After studying this chapter, the reader should be able to:

• Discuss the use of light and electron microscopy
• Discuss ways in which tissues can be dissociated and subcellular fractions isolated.
• Describe the ways in which chromatography and electrophoresis can be used to analyze cellular macromolecules.
• Trace cellular events with radioisotopes and antibodies.
• Outline the most important techniques and applications of genetic engineering.

1. General information
1. Light microscopy can resolve objects that are more than 0.2 m m apart
2. The cellular ultrastructure can be seen with both transmission electron microscopes (TEM) and scanning electron microscopes (SEM)
3. The normal resolution of TEM is 2 nm, or 100 times smaller than with a light microscope
4. For both light and electron microscopy, various techniques of fixation, embedding, sectioning, and staining are available
2. Light microscopy
1. Typical mammalian cells, at 10 to 20 m m in diameter, are colorless and translucent
2. Bacteria and mitochondria, at 0.5 m m in size, are the smallest objects that can be seen under the light microscope
1. The limit of resolution is the limiting separation at which two objects can still be seen as distinct
2. The limit of resolution depends on the wavelengths of visible light, from violet (0.4 m m) to deep red (0.7 m m)
3. For light microscopy, the limit of resolution is 0.2 m m
3. Because tissues have to be fixed, sectioned, and stained, various techniques of specimen preparation are needed for light microscopy
4. Fixation is the immobilization, killing, and preservation of the sample
1. Common fixatives are reactive aldehydes, such as formaldehyde and glutaraldehyde
2. By fixation, cells are made permeable to staining reagents; the fixatives also crosslink cellular macromolecules so that they are stabilized
5. Because most tissues are too big for light microscopy, they must be sectioned before observation
1. Because tissues remain soft and fragile after fixation, they must be embedded in a supporting material for sectioning
2. Common embedding materials are waxes and resins
3. Tissues are sectioned in an instrument known as a microtome, with which 1-10 m m thick sections can be cut
4. Sections can also be cut in a cryostat, in which frozen sections can be cut without prior fixation
6. Staining makes cells visible under the microscope by addition of an appropriate dye
1. Organic dyes developed for the textile industry were found to stain biological specimens
2. Stains such as Coomassie blue and hematoxylin are still used in histology today
7. In addition to the standard bright field light microscopy, other variations are available
1. Live cells can be observed with these special techniques
2. By phase-contrast microscopy and Nomarski differential-interference-contrast microscopy, unstained cells and specimens can be seen; their various subcellular constituents, with different densities, cause light interference effects
3. In dark field microscopy, light is scattered by cellular components and cells are seen as illuminated objects against a dark background
8. Using a fluorescence microscope, fluorescent dye-stained cellular structures can be detected
1. A fluorescent molecule absorbs light at one wavelength and emits it at a longer wavelength
2. Common fluorescent dyes are fluorescein, which emits an intense yellow-green fluorescence, and rhodamine, which emits a deep red fluorescence
3. Molecules such as antibodies can be tagged (or coupled) with a fluorescent dye and used to visualize specific cellular structures
9. For light microscopy, video image processing can be used
1. Time lapse pictures can be taken by microcinematography
2. Computer-assisted image intensification systems can retrieve images and remove background "noises"
10. A new development is confocal microscopy, which offers increased resolution, higher contrast, and narrower depth of field
1. Confocal microscopy can be used to optically section specimens; this is faster than physical sectioning and avoids potential artifacts associated with physical sectioning
2. Confocal microscopy yields a better three-dimensional reconstruction and even provides a stereo image
3. With proper fluorescent dyes and probes, dynamic changes in the physiology of living cells can be studied by confocal microscopy
3. Electron microscopy
1. Cellular fine structures must be studied with electron microscopes (TEM/SEM)
1. This process uses a beam of electrons rather than a beam of light to reach the specimen that has been placed in a vacuum to produce an image
2. A vacuum is needed because molecules in air can scatter the electrons
2. Sample preparation is different for EM than for light microscopy
1. Tissues are fixed with glutaraldehyde and osmium tetroxide
2. Tissues are dehydrated in an alcohol series and embedded in a resin polymer
3. Thin sections of 50-100 nm are cut with a diamond knife in a microtome
4. Sections are placed on metal grids for viewing
5. Serial thin sections can yield a three-dimensional reconstruction of cellular fine structures
3. Specimen treatment with heavy metals such as osmium reveals cellular structures
1. Alternatively, other electron-dense markers, such as colloidal gold particles, may be used
2. By this process, for example, an antibody can be coupled to gold particles for ultrastructural localization
4. Like TEM, SEM can also provide a three-dimensional image of the specimen's surface
1. After being coated with a thin layer of heavy metal, a fixed specimen is scanned with a beam of electrons
