China
Africa
Explore chapters and articles related to this topic
Cancer Informatics
Published in Trevor F. Cox, Medical Statistics for Cancer Studies, 2022
Now we can match the probes to genes. The gene library for our microarray data is “pd.hugene.1.0.st.v1”, which can be downloaded from the Bioconductor website. The function oligo::getNetAffx() will sort out the gene annotation. Once annotated, for each gene, we take the median of the expression values for all the probes matching to the particular gene. For example, the gene, “PTEN” has two probesets, 7928959 and 8160718 associated with it, and so the median or mean of the two expression values is used for the gene. We are left with 23,307 genes that have expression values.
A Survey of Newer Gene Probing Techniques
Published in Victor A. Bernstam, Pocket Guide to GENE LEVEL DIAGNOSTICS in Clinical Practice, 2019
An alternative approach is based on two complete sets of small inserts, and complete digest DNA libraries for each of the 24 human chromosomal types constructed at the National Laboratory Gene Library Project. The available information is used in genome mappingin the search for RFLP markers linked to various genetic diseasesas a source of unique sequence probes for “chromosome painting”, by fluorescent ISH (FISH)
Vector Technology of Relevance to Nitrogen Fixation Research*
Published in Peter M. Gresshoff, Molecular Biology of Symbiotic Nitrogen Fixation, 2018
Reinhard Simon, Ursula B. Priefer
Of course, they should also allow a sufficiently high efficiency of introduction into the host cells. Therefore, versatile broad host-range cloning vectors should be transmissible by conjugation into a wide range of bacterial species. A high frequency of transfer is not only advantageous for the introduction of single recombinant molecules, but also a prerequisite for mobilization of a large number of clones, e.g., screening a gene library.
An overview of advancement in aptasensors for influenza detection
Published in Expert Review of Molecular Diagnostics, 2022
Varsha Gautam, Ramesh Kumar, Vinod Kumar Jain, Suman Nagpal
In the work by Kwon et. al., the gene library was developed using a 1015 ssDNA pool comprised of random 30-mer sequence marked by the presence of designed primers. They develop the following three aptamers RHA0006, RHA0385, and RHA1635 using SELEX. SELEX was performed as follows: the 20 μL ‘TALON Super flow Metal Affinity’ resin solution and 25 μg HA protein solution were prepared in 200 μL and incubated at room temperature for half an hour. The ssDNA library was pre-absorbed into the TALON resin mixture with tRNA and bovine serum albumin (acetylated). The unbound ssDNA was eluted and mixed with 100 μL HA coated beads followed by incubation (1.5 hr) and washing (3 times). PCR with biotin-conjugated reverse primers was carried to make up a total volume of 400 μL dsDNA. To obtain ssDNA aptamers, dsDNA was purified and incubated with SA-beads in hybridization buffer and washed (2 times) in the buffer. ssDNA bounded beads were eluted followed by PCR, and biotinylated and non-biotinylated PCR products were separated. They carried 10 rounds of SELEX.
Antibody discovery and engineering by enhanced CRISPR-Cas9 integration of variable gene cassette libraries in mammalian cells
Published in mAbs, 2019
Cristina Parola, Daniel Neumeier, Simon Friedensohn, Lucia Csepregi, Mariangela Di Tacchio, Derek M. Mason, Sai T. Reddy
We recently developed a mammalian cell platform, referred to as plug-and-(dis)play (PnP) hybridomas, in which the genome of hybridomas is reprogrammed by CRISPR-Cas9 HDR for the expression of a recombinant antibody of choice.27 Here, we present an innovative workflow for the generation, display and selection of full-length IgG libraries using this system. We take advantage of an improved HDR plasmid donor format that uses in situ linearization to substantially increase Cas9-driven integration efficiency. In one application, we used this approach to generate a full-length IgG immune library by cloning the VL and VH genes from the plasma cells of a mouse immunized with the model antigen ovalbumin (OVA). Screening of this combinatorial variable gene library resulted in the isolation of more than 40 unique antigen-binding variants. Next, we used our enhanced Cas9 HDR approach to perform affinity maturation of a previously known antibody that recognizes the model antigen hen egg lysozyme (HEL).27 In this case, a random mutagenesis library was generated by error-prone (EP) PCR on the VH region, which was then cloned and integrated in PnP cells by HDR. With a library size of <104, five improved variants were successfully isolated by screening, each of them exhibiting affinities toward HEL in the low picomolar range. The results presented here show that enhanced Cas9-driven HDR can be used for important applications in antibody discovery and engineering.
Bacterial extracellular vesicles in biofluids as potential diagnostic biomarkers
Published in Expert Review of Molecular Diagnostics, 2022
Kar-Yan Su, Jie-Yi Koh Kok, Yie-Wei Chua, Shearn-Dior Ong, Hooi Leng Ser, Priyia Pusparajah, Pui San Saw, Bey Hing Goh, Wai-Leng Lee
Investigations on distinct bacterial compositions have commonly been performed through metagenome analysis of BEVs. Bypassing the needs of cultivation, taking the metagenomic approach enables thorough analysis to evaluate the important role of BEVs in human health via comparison of BEV compositions between patients and healthy individuals using statistical analyses. Analysis of diversity and abundance of bacterial compositions in the EVs found in biofluids were generally performed via next-generation sequencing (NGS) of V3-V4 hypervariable regions in 16S rRNA metagenomic analysis [7,10–16]. Indigenous 16S rDNA was selected for sequencing in order to exclude the host-cell-derived EVs. While studies have indicated various primer pairs that could be used in metagenomic studies, the V3-V4 regions are commonly analyzed as these regions consist the greatest nucleotide heterogeneity, which provides the largest discriminatory power [8,17,18]. V3 and V4 amplicons have been reported to be fully recovered using the Illumina MiSeq platform, which can be used for sequencing up to 600 nucleotides from both ends of an amplicon [7,10–16,19]. Generally, 16S rRNA gene amplicon metagenomic analyses were conducted on bacterial DNA by performing PCR amplification of V3-V4 hypervariable regions in 16S rRNA genes with specific primers [7,10–16]. The PCR products were then used to construct a 16S rRNA gene library following MiSeq System guidelines. Analysis of bacterial composition in the EVs would be carried out following biomarkers selection based on relative abundances at the genus level. A diagnosis model of biomarkers could then be developed using multiple-regression analysis and machine learning algorithms [7,10–16].