Biotechnology/Recombinant DNA Techniques

See also Measurements

Web Sites:    Bioprotocols.com (protocols contributed by scientists by over 125 academic institutions)

  UK NEKAS (gives advice to clinical laboratories around the world on the quality of analytical performance)

International Mouse Strain Resource   

Gel Electrophoresis: This is a basic technique and has many applications including:

  • DNA Footprinting: has been instrumental in the identification of the binding sties for DNA binding proteins on enhancers and promoters. (1) an enhancer or promoter sequence is first labeled at the 5' end with 32P. (2) the labeled DNA is then divided into 2 factions. One fraction is incubated with an extract containing a DNA binding protein, the other is left alone. (3) both fractions are then digested with a nuclease or a chemical that makes random cuts in the phosphodiester bonds of the DNA. (4) the 2 fractions are run on a gel. The fraction which contains the binding proteins will have certain fragments missing from its gel lane. These missing fragments or "footprints" are due to the fact that cleavage did not occur due to the protection of the binding protein.

  • Electrophoretic Mobility Shift Assay:

  • Analysis of Proteins (Proteomics):

  • Analysis of DNA: The length and purity of DNA molecules can be determined by the same types of gel electrophoresis methods used for the analysis of proteins. Since each nucleotide in the DNA already carries a single negative charge, there is no need to add the negatively charged detergent SDS that is required to make protein molecules move uniformly toward the positive electrode. Agarose is used to separate large DNA molecules. For DNA fragments less than 500 nucleotides long, polyacrylamide gels must be used.

Restriction Enzymes: One of the biggest discovers in the field of molecular biology occurred in the 1970s with the discovery that a variety of bacteria produce enzymes, called restriction endonucleases, that degrade foreign DNA (e.g., bacteriophage DNA) but do not cleave the bacterial cell DNA which contain methylated residues. A restriction endonuclease cuts both DNA strands at a specific point. Some of these enzymes cut on the central axis within the restriction site thereby generating "blunt" ends whereas other enzymes cut the DNA off center from the central axis of the recognition site thereby producing staggered fragments having short single stranded overhangs called "sticky ends" extending from one of the strands of the double stranded fragment.  This sticky end is complementary to any other DNA molecule which is cut with the same restriction enzyme thereby allowing the joining of 2 such DNA molecules. This allows a number of applications:

  • Large DNA fragments may be more suitably cloned into cosmid vectors as well as even yeast chromosomes.

  • Fusion proteins: Using restriction enzymes and vectors, one can create fusion proteins. This has been very useful for determining signal sequences on proteins. For example, one can attach a signal sequence to the sequence that codes for some protein of interest. After the cDNA encoding this fusion protein is transfected into cells, the location of the fusion protein can be determined immunostaining or by cell fractionation.

  • Restriction Mapping: By cleaving a DNA sample with 2 or more restriction enzymes (alone and in combination), it is possible to determine the location of the restriction sites and relative distances between such sites to create a restriction map. In one type of restriction mapping, called chromosomal walking, a particular mRNA is isolated and converted with reverse transcriptase into cDNA which is then cloned. Large framents of chromosomal DNA are also cloned in cosmid vectors and then screened with the cDNA probe. Each cosmid clone shown to hybridize with a cDNA probe is then digested by several restriction enzymes to create a restriction map. The most distal 5' and 3' ends of the cosmid clones are then subcloned and used as probes to identify still more cosmid clones to extend the restriction map in both directions.

  • Cloning of cDNA:  

  • Cloning of genomic DNA:

  • Southern Blotting: This is a technique developed by E. M.Southern that can be used to identify genes of interest. (1) DNA is first cut with restriction enzymes and the fragements are separated according to size by electrophoresis on an agarose gel. (2) The gel is then soaked in NAOH to denature the dsDNA, and the resulting ssDNA graments are transferred onto a nitrocellulose or nylon filter by capillary action. (3) the filter is incubated with an appropriate radiolabeled probe specific for the gene sequence of choice. (4) If the gene of interest exists, the probe will hybridize with that ssDNA fragment and the fragment can be visually detected by autoradiography.