2. The resolution of SEM is 10 nm, as compared to 2 nm for TEM
5. Both TEM and SEM provide a close look at cellular fine structures and subcellular organelles

1. General information
1. Experimental techniques for studying living cells are available
2. Cells can be isolated from a tissue and cultured in the laboratory
3. Cellular organelles can be isolated by centrifugation

1. Cellular probes and cell manipulation
1. Living cells can be analyzed experimentally
1. Ion-selective electrodes can be used to measure the intracellular concentration of inorganic ions, such as H⁺, Na⁺, K⁺, Cl^-, Ca⁺⁺ and Mg⁺⁺; pH can also be determined
1. Ca⁺⁺ is an important inorganic molecule, and probes are available to detect free Ca⁺⁺ changes inside living cells
2. Intracellular Ca⁺⁺ levels can be measured with the synthetic fluorescent Ca⁺⁺ indicators fura-2 and quin-2 and the bioluminescent protein aequorin, which comes from jellyfish
2. "Caged" molecules can be used to study intracellular dynamics
1. A molecule of interest is synthesized as a precursor and introduced into the cell; the molecule can then be activated only at a chosen site by focusing a spot of light on that location
2. Inactive photosensitive precursors are called "caged" molecules
3. "Caged" precursors for many small molecules, such as Ca⁺⁺ and GTP, are available
2. For molecules that are membrane impermeant, such as proteins and nucleic acids, three methods can be used to introduce macromolecules into mammalian cells
1. By microinjection, materials of interest can be injected into cells with a glass micropipet
2. By electroporation, application of a brief, intense electric shock causes transient pore formation on the cell membrane, allowing materials of interest to enter the cytosol
3. By membrane vesicle fusion, materials of interest can be enclosed in lipid vesicles (known as liposomes) and fused with the target cell membrane using chemicals such as polyethylene glycol (PEG) or viruses such as the Sendai virus
3. By the technique fluorescent analog cytochemistry, a purified protein is coupled to a fluorescent dye and then microinjected into a living cell; the fate of the labeled protein can be followed in a fluorescence microscope as the cell divides and differentiates
4. Alternatively, the jellyfish green fluorescent protein (GFP) can be used to construct a fusion protein with the cellular protein of interest by recombinant DNA technology, and this can be used to trace the protein in the living cell

3. Cell culture
1. Mammalian cells can be dissociated from tissues
1. In the presence of proteinases such as trypsin and collagenase and chelators such as ethylenediamine tetraacetic acid (EDTA), which chelates Ca⁺⁺, extracellular proteins that bind cells together can be degraded and then cell-cell interactions can be disrupted
2. Normally, a tissue is composed of many differentiated cell types; it is possible to separate and isolate the particular cell types of interest
3. If there is a unique cell surface marker on the cell type, an antibody can be produced and used to isolate that cell type in a fluorescence-activated cell sorter; by a process called flow cytometry, a laser beam detects and selects the cells with the fluorescent-dye tagged antibody bound to the cell surface
4. Isolated cells can be cultured in vitro ("in glass")
2. A primary culture (also known as an explant) is a group of cells directly derived from a tissue
1. A cell strain is a cell culture that is not permanent
1. For example, the WI-38 strain of embryonic human fibroblasts will undergo about 50 population doublings before dying
2. This phenomenon, first noted by Leonard Hayflick in 1961, is called the Hayflick limit
2. A cell line is a cell culture that can be propagated permanently (e.g., mouse 3T3 fibroblasts grow and form a confluent monolayer in a tissue culture flask)
3. A transformed cell line is a cell culture that can cause tumors if injected into appropriate animals (e.g., HeLa cells are human epithelial cells derived from a carcinoma of the uterine cervix that have been grown continuously in the laboratory since 1952; HeLa is from initials of the patient, Henrietta Lacks)
4. For the in vitro culture of mammalian cells, tissue culture media are necessary
1. A typical tissue culture medium consists of amino acids and glucose as nutrients, vitamins, salts, antibiotics (e.g., penicillin and streptomycin), and the indicator dye phenol red (a pH indicator that changes from red to orange-yellow as the medium turns acidic due to cell metabolism)
2. Another essential ingredient is a source of serum (such as fetal calf serum); serum provides necessary growth factors
3. Serum in a tissue culture medium, which is used to provide growth factors, can be replaced by specific growth factors and other known components (such as selenium [Se], a micronutrient); a serum-free medium is called a defined medium, since all components are known
4. Individual differentiated cell types have different requirements for growth factors and other components
5. Mammalian cells are grown in an incubator at 37^oC, with 95% air and 5% CO₂
5. In cell cloning, all daughter cells are descendants of the same parent cell; this process provides cells that are genetically uniform
6. In cell fusion, cells can be fused by chemicals (polyethylene glycol, PEG) or viruses (Sendai virus)
1. A fused product from two cells that contains two nuclei is a heterokaryon
