Molecular Tools for Studying Genes and Gene Activity (CMB part 3)

Methods for purifying proteins and nucleic acids are fundamental in molecular biology. DNA, RNA, and proteins of varying sizes can be effectively separated using gel electrophoresis. Agarose is the most commonly used gel for nucleic acid electrophoresis, while polyacrylamide is typically employed for protein electrophoresis. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) separates polypeptides based on their sizes. For higher resolution, two-dimensional gel electrophoresis is utilized, combining isoelectric focusing in the first dimension with SDS-PAGE in the second. Ion-exchange chromatography is another technique that separates substances, including proteins, according to their charges, often employing positively charged resins like DEAE-Sephadex.Labeled DNA or RNA probes can be hybridized to DNAs with identical or very similar sequences on a Southern blot. Modern DNA typing employs Southern blots and multiple DNA probes to detect variable sites in individual organisms, including humans. Additionally, labeled probes may be hybridized to entire chromosomes to identify specific genes or DNA sequences, a process known as in situ hybridization, or fluorescence in situ hybridization (FISH) when fluorescently labeled probes are used. Proteins in complex mixtures can be detected and quantified using immunoblots, or Western blots, where proteins are electrophoresed, transferred to a membrane, and probed with specific antibodies detected via labeled secondary antibodies or protein A.The Sanger DNA sequencing method relies on dideoxy nucleotides to terminate DNA synthesis, producing DNA fragments of varying sizes that can be analyzed by electrophoresis. The last base of each fragment is determined by the specific dideoxy nucleotide used to terminate the reaction, enabling fragments to be ordered by size, with each one being a single, known base longer than the previous.

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