  • Northern Blotting: This procedure is just like southern but it is used to detect mRNA molecules rather than DNA fragments.  It is useful to detect how much of a specific mRNA is expressed in cells under different conditions. Increased levels of mRNA will bind more of the labeled DNA probe. (1) mRNA is first denatured to ensure it is linearized. (2) the mRNA molecules are then separated by size by electrophoresis and transferred to a nitrocellulose filter to which the mRNA will adhere. (3) the filter is incubated with a labeled DNA probe and then subjected to autoradiography.

  • PCR: This powerful technique has been equally important as the discovery of restriction enzymes for the field of molecular biology. In the real-time PCR system, the 5' to 3' exonuclease activity of Taq plymerase leads to release of the reporter dye from the vicinity of the quencher dye, resulting in increased reporter fluorescence. The fluorescence intensity is correleated with the amount of target DNA and is detected using an automated fluoreometer.

  • DNA subtractive hybridization was used to isolate chains from the T cell receptor. In this procedure, B cell mRNA was hybridized to T-cell mRNA. Te unhybridized cDNA remaining after this step represented mRNA that was unique to the T cell, including the mRNA encoding its T-cell receptor.

Production of Monoclonal Antibodies:

Monoclonal antibodies may be produced by using the basic method developed by Kohler and Milstein, reported in Nature, 256:495-97 (1975). This procedure involves 3 main steps:

  • 1st, B-cell hybridomas are formed by fusing primed B cells and myeloma cells. This procedure involves 1) priming a mouse with a given antigen. 2) fusing spleen cells from that primed mouse with mouse myeloma cells using plyethylene glycol. The myeloma cells have the immortal growth properties of a cancer cells but do not secret their own antibody gene product. The myeloma cells also lack the ability to produce an enzyme known as HGPRT which is necessary in the synthesis of nucleotides by the salvage pathway. 3) Growth of the cells on a HAT medium which contains aminopterin which blocks the synthesis of nucleotides by the de novo pathway. When the de novo pathway is blocked, cells try to synthesize nucleotides by the salvage pathway. Fused hybridomas will be able to do this since they will contain the full complement of necessary enzymes (spleen cells which are HGPRT+ ) but myeloma cells which have not fused will not be able to grow. Unfused spleen cells do not need to be selected at all because they are terminal cells.
  • 2nd, the resulting clones are screened for those clones which secrete the antibody with the desired specificity. The supernatant of each hybridoma culture contains its secreted antibody and can be assayed for a particular antigen specificity as by ELISA (antigen that reacts with the desired antibody is bound to microtiter wells).
  • Once the hybridoma secreting a monoclonal antiboyd of the desired specificity has been identified, it is growth in tissue culture flasks or can be grown in the periotneal cavity of histocompatible mice.

DNA Microarrays: can be used to monitor the expression of thousands of genes simultaneously. To prepare the microaaray, DNA framents, each corresponding to a gene, are spotted onto a slide by a robot. mRNA can be collected from two different cell samples for a comparison of their relative levels of gene expression. These samples are converted to cDNA and labeled, one with a red fluorochrome and the other with a green fluorochrome. The labeled samples are mixed and then allowed to hybridize to the microarray. After incubation, the array is washed and the fluorescence scanned. Red spots will indicate that the gene in sample 1 is expressed at a higher level than sample 2 and green vice versa. Yellow spots reveal genes that are expressed at equal levels in both cell samples.

Gene Therapy

Measures of T cell Immunity

Transfection of Cells: There are viral and non-viral methods of getting material like DNA into cells.

Methods of Viral transfection include (1) Retrovirus  (2) Adenovirus (3) Adeno-associated virus.

Non-viral methods of transfection include (1) liposomes (2) particle bombardment (3) direction injection and (4) electroporation.            

Click here for Techniques with Bacteria

Experimental Animal Models

Research in biotechnology and immunology requires the use of various animal models. The fact that something can be done in the test tube (in vitro) with a cell line above, does not mean that it can be done in vivo in a living animal. For example, if large amounts of antiserum are needed, a rabbit, goat, sheep or horse might be the best animal. If one is working on a vaccine, the animal chosen must be susceptible to the infectious agent you are studying. Many animals, for example, are not susceptible to HIV infection. Such animals would thus not be good research models to study the efficacy of an HIV vaccine.  Mice are the most common type of research animal used. They are easy to handle and have a rapid breeding cycle. Their immune system has also been extensively characterized.   For some protocols on the use of mice click here.