2. Eventually the two nuclei fuse; the resulting product is called a somatic cell hybrid
3. In a typical cell fusion between human and rodent (mouse or hamster) cells, there is a selective loss of human chromosomes in the hybrids
1. This phenomenon has been applied in human gene mapping
2. For example, using a panel of hybrid cells retaining different human chromosomes, the gene of the human proteinase cathepsin B has been detected only in hybrids with human chromosome 8; thus this human gene is located in chromosome 8

4. Organelle fractionation
1. Subcellular organelles can be isolated from tissues or cultured cells
1. Cells are disrupted by homogenization, in an appropriate medium that maintains proper isotonicity (or osmolarity)
2. With either a hand- or motor-driven homogenizer (basically a pestle and a tight-fitting tube), the tissue is homogenized by vertical strokes and rotation of the pestle
3. The cell membrane is broken and the subcellular organelles are released into the homogenization medium
2. In centrifugation, particles sediment according to their size, density, and shape
3. Different subcellular fractions can be separated from such a homogenate by differential centrifugation
1. A low-speed centrifugation, 800-1,000 X gravity (g), brings down the nuclei
2. A medium-speed centrifugation, 20-30,000 X g, brings down mitochondria, lysosomes, and peroxisomes
3. A high-speed centrifugation, 100,000 X g, brings down microsomes (membrane vesicles) plus a soluble fraction (ribosomes, proteins)
4. Organelles can also be separated by equilibrium density-gradient centrifugation
1. A medium with varying density (such as 5-20% sucrose in a buffer) is made or generated in a centrifugation tube
2. Different organelles will sediment into regions (as bands) where the density of the organelles is the same as the density of the medium

1. General information
1. Biochemical methods are used to analyze cellular macromolecules
2. Techniques of chromatography and electrophoresis allow for the study of nucleic acids and proteins
3. Quantitation of macromolecules can be achieved by spectroscopy
4. The three-dimensional structure of individual macromolecules can be detected by X-ray diffraction or nuclear magnetic resonsance

2. Chromatography
1. Chromatography is the separation of molecules by partitioning them between two media
1. One medium, or phase, is stationary; and the other is moving
2. In column chromatography, using a solid medium through which a flow of liquid is passed results in a differential migration of macromolecules such as proteins
1. Proteins pass through a column containing porous solid matrixes
2. Due to protein-matrix interaction, different proteins are retarded to different extents
3. By this approach, different proteins can be separated and collected (or eluted) from the column
2. Macromolecules such as proteins are separated by size (gel filtration or size exclusion chromatography), charge (ion-exchange chromatography), and binding capacity (affinity chromatography)
3. Electrophoresis
1. Electrophoresis is the movement of charged molecules in an electric field
2. Cellular macromolecules, such as proteins, can be analyzed by electrophoretic techniques
1. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separates proteins in a solid support according to size
2. SDS is an ionic detergent that unfolds proteins; it is negatively charged and binds to the polypeptide chain
3. Polyacrylamide is a synthetic gel for protein separation
4. b -mercaptoethanol is used to reduce the protein disulfide bonds
5. After electrophoresis, separated protein bands can be visualized using dyes such as Coomassie blue or techniques such as silver stain, which can detect 10 ng protein in a gel band or gel spot
3. Western blotting (protein blotting) is the transfer of proteins from a gel into a membrane support (such as nitrocellulose paper) and the detection of the protein of interest among the transferred proteins by the use of an antibody
4. Isoelectric focusing (IEF) is the separation of proteins in a solid support according to their charge
5. Two-dimensional gel electrophoresis (2-D gel) is the combination of IEF and SDS-PAGE
6. Both polyacrylamide gel and agarose gel electrophoresis are available for the separation of nucleic acids
1. Agarose is used to separate the larger nucleic acids, polyacrylamide is used to separate the smaller ones
2. Different buffers are used to separate nucleic acids and proteins
3. The separated nucleic acids can be visualized by stains such as ethidium bromide, using UV light
4. Other biochemical methods
1. Proteins separated by chromatographic and electrophoretic techniques can be further analyzed and characterized
1. A peptide map of the protein of interest can be generated (e.g., using an enzyme such as trypsin that cleaves after Lys or Arg residues in a protein, or a chemical such as cyanogen bromide that cleaves after Met in a protein)
2. The amino acid sequence of a purified protein can be determined in an instrument called automatic amino acid sequenator
2. Macromolecules such as proteins and nucleic acids can be quantitated by spectroscopy (also known as spectrophotometry)
1. Spectroscopy is an analytical method of measuring the amount of light absorbed by a substance in solution
2. Using a UV spectrophotometer, protein quantitation can be measured at 280 nm, nucleic acids, at 260 nm
3. The structure of macromolecules can be determined by X-ray diffraction and two-dimensional nuclear magnetic resonance (NMR)
