for medical staff to read the results. Therefore, it is a particularly valuable method to handle major and urgent MDR-TB diagnostics.

Carbon nanodots combined with loop-mediated isothermal amplification (LAMP) for detection of African swine fever virus (ASFV).

The spread of African swine fever virus (ASFV) caused huge economic costs, so early detection is particularly important. Here, we established a fluorescence biosensor based on carbon nanodots (CNDs) and loop-mediated isothermal amplification (LAMP) to ultra-sensitively detect ASFV. LAMP with high efficiency produced a large amount of pyro phosphoric acid and caused pH change in a short time. CNDs with strong light stability had a large fluorescence response at the emission wavelength of 585.5 nm to small pH change by the excitation wavelength of 550 nm. The biosensor realized "turn-off-on" mode for ASFV detection with the detection limit as low as 15.21 copies µL-1. In addition, the biosensor had high accuracy in the actual sample assay. Therefore, the biosensor achieved rapid, sensitive, low-cost, and simple detection for ASFV. Moreover, the biosensor broadened the detection pathway of LAMP as a tool with great development prospect.

Pathway engineering of Escherichia coli for one-step fermentative production of L-theanine from sugars and ethylamine.

L-theanine is the most abundant free amino acid in tea that offers various favorable physiological and pharmacological effects. Bacterial enzyme of γ-glutamylmethylamide synthetase (GMAS) can catalyze the synthesis of theanine from glutamate, ethylamine and ATP, but the manufacturing cost is uncompetitive due to the expensive substrates and complex processes. In this study, we described pathway engineering of wild-type Escherichia coli for one-step fermentative production of theanine from sugars and ethylamine. First, the synthetic pathway of theanine was conducted by heterologous introduction of a novel GMAS from Paracoccus aminovorans. A xylose-induced T7 RNA polymerase-P T7 promoter system was used to enhance and control gmas gene expression. Next, the precursor glutamate pool was increased by overexpression of native citrate synthase and introduction of glutamate dehydrogenase from Corynebacterium glutamicum. Then, in order to push more carbon flux towards theanine synthesis, the tricarboxylic acid cycle was interrupted and pyruvate carboxylase from C. glutamicum was introduced as a bypath supplying oxaloacetate from pyruvate. Finally, an energy-conserving phosphoenolpyruvate carboxykinase from Mannheimia succiniciproducens was introduced to increase ATP yield for theanine synthesis. After optimizing the addition time and concentration of ethylamine hydrochloride in the fed-batch fermentation, the recombinant strain TH11 produced 70.6 â€‹g/L theanine in a 5-L bioreactor with a yield and productivity of 0.42 â€‹g/g glucose and 2.72 â€‹g/L/h, respectively. To our knowledge, this is the first report regarding the pathway engineering of E. coli for fermentative production of theanine. The high production capacity of recombinant strain, combined with the easy processes, will hold attractive industrial application potential for the future.

DNA Sequencing Data Analysis.

Among various biological data, DNA sequence is doubtlessly a fundamental datum. By obtaining particular DNA sequence data and analyzing, biologists get to understand life science more precisely. This chapter is an overview of DNA sequencing technology and its data analysis methods, providing information about DNA sequencing, several different methods, and tools applied in data analysis. Both advantages and disadvantages are discussed.