Methods in molecular biology最新文献

Nucleotide sequence analyses provide insights into changes that might have an impact on proteins and their function. With the rapid accumulation of sequence data, it is now possible to recover the evolutionary history of most genes at the population level to species and beyond. Those sequences can be compared for substitutions that might change or not change the encoded protein and its function, but they can also help to estimate evolutionary relationships. These hypotheses, as phylogenetic trees, provide visual and statistical guidance for characterizing the degree of relatedness among biological entities. In a phylogenetic tree, ancestor-descendant relationships are represented by connections, and closely related entities share most of these links, which represent their evolutionary closeness. In this chapter, I outlined a method to retrieve and label nucleotide sequences of the cytokine IL17A gene, align them to identify substitutions in homologous sites, estimate phylogenetic trees with support values, and visualize these trees as images. The methodology outlined here uses free software packages in the R environment and the Python language.

The whole genome amplification (WGA) allows new clinical applications with minimal genetic material, such as in the genetic diagnosis of Mendelian diseases in embryos before implantation (Preimplantation Genetic Test for Mendelian Abnormalities, PGT-M). This approach allows couples to avoid the transmission of Mendelian disease by undergoing assisted reproduction treatment through in vitro fertilization (IVF). First, Preimplantation Genetic Testing for Aneuploidy (PGT-A) is used to identify chromosomal aneuploidies in IVF-generated embryos. Then, or in parallel, euploid embryos can be screened for specific diseases caused by variants in a single gene to achieve the conception of offspring free of a specific monogenic disease.Here, we detail the WGA preparation and two downstream usages: (1) preparation of PCR fragments for Sanger sequencing, exemplifying the diseases we detected for healthy embryo selection and transfer in IVF, and (2) detection of chromosome Y for embryo sex diagnosis.

Resistance against common treatment regimens like chemotherapy is a major concern in current cancer research and clinical disease management. Moreover, in vitro cell culture models cannot always adequately represent the tumor situation in vivo, hampering the analysis of the underlying mechanisms. Efforts have been made to establish three-dimensional (3D) cancer cell models, such as spheroids or organoids, to mimick the in vivo situation of the tumor more closely. Here, we describe the establishment of a 3D spheroid model of head and neck squamous cell carcinoma (HNSCC), allowing for the study of chemoresistance. The analysis of relevant phenotypic readouts using a high-content screening platform enables the objective evaluation and monitoring of spheroids during chemotherapeutic treatment. This model also represents a starting point for the evaluation of a precision medicine-based management of HNSCC patients.

Single-cell RNA sequencing is widely used in developmental biology, immunology, cancer research, and clinical applications, providing a scalable and reliable approach for single-cell transcriptomics. The Chromium Next GEM Single Cell 3' Reagent kits by 10× Genomics provide an advanced method for generating single-cell gene expression libraries using microfluidic partitioning and barcoding technology. These kits enable the profiling of thousands of individual cells in a single experiment by encapsulating single cells with uniquely barcoded Gel Beads-in-Emulsion (GEMs). Reverse transcription (RT) occurs within each GEM, producing barcoded cDNA, which is subsequently purified, amplified, and converted into a dual-indexed sequencing library. The workflow consists of four major steps: GEM generation and barcoding, post-GEM-RT cleanup and cDNA amplification, 3' gene expression library construction, and sequencing. Quality control measures, including SPRIselect bead cleanup, Bioanalyzer/TapeStation validation, and PCR optimization, ensure high-quality sequencing results. The final library is compatible with Illumina sequencing platforms, allowing researchers to analyze cellular heterogeneity, gene expression dynamics, and rare cell populations. This protocol is based on the 10× Chromium Single Cell 3' Reagent Kits user guide (v3.1-Dual Index), which can be downloaded from the 10× Genomics website ( https://www.10xgenomics.com ). It is recommended to refer to the original official guide for more details when carrying out the experiments with these kits, ensuring you stay updated with any revisions or updates.

In the clarification of sexual assault cases, forensic DNA profiling of single sperm cells can be desirable if only a few sperm cells are present in intimate swabs analyzed, or if sperm cells from more than one individual are present. However, the DNA content of a single sperm cell is far below the sensitivity limits of current standard forensic methods and thus does not allow for reliable DNA profiling. Here, we describe a protocol for micromanipulation of single sperm cells that is based on using an adhesive-coated tungsten needle to pick individual cells that have been spread on an agarose plate. Forensic DNA typing is thereafter accomplished by subjecting the extracted DNA to multiple strand displacement amplification (MDA) prior to analysis. For MDA, the QIAGEN REPLI-g Single Cell kit is used, and DNA profiles are analyzed using the Promega PowerPlex ESX 17 kit followed by capillary electrophoresis. Using this protocol, over 80% complete haploid DNA profiles can be obtained from the majority of single sperm cells picked.

