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Biology LibreTexts

8: Basic Techniques

  • Page ID
    7856
  • [ "article:topic-guide", "authorname:ahern2" ]

    The environment of a cell is very complex, making it difficult to study individual reactions, enzymes, or pathways in situ. The traditional approach used by biochemists for the study of these things is to isolate molecules, enzymes, DNAs, RNAs, and other items of interest so they can be analyzed independently of the millions of other processes occurring simultaneously. Today, these approaches are used side by side with newer methods that allow us to understand events inside cells on a larger scale- for example, determining all the genes that are being expressed at a given time in specific cells. In this section we take a brief look at some commonly used methods used to study biological molecules and their interactions.

    • 8.1: Cell Lysis
      To separate compounds from cellular environments, one must first break open (lyse) the cells. Cells are broken open, in buffered solutions, to obtain a lysate. There are several ways of accomplishing this.
    • 8.2: Fractionation and Chromatography Techniques
      Fractionation of samples, as the name suggests, is a process of separating out the components or fractions of the lysate. Fractionation typically begins with centrifugation of the lysate. Using low-speed centrifugation, one can remove cell debris, leaving a supernatant containing the contents of the cell. By using successively higher centrifugation speeds (and resulting g forces) it is possible to separate out different cellular components, like nuclei, mitochondria, etc., from the cytoplasm.
    • 8.3: Electrophoresis
      Electrophoresis uses an electric field applied across a gel matrix to separate large molecules such as DNA, RNA, and proteins by charge and size. Samples are loaded into the wells of a gel matrix that can separate molecules by size and an electrical field is applied across the gel. This field causes negatively charged molecules to move towards the positive electrode. The gel matrix, itself, acts as a sieve, through which the smallest molecules pass rapidly, while longer molecules are slower-movi
    • 8.4: Detection, identification and quantitation of specific nucleic acids and proteins
      One way to detect the presence of a particular nucleic acid or protein is dependent on transferring the separated molecules from the gels onto a membrane made of nitrocellulose or nylon to create a “blot” and probing for the molecule(s) of interest using reagents that specifically bind to those molecules. The next section will discuss how this can be done for nucleic acids as well as for proteins.
    • 8.5: Transcriptomics
      Consider a matrix containing all of the known gene sequences in a genome. To make such a matrix for analysis, one would need to make copies of every gene, either by chemical synthesis or by using PCR. The strands of the resulting DNAs would then be separated to obtain single-stranded sequences that could be attached to the chip. Each box of the grid would contain sequence from one gene. One could analyze the transcriptome - all of the mRNAs being made in selected cells at a given time.
    • 8.6: Isolating Genes
      Methods to isolate genes were not available till the 1970s, when the discovery of restriction enzymes and the invention of molecular cloning provided, for the first time, ways to obtain large quantities of specific DNA fragments, for study. Although, for purposes of obtaining large amounts of a specific DNA fragment, molecular cloning has been largely replaced by direct amplification using the polymerase chain reaction described later, cloned DNAs are still very useful for a variety of reasons.
    • 8.7: Polymerase Chain Reaction (PCR)
      The polymerase chain reaction (PCR) allows one to use the power of DNA replication to amplify DNA enormously in a short period of time. As you know, cells replicate their DNA before they divide, and in doing so, double the amount of the cell’s DNA. PCR essentially mimics cellular DNA replication in the test tube, repeatedly copying the target DNA over and over, to produce large quantities of the desired DNA.
    • 8.8: Reverse Transcription
      In the central dogma, DNA codes for mRNA, which codes for protein. One known exception to the central dogma is exhibited by retroviruses. These RNA-encoded viruses have a phase in their life cycle in which their genomic RNA is converted back to DNA by a virally-encoded enzyme known as reverse transcriptase. The ability to convert RNA to DNA is a method that is desirable in the laboratory for numerous reasons.
    • 8.9: FRET
      The fluorescence resonance energy transfer (FRET) technique is based on the observation that a molecule excited by the absorption of light can transfer energy to a nearby molecule if the emission spectrum of the first molecule overlaps with the excitation spectrum of the second.  This transfer of energy can only take place if the two molecules are sufficiently close together (no more than a few nanometers apart.
    • 8.10: Genome Editing (CRISPR)
      The development of tools that would allow scientists to make specific, targeted changes in the genome has been the Holy Grail of molecular biology. An ingenious new tool that is both simple and effective in making precise changes is poised to revolutionize the field, much as PCR did in the 1980s. Known as the CRISPR/Cas9 system, and often abbreviated simply as CRISPR, it is based on a sort of bacterial immune system that allows bacteria to recognize and inactivate viral invaders.
    • 8.11: Protein Cleavage
      Because of their large size, intact proteins can be difficult to study using analytical techniques, such as mass spectrometry. Consequently, it is often desirable to break a large polypeptide down into smaller pieces. Proteases are enzymes that typically break peptide bonds by binding to specific amino acid sequences in a protein and catalyzing their hydrolysis.
    • 8.12: Membrane Dynamics (FRAP)
      Understanding the dynamics of movement in the membranes of cells is the province of the Fluorescence Recovery After Photobleaching (FRAP) technique. This optical technique is used to measure the two dimensional lateral diffusion of molecules in thin films, like membranes, using fluorescently labeled probes. It also has applications in protein binding.

    Thumbnail: A western blot. Image used with permission (CC BY-SA 3.0; Magnus Manske).