1. The double helix structure of DNA was first proposed by James Watson and Francis Crick in 1953 based on X-ray diffraction pattern
2. 2D-NMR has been useful in deducing the structure of small proteins (up to 20 Kd in size) and protein domains
4. Function of macromolecules can be investigated in cell-free systems
1. In vitro translation, such as the rabbit reticulocyte lysate system, can be used to study eukaryotic protein synthesis
2. Bacterial in vitro transcription-translation system can be used to study expression of cloned genes

1. General information
1. Classically, both radioisotopes and antibodies have been used as tracers to follow specific proteins
2. Recent advances in immunology have led to the production of monoclonal antibodies
3. Some monoclonal antibodies have catalytic functions
2. Radioisotope techniques
1. A radioisotope is an unstable isotope (form) of a chemical element which undergoes spontaneous disintegrations, i.e., transformations to a more stable form
1. Disintegration involves the release of energetic particles or radiant energy, which can be quantitively measured
2. Half-life is the time required for 50% of the atoms of an isotope to disintegrate
3. The common isotopes used in cell biology and their half lives are ³H (12.4 yr), ¹⁴C (5570 yr), ³²P (14 d), ³³P (25.5 d), ³⁵S (87 d), and ¹²⁵I (60 d)
2. Radioisotopes offer high sensitivity; e.g., 1000 molecules in a sample can be detected
3. Radioisotopes are incorporated into precursors of small molecules for macromolecule synthesis, e.g. ³⁵S-methionine (protein synthesis), ³H-uridine (RNA synthesis), ³H-thymidine (DNA synthesis), and ³²P-ATP (DNA sequencing)
4. In pulse-chase experiments, cells are given a radioisotope precursor for a short period (pulse) and then are incubated in the absence of the radioisotope (chase) in order to follow the fate of the molecule of interest through different subcellular compartments, or to different chemical forms
5. In autoradiography, the presence of radioisotopes (or labeled materials) is traced to specific subcellular localizations (such as in light or electron microscopy), or to specific synthetic activities (such as proteins synthesized after a stimulation as detected by 2-D gel)
3. Antibody techniques
1. Traditionally, antibodies have been generated in rabbits or, if larger quantities are needed, in goats
1. The antigen or molecule of interest is injected into the animal (usually with an adjuvant)
2. An adjuvant is a compound used to enhance the immune response; perhaps the most famous is Freund's adjuvant, a water-in-oil emulsion containing heat-killed tuberculin bacilli
3. Additional (or booster) shots are administered
4. Animals are bled and antibodies are purified from the blood; since there are many other antibodies, an antigen column is often used to obtain an antibody against a single target
5. Although a single antigen (or immunogen) is used, many lymphocytes (or B cells) are stimulated to produce antibodies; these antibodies are polyclonal, meaning that different antibodies that recognize and bind to different parts of the antigen are generated
2. Antibodies serve as tracers in cellular studies
1. Antibodies can be tagged by fluorescent molecules, such as fluorescein and rhodamine
2. Antibodies can also be tagged by molecules such as biotin (a water soluble vitamin); biotin-coupled antibodies are detected by avidin (an egg protein) or streptavidin (a bacterial protein)
3. Detection molecules such as avidin can be conjugated to marker enzymes; common ones include alkaline phosphatase (AP) and horseradish peroxidase (HRP)
4. Enzyme-substrate hydrolysis yields an insoluble product for subcellular localization and signal amplification
3. Enzyme-linked immunosorbent assay (ELISA) is a method used to detect and quantitate the amount of target antigen in a specimen
4. Monoclonal antibodies recognize single sites of the target antigen
1. The hybridoma technology for producing monoclonal antibodies was first developed by Georges Kohler and Cesar Milstein in 1975
2. Instead of rabbits, mice are used
3. The antigens of interest are injected and booster shots administered
4. The immunized mouse is sacrificed and its spleen is dissected
5. The spleen lymphocytes, which contain B cells that produce antibodies, are isolated and fused with mouse myeloma cells
1. Mouse myeloma cells, derived from B cell tumors, are immortal but do not produce antibody
2. Fusion is accomplished by PEG or Sendai virus
6. Cell fusion products are cultured in a selective medium, such as hypoxanthine-aminopterin-thymidine (HAT) medium
7. Tumor cells are killed in the HAT medium; since unfused B cells are not immortal, only the hybrids will survive
8. Such somatic hybrids, products of a single B cell and a myeloma cell, are known as hybridomas; hybridomas produce monoclonal antibodies, antibodies that recognize only a single site on the antigen
5. Using the hybridoma technology and appropriate antigens, Richard Lerner in 1986 generated monoclonal antibodies that have catalytic functions; these antibodies that can act as enzymes are called abzymes
6. Besides being useful as biological tracers, antibodies are useful clinical reagents; e.g., an antibody linked to a toxin can target to specific tumor cells and kill the cells; Herceptin is a commercial antibody for treating some breast cancer

1. General information
1. Gene cloning and genetic engineering techniques have made an impact in all areas of life sciences, from basic research to practical applications