Inbred Mouse:

Immunologists often work with genetically identical animals produced by inbreeding. This is advantageous in that one can control variation caused by differences in the genetic backgrounds of the mice. Repeated inbreeding for 20 generations usually yields an inbred strain whose progeny are homozygous at more than 98% of all loci. Hundreds of different inbred strains of mice are available. These strains are identified by a series of letters and/or numbers. Most of these strains are purchased by suppliers such as Jackson Laboratory in Bar Harbor, Maine.

Some common inbred mouse strains include : (1) balb/c (very docile) (2) AJmice (more aggressive) (3) black (aggressive) and (4) DBA/2 (very aggressive).        Mouse genetics unit   

Adoptive-Transfer Systems: In adoptive-transfer systems, the immune cells of the recipient mouse is inactivated by exposing the host to x-4ays which can kill almost all of its lymphocytes. If the host's hematopoietic cells might influence the adoptive-transfer experiment, then even higher x-ray levels are used to eliminate the entire hematopoietic system. Thereafter the lymphocytes form the spleen of a donor can be studied without interference from the host lymphocytes.

Scid Mouse: "Scid" stands for severe combined immunodeficiency disease. Scid mice fail to develop mature T and B cells and are thus severely compromised immunologically. The absence of these cells enables SCID mice to accept foreign cells and grafts from other strains of mice and other species. An autosomal recessive mutation resulting in SCID developed spontaneously in a strain of mice called CB-17.

Scid mice have been very useful in the study of human lymphocytes. For example, one can transplant human thymus and lymph node tissue under the kidney capsule of the SCID mouse and inject the mouse with human fetal liver cells (stem cells). Those stem cells will then migrate to the human thymus where they will mature into T cells.

Transgenic Mice: are mice which have a gene insertion, deletion or replacement. The foreign or modified genes that are added are called "transgenes." There are two main methods to create transgenic mice. The first relies on random insertion of the gene into the mouse. The second rlies upon homologous recombination.

IMMUNOASSAYS        Flow Cytommetry

MICROSCOPY

Light Microscopy

The wavelenght of visible light ranges from about 0.4 um to 0.7 um. This means that the best possible detail which a light microscope can resolve is 0.2 um. In a bright field microscope an image is obtained by the simple transmission of light through a cell. In a dark field microscope, the illuminating rays of light that are scattered by the various components of a cell are directed from the side so that only scattered light enters the microscope lenses. The cell appears as a bright object against a dark background.

Phase contrast microscopes and differential interference contrast ("Nomarsky") microscopes take advantage of the fact that the phase of a light wave is changed as it passes through a cell. Light that passes through a dense part of the cell like the nucleus, for example, is retarded and the light phase is consequently shifted relative to light that passes through a thinner region of the cell such as the cytoplasm.

A Fluorescence microscope is similar to an ordinary light microscope except that illuminating light is passed through 2 sets of filters. The first filter is selected so that it passes only the wavelenghts that excite a particular fluorescent dye, while the second filter blocks out this light and passes only those wavelenghts emitted when the dye fluoresces. Fluorescent molecules absorb light at one wavelenght (higher energy) and emit it at another longer wavelenght (lower energy). If a compound is illuminated at its absorbing wavelenght and then viewed through a filter that only allows light of the emitted wavelenght to pass, it will glow against a dark background. The same number of molecules of an ordinary stain viewed conventionally would be practically invisible.

One more complex application of fluorescence is fluorescence resonance energy transfer where two molecules (proteins) of interest are each labeled with a  different fluorochrome so that the emission spectrum of one fluorochrome overlaps with the absorption spectrum of the other. If the two proteins bind the energy of the absorbed light is transferred from one to the other.

Confocal microscopy is used to get a 3 dimensional picture of a cell or tissue by focusing a laser at a specific depth in the specimen. Multiphoton microscopes which are very expensive use principles of confocal microscopy with infared lasers which finely focus light onto the specimen and is less damages to living tissues.

APOPTOSIS

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