Tip-enhanced Raman spectroscopy (TERS) has been widely used for the nanoscale chemical and structural analysis of biomolecules. TERS combines indeed the high molecular sensitivity of Raman spectroscopy with the high spatial resolution of scanning probe microscopies (SPM), by exploiting the intense electromagnetic field generated at the apex of a metal SPM tip at nanometer distance from the sample. While TERS studies on ribonucleic acid (RNA) nucleobases started more than two decades ago, several major scientific advances like the advent of a compelling TERS-based RNA sequencing method and the description of amyloid-like RNA-induced tau fibrils potentially implicated in Alzheimer's disease have been only achieved recently. In this chapter, after presenting these promising advances, we provide technical and methodological information allowing TERS maps of such RNA samples to be obtained, processed, and correctly interpreted in order to properly describe the chemical composition of RNA components and the structure of tau proteins interacting with them.

Gene regulatory proteins, such as transcription factors (TFs), bind to DNA and orchestrate spatial and temporal gene expression patterns. This regulation relies on dynamic and coordinated protein-protein interactions, either through direct pairwise interactions or as part of multiprotein complexes. Therefore, the development of methods to assay these interactions within the cellular context is crucial. Förster resonance energy transfer combined with fluorescence lifetime imaging microscopy (FRET-FLIM) is a powerful quantitative imaging technique for detecting protein-protein interactions in vivo. This approach involves labeling the proteins of interest with fluorescent tags and measuring changes in the fluorescence lifetime of the donor fluorophore. The reduction in donor lifetime in the presence of the acceptor fluorophore provides direct evidence of a physical interaction between the proteins under study. In this chapter, we present a detailed and simple protocol for the acquisition and analysis of FRET-FLIM data using the Leica STELLARIS 8 FALCON FLIM Microscope® system, illustrated with two interacting transcription factor proteins as an example.

Plant growth relies on flexible gene regulation to adapt to environmental changes. This process is ultimately controlled by transcription factors (TFs), which bind to specific DNA motifs, known as TF-binding sites (TFBS), located in the gene regulatory regions to regulate their expression. These interactions play crucial roles in plant development and responses to environmental cues, as well as in plant evolution and domestication, making both cis- (i.e., TFBS) and trans-regulatory factors as potential molecular targets in plant breeding for traits such as yield, quality, and stress resilience. These biotechnological approaches require precise knowledge of the target gene sets and TFBS specifically recognized by TFs. Recent advances in high-throughput sequencing techniques have enabled precise identification of TF target genes, especially thanks to methodologies that combine the main features of both in vitro and in vivo approaches. However, small scale and targeted approaches are still needed to evaluate the relative contribution of specific nucleotide positions in TF recognition. In this chapter, we describe a modified version of DNA Affinity Purification sequencing (DAP-seq) that replaces genomic DNA with a PCR-generated library of TFBS variants. This approach, termed targeted-DAP, allows a flexible and quantitative analysis of TF-binding using next-generation sequencing. Additionally, expressing TFs in Escherichia coli provides an economical source of proteins, enabling scalable and cost-effective analysis of DNA-binding specificities. We showed the benefits of this technique to demonstrate the contribution of the genomic context around the TFBS for specific recognition of a bHLH TF. Development of targeted DAP-seq would be of interest for the evaluation of nucleotide variation-either allelic or generated by CRISPR/Cas-within TFBS in TF recognition with predictable consequences on plant phenotypes.

We recently described a method for PCR-independent sequence determination of a set of seven taxonomic markers for phytoplasmas and determined its utility for high-resolution strain differentiation of phytoplasma groups, including 16SrI, 16SrIII, 16SrX, and 16SrXII. Here we describe a protocol for high-throughput strain characterization with multiplexed hybridization reactions and a data analysis method that assembles five protein-coding gene sequences and concatenates them into a single phylogenetic marker of approximately 7.7 kb. In combination with the RFLP typing of the determined 16S and cpn60 sequences, this method provides detailed sequence data on phytoplasmas of known or unknown taxonomic affiliations and can differentiate closely related but distinct phytoplasmas within the same group, for example, readily differentiating the ribosomal subgroup 16SrI phytoplasmas 'Candidatus Phytoplasma asteris' and 'Candidatus Phytoplasma triticii'.

The current "gold standard" for the identification of MHC-restricted peptides is conventional immunoprecipitation/mass spectrometry (MS); however, this approach requires a relatively large amount of sample, complex purification procedures, and computational analyses to assign peptides to individual alleles. Here, we provide instructions for the expression, purification, and MS identification of MHC-presented peptides of human and non-human, classical and non-classical, class I and class II proteins using a readily expressible, soluble, and easily purifiable single-chain dimer construct. This procedure enables the identification of up to tens of thousands of allele-specific peptides per run for peptidomics, ligandomics, therapeutic targeting, and motif analyses. Also included are instructions for construct design, streamlined lentiviral transfection and transduction, and computational methods for high-throughput processing of MS results, yielding high confidence peptide lists, motifs, and multiple analytics.