2. Human or other genes of interest have been expressed in a variety of cells, including bacteria, bacteriophages, yeasts, insect cells, and mammalian cells
3. Genes can be expressed or deleted from experimental animals, in transgenic flies, worms, mice, and even goats and pigs
4. One technique known as the polymerase chain reaction (PCR) has found applications in many fields, from diagnostic microbiology to paleontology
2. Restriction enzymes
1. A restriction enzyme (or endonuclease) cuts DNA at a specific site
1. Because the DNA macromolecule is long, it is difficult to handle
2. The availability of restriction enzymes makes the DNA molecule easier to study
2. Restriction endonucleases cleave DNA into fragments and the cut DNA can then be analyzed by agarose gel electrophoresis and ethidium bromide (or SYBR Green I) staining
1. For simple DNA, such as cloned plasmids or small viral genomes (like the simian virus SV40), specific DNA bands are seen; by judicious use of different restriction enzymes, the different restriction fragments can be aligned into a restriction map
2. For complex genomes such as mammalian DNA with many different DNA sequences, what is seen is a smear of DNA along the whole lane of the gel; in such cases, specific DNA probes are needed to select for the DNA fragment of interest by the process of nucleic acid hybridization
3. Restriction enzymes are isolated chiefly from microorganisms, including bacteria and archaebacteria
1. To date, over 10,000 microbes have been examined and over 2,500 restriction enzymes have been found
2. The 2,500 or so enzymes exhibit about 200 different specificities; isoschizomers are different restriction enzymes that recognize the same DNA sequence
3. Although the enzymes commonly recognize a sequence of 4-6 nucleotides in length, some have recognition sequences of 8 or more nucleotides
4. The recognized sequence is a palindromic DNA sequence; thus, at the recognition site, the nucleotide sequences of double strand (ds) DNA are the same for the two strands
5. The common restriction enzymes used in recombinant DNA experiments are Type II restriction endonucleases
1. Type II restriction endonucleases act as homodimers and bind, recognize, and then cut the recognized nucleotide sequence
2. Type I restriction enzymes recognize nucleotide sequences but cut at a different site
3. Some enzymes recognize and cleave the DNA into staggered or cohesive ends; thus, the restriction endonuclease Escherichia coli restriction enzyme 1 (EcoRI) recognizes the sequence GAATTC and the cut is G/AATTC, giving a 5' overhang (i.e., the single strand, AATT, is the 5' strand)
4. Some give 3' overhangs; e.g., the PstI (from Providencia stuartii) site is CTGCA/G (the single strand, ACGT which is complememtary to TGCA, is the 3' strand)
5. Some give flush (or blunt) ends; e.g., SmaI (Serratia marcescens) cuts at CCC/GGG
6. The enzyme XmaI (Xanthomonas malvacearum) is an isoschizomer of SmaI, cutting at C/CCGGG and giving a 5'overhang
7. The enzyme TaqI (Thermus aquaticus) recognizes a 4 nucleotide sequence T/CGA, the enzyme NotI (Nocardia otitidis-caviarum) recognizes a 8 nucleotide sequence GC/GGCCGC, and both give 5'overhangs
6. The restriction fragments can be used as DNA probes, as source materials for DNA cloning, for DNA rapid sequencing, and for other purposes
4. In nature, restriction endonucleases may protect the microbes
1. Bacteria use these enzymes to cut DNA of invading viruses
2. To protect the bacteria's own DNA from cutting, the bacterial DNA is modified by DNA methylation, via the action of sequence-specific DNA methyltransferases
3. The sequence specific restriction endonuclease and its corresponding methyltransferase form the microbe's restriction-modification system
4. Restriction enzymes are widespread in nature; enzymes of the same specificity occur in different species and enzymes of different specificities occur in different isolates of the same species
3. Molecular cloning
1. Molecular cloning or gene cloning is a rapid method for generating many copies of a DNA fragment
1. By recombinant DNA technology, any gene can be copied (or cloned) in bacteria or bacteriophages, making available unlimited, identical copies of the gene for further analysis, such as DNA sequencing
2. With the addition of appropriate signals for protein expression, it is possible to express the gene product in bacteria (e.g., bacterial expression of human insulin)
2. To express foreign DNA (from human or other sources) in bacteria and phages, cloning vehicles or cloning vectors are needed
1. Bacterial plasmids are small, circular, double strand DNA that occur naturally and often confer antibiotic resistance to the host bacterium (the bacterium has its own chromosomal DNA)
2. They contain sequences (origins of replication) that allow them to be replicated in the cell
3. Bacterial plasmids can be adapted to be cloning vectors by the addition of appropriate restriction sites for DNA cloning, the deletion of non-essential DNA fragments not relevant for plasmid replication, and the inclusion of appropriate antibiotics in resistance genes for the selection of recombinant colonies
4. One famous plasmid vector is pBR322
1. In its name, p stands for plasmid and BR denotes the inventors Francisco Bolivar and Raymond Rodriguez
2. First reported in 1977, it is 4361 nucleotides in size and confers tetracycline and ampicillin resistance upon its bacterial host cell
5. Other common cloning vectors include the ones derived from the bacteriophages lambda and M13
3. With appropriate cloning vectors, it is possible to make a library of the entire human genome (or any other species of interest) in recombinant bacteriophages
1. Such a collection of phages is called a genomic DNA library of a particular species
2. Normally, lambda phage-derived vectors, such as lambda EMBL3 (for European Molecular Biology Laboratory and related vectors), are used because they can incorporate larger fragments of foreign DNA (up to 20-25 kilobases [Kb]), whereas plasmid vectors only take smaller-size foreign DNA inserts
1. Both the vector and human DNA are cut with the same restriction enzyme (or ones that recognize the same sequence)
2. A ligation (or linking) reaction allows the combination of the foreign and vector DNA
3. An in vitro packaging reaction then allows the recombinant DNA molecules to be made into recombinant phages
4. The phages are used to infect the bacterial host (E. coli) and appear as plaques on a lawn of host bacteria on an agar plate
3. A genomic DNA clone may contain both coding and non-coding segments of a gene of interest
1. Most eukaryotic genes are interrupted by non-coding segments known as introns
2. Both exons and introns may be included (exons code for segments of mRNA)
3. Alternatively, it may contain other gene regulatory regions
4. Because most differentiated cell types retain the same genome, the genomic DNA library is the same, irrespective of the source of DNA (except in lymphoid cells)
4. In contrast, a cDNA library is different for each cell type or each tissue
1. Each cell type or tissue expressed its own set of mRNAs; cDNAs are the complementary DNAs copied from these mRNAs
2. Since mRNA is the processed form of its gene (minus introns), it is smaller than the original gene; both plasmid (such as pBR322) and phage (such as lambda gt11 and derivatives) cloning vectors are used for cDNA construction
1. First, total RNA is isolated from the cells or tissues of interest
2. Then, poly A+ RNA is isolated from the total RNA (most mRNA contains a poly A tail at the 3'end) by using an oligo dT cellulose or related affinity chromatography technique
3. Copies of mRNA are made into cDNA using the viral enzyme reverse transcriptase, which is currently available from avian myeloblastosis virus (AMV), Moloney murine leukemia virus (MMLV), or a cloned version from these RNA viruses
4. The mRNA is degraded by an alkali treatment; the complementary strand is made by the enzyme DNA polymerase
5. The ds cDNA is ligated to the plasmid vector using the enzyme DNA ligase; phage vector may also be used and steps such as the addition of oligonucleotide linkers may be included to ensure restriction site compatibility
6. The recombinant plasmids are used to transform the host bacteria (E. coli) and thus to generate a cDNA library; recombinant plasmids confer antibiotic resistance on the bacteria
5. With the availability of genomic or cDNA libraries, methods have been developed to screen for the gene of interest
1. Using labeled nucleic acid as probes, the screening process is called colony hybridization for plasmid vectors and plaque hybridization for phage vectors
2. If the cDNA library has been made with expression vectors such as lambda gt11, an antibody against the gene of interest can be used to screen the protein product on membrane filters, using a technique such as ELISA
6. In addition to the common plasmid and bacteriophage cloning vectors, others are available for specific purposes; these include cosmids and yeast vectors (yeast artificial chromosomes [YACs]) for large fragments of DNA, baculovirus-derived vectors for the expression of proteins and glycoproteins in insect cells, and shuttle vectors for expressing cloned genes in both bacteria and mammalian cells
4. Hybridization and sequencing of nucleic acids
1. Based on the ability of a nucleic acid sequence to bind to its complementary sequence, nucleic acid hyridization can identify specific sequences of DNA and RNA with great accuracy and sensitivity
2. Colony and plaque hybridizations are nucleic acid hybridizations used to screen for recombinant clones of interest
1. Recombinant bacterial colonies or phage plaques on the bacterial lawn are grown on agar plates
2. Duplicates of the colonies or plaques are transferred from the plates to membrane filters made of nitrocellulose or nylon
3. Appropriate probes are then used to screen the filters for the clones of interest
4. From the selected clone on the filter, the corresponding colony or plaque is then isolated from the original agar plate and the recombinant clone can be expanded, stored, and further studied
5. Different types of probes can be used for screening
1. Nucleic acid probes may vary in length from 15 nucleotides (oligonucleotides) to thousands of nucleotides
2. The shorter oligonucleotides may be synthesized based on the known amino acid sequence of the gene of interest
3. The longer probes may be a cloned cDNA or genomic sequence of the same gene of interest from another species; sequence divergence must be considered in inter-species gene screening
6. Nucleic acid hybridization is performed between the probe and recombinant DNA on the filter
1. The recombinant DNAs from lysed plaques or colonies are denatured into single strands by alkali treatment
2. The nucleic acid probe is also denatured (normally by boiling to dissociate it into single strands)
3. The probes must be tagged so that they are recognizable; e.g., radioactive probes can be tagged with ³²P-dCTP and nonradioactive probes with hapten labeled precursors, such as DIG or digoxigenin-dUTP and biotin-dATP
4. Bound nucleic acid probes can be recognized by autoradiography with X-ray films or by an enzyme-linked antibody that recognizes the haptens
3. In situ hybridization is nucleic acid hybridization used to localize the sites of gene expression in cells and tissues by light and electron microscopy
4. Southern blot hybridization (DNA blot hybridization) is a type of nucleic acid hybridization used to identify DNA sequences of interest
1. Southern blot hybridization was named after Edwin Southern, who first devised the technique in 1975
2. In the first step, DNA, perhaps after restriction by enzyme digestion, is separated by agarose gel electrophoresis
3. DNA in the gel is denatured and broken into smaller fragments by alkali and acid treatments
4. DNA is then transferred from the gel to a membrane filter made of nitrocellulose, nylon or other material by blotting it with an appropriate buffer; the original technique employed capillary action, but electric transfer has also been used
5. Through nucleic acid hybridization, a DNA probe is used to hybridize to the DNA band of interest among all the transferred and immobilized DNA bands on the filter
5. Similarly, northern blot hybridization (RNA blot hybridization) detects RNA species of interest using an appropriate DNA probe
1. The term "northern" was initially used to distinguish the technique from that developed by Southern; it has no technical significance
2. Total RNA or poly A+ RNA is separated by gel electrophoresis in a different system, such as the formamide formaldehyde gel
6. The Southern, northern and western blot (the protein transfer discussed earlier) methods allow sensitive detection of DNA, RNA and protein
7. For the study of nucleic acids, one important advance is the rapid sequencing of DNA
1. With the availability of cloned DNA fragments, it became possible to sequence the cloned DNA by rapid methods
2. Two sequencing methods have been developed; Fred Sanger invented the enzymatic method in 1975 and Allan Maxam and Walter Gilbert invented the chemical method in 1977
3. By using the enzymatic rapid DNA sequencing method, the genome of the bacteriophage phi X174 (5386 base pairs) was completely sequenced in 1977
8. The enzymatic sequencing method involves DNA synthesis reaction in vitro
1. Components may include: the cloned DNA template, such as the ssDNA cloned in M13 phage vector or denatured plasmid DNA; radioactive precursors, such as ³⁵S-dATP; a primer, such as the oligonucleotide that binds close to the cloning site of the cloning vector, and DNA polymerase, such as the cloned DNA polymerase from phage T7
2. Four separate reactions of DNA synthesis are performed, each with a different dideoxynucleotide included in the reaction mixture; incorporation of such a modified nucleotide precludes further elongation of the newly synthesized DNA chain, stopping DNA synthesis
3. A proper ratio of deoxynucleotide and dideoxynucleotide is used, so that statistically synthesized single-strand (ss) DNA of different sizes can be obtained
4. The products are analyzed by high resolution polyacrylamide gel electrophoresis, which can differentiate the size of the ssDNAs one base shorter than each other, and autoradiography
5. Reading of the autoradiographic pattern yields the DNA sequence of the ssDNA
9. The chemical sequencing method involves the electrophoretic analysis of ³²P end-labeled ssDNA after specific reactions
1. The labeled ssDNA is purified and divided into four portions for four separate chemical reactions; each will recognize and cut the ssDNA after a different, specific base G, A, T, or C
2. Under appropriate experimental conditions, with many copies of end-labeled ssDNA available, each copy of ssDNA can be made to have only one single cut by the chemical reagent
10. Rapid DNA sequencing methods have been adapted for automation and nonradioactive precursors, such as fluorescent nucleotides, are available for this purpose
5. Polymerase chain reaction (PCR)
1. The technique of polymerase chain reaction (PCR) was developed by Kary Mullis and first reported in 1985
2. PCR is a powerful method for copying or amplifying DNA fragments of interest in vitro, as opposed to a process of in vivo cloning that uses microorganisms such as bacteria
3. The use of thermophilic DNA polymerase, including Taq DNA polymerase from Thermus aquaticus, and the Vent DNA polymerase from the archaebacterium Thermococcus litoralis, has simplified the PCR process, as has the development of a thermocycler
4. Reagent components include 2 primers (a 5' sense primer and a 3' antisense primer), the DNA of interest (template), the enzyme, the nucleotide precursors, and a buffer
5. The basic reaction involves repetition of 3 steps: DNA denaturation, such as the application of heat at 95^oC for 1 min; primer annealing, such as the application of heat at 37^oC for 1 min; and primer extension or elongation, such as the application of heat at 72^oC for 1 min
6. After the steps have been repeated for 15-50 cycles, the amplified DNA can be analyzed by gel electrophoresis, cloning, or sequencing
7. Many applications have been developed, including detecting infections and mutations, monitoring cancer therapy,analyzing forensic samples, and studying ancient DNA, such as that of the quagga, an extinct member of the horse family
6. Applications
1. Recombinant DNA techniques permit reverse genetics
1. From a 2-D gel protein spot, it is possible to isolate the protein and determine its partial amino acid sequence
2. If the protein sequence of a gene of interest is known, it is possible to chemically synthesize the oligonucleotide probe and screen for the recombinant clone from gene libraries (from a partial cDNA clone, to a complete cDNA clone, to a genomic DNA clone)
3. Sequencing of the clone yields the complete sequence of the gene of interest
4. It is possible to perform in vitro mutagenesis to alter the sequence of the gene
5. The altered (or mutated) gene can be put back into cells or organisms in order to study its function (e.g., transgenic animals)
2. Scientifically useful transgenic animals include transgenic flies (Drosophila melanogaster), worms (Caenorhabditis elegans), and mice; commercially important transgenic animals include farm animals (such as transgenic pigs expressing human hemoglobin, and transgenic sheep expressing human anti-hemophilic Factor IX protein in their milk)
3. Generation of transgenic mice involves several steps
1. A cloned gene of interest is microinjected into the male pronucleus of the fertilized mouse egg
2. The egg is allowed to develop into an embryo in tissue culture and then transferred into a pseudo-pregnant surrogate mother
3. After birth, the animals are checked for the expression of the transgene; additional mice are bred to maintain the gene of interest in the germ line
4. In 1999, Joe Tsien and colleagues produce transgenic mice with NMDA (N-methyl-D-aspartate) receptor, yielding "smarter" mice
5. Development of a "knockout mice" generation involves even more steps (deletion of the gene of interest from mice, also known as targeted gene replacement, by replacing the normal gene in mice with a mutant gene)
1. The first step is the manipulation of mouse embryonic stem (ES) cells; the gene of interest is disrupted (or replaced) in these cells by the use of cloning vectors carrying the mutant gene and appropriate markers, such as the antibiotic resistance gene for the purpose of selection
2. A rare homologous recombination event, the replacement of the normal gene with the mutant gene at the gene locus, must occur
3. The rare ES cell with the targeted gene mutation is selected, cultured, and microinjected into the mouse embryo when it is at the blastocyst stage
4. After embryos are developed in surrogate animals, as described before, offspring expressing the mutant gene in the germ line are selected
5. Because breeding leads to daughter mice that have the mutation in both copies of the gene of interest, the effect of gene loss on cell and animal function can be investigated
6. Transgenic and knockout mice can provide useful animal models of human genetic diseases
1. For example, knockout mice of the cystic fibrosis (CF) gene were reported in 1992 and 1993, only a few years after the discovery of the human gene in 1989
2. The cystic fibrosis transmembrane conductance regulator (CFTR) gene is large with 250 Kb and codes cyclic AMP-dependent chloride channel, a protein of 1480 amino acids
3. The knockout mice show symptoms that are similar to those of human CF, including defective epithelial Cl^- transport, mucus accumulation in the intestine, and early death due to intestinal obstruction
4. The human hereditary breast cancer gene BRCA1 gene was cloned in 1994; and knockout mice model was made in 1996
5. BRCA1 gene codes for a transcription factor that regulates cell growth; knocknout mice die during embryogenesis
7. Some recombinant DNA products have led to controversy, e.g. the approval in 1994 by United States Food and Drug Administration (FDA) of the use of recombinant bovine somatotrophin (BST), a growth hormone, to boost milk production in cows
8. In 1999, GM (genetically modified) foods raises concerns in Europe (over United States use of agricultural biotechnology, e.g. corn with gene coding for insecticide)
9. DNA or gene chips allow the analysis of thousands of genes on microscopic slides; such cDNA microarray set-ups are available for studies of gene expression patterns
10. Advances in recombinant DNA technology have led to human gene therapy
1. The world's first approved gene therapy was performed in 1990 by French Anderson and colleagues
2. The researchers isolated blood cells from two young girls with severe combined immunodeficiency (SCID), also known as "bubble boy disease"
3. Because one form of SCID involves a defect in the enzyme adenosine deaminase (ADA), the researchers added the normal ADA gene to isolated stem cells and then returned the cells to the patients
4. In 1994, the treated girls Cynthia Cutshall and Ashanthi DeSilva are serving as "research ambassadors" for the March of Dimes-Birth Defects Foundation
5. Method of delivery is a concern; modern vectors under study include retroviruses, adenoviruses, adeno-associated viruses; liposomes and "naked" DNA.

Study Activities

1. Describe the steps and methods that could be undertaken to isolate a cell type of interest, given a tissue and an antibody to a unique cell surface protein of that cell type.
2. Given an antibody and a cDNA clone of murine liver protein albumin, analyze its gene expression during mouse development by Southern, northern, and western blots.
3. Assuming access to the cloned human diseased gene and its normal counterpart, design experiments to generate an animal model of this human disease.