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LECTURE 2: WORKING WITH NUCLEIC ACIDS

I. Detection:

Nucleic acids can be detected by several methods, including: UV absorption, intercalation of dyes, incorporation and detection of radiolabel, and hybridization.

  1. The aromatic bases of nucleic acids absorb ultaviolet (UV) light with a maximum absorption occurring at a wavelength of 260 nm. Absorption is proportional to concentration: a 50 ug/mL solution of DNA will have an A260 = 1. For RNA, an A260 = 1 indicates a concentration of 40 ug/mL.
  2. Various dyes can be intercalated into the structure of nucleic acids (p. 139, Figure 17.7). The most commonly used in the lab is ethidium bromide, a flat molecule that can insert between adjacent bases. Nucleic acids "stained" with ethidium bromide can be visualized under UV light. Two other intercalating dyes are acriflavine and acridine orange.
  3. Radiolabel in the form of 32P can be inserted into nucleic acids.
    1. Internal labeling inserts 32P throughout the molecule by performing DNA replication in the presence of a 32P-labeled substrate. It requires a template nucleic acid, an oligonucleotide primer for intitiation, a DNA polymerase enzyme to construct the labeled nucleic acid and the substrate deoxynucleotide triphosphates (dNTPs). At least one of the dNTPs is labeled with 32P in the alpha position, which will be donated to the nucleic acid during DNA replication.
    2. End labeling is performed using polynucleotide kinase. This enzyme uses gamma-labeled 32P-ATP as a substrate to phosphorylate the 5' hydroxyl group of nucleic acids - i.e. it adds a single 32P-phosphate to the 5' "end" of the molecule.
    3. Radiolabeled nucleic acids can be detected by three methods: a. Autoradiography involves the exposure of photographic film by a radioactive source. b. Geiger counters monitors the ionization of gases when radioactive particles interact with them. c. Scintillation counting measures the light emission of molecules that fluoresce upon interaction with radioactive material. This last method allows for accurate quantitation of radioactivity.
  4. Hybridization (page 140) involves the annealing and detection of a complementary, labeled, single-stranded nucleic acid probe to a target sequence. There are several steps:
    1. First, the DNA is denatured - heat is used to separate the two strands of nucleic acid
    2. The DNA is applied to a nitrocellulose filter which binds single-stranded nucleic acids
    3. Non-specific DNA is used to coat the remaining portions of the filter
    4. A radiolabeled probe is added - a single-stranded nucleic acid with sequence complementary to the target
    5. The temperature is reduced slowly to allow complementary base pairing between the probe and the target nucleic acid
    6. Excess probe is washed off and the filter is dried
    7. Radiolabel is detected by autoradiography
  5. Hybridization (continued) - a variation
    1. Electrophoresis - DNA may be fractionated by size by gel electrophoresis prior to hybridization
    2. DNA can be transferred from gels to nitrocellulose by capillary action or by applying a potential difference (electroblotting)
    3. Probing is done as described above and "bands" that contain the target sequence are detected by autoradiography
    4. E. Southern (1975) first described this method of hybridization. "Southern blotting" refers to the detection of DNA targets by this procedure. In a related technique, "Northern blotting", RNA targets are detected by hybridization. .

Examples of uses for hybridization:

  1. Detection of an organism e.g. detecting an organism encoding a gene for a toxin
  2. Evolutionary classification based on 16S and 18S rRNA sequences
  3. Counting and identifying organisms in natural samples (Figure 17.7)

II. PURIFICATION OF DNA (p. 137)

  1. Precipitation and Extractions:
    1. First, cells are lysed using the enzyme lysozyme
    2. The slurry is treated with sodium hydroxide followed rapidly with acid and centrifugation is used to remove cell debris while the nucleic acids remain in solution
    3. Nucleic acids can be precipitated by treatment with ethanol and high salt at low pH; the precipitate is collected by centrifugation and dissolved in buffer
    4. RNA can be removed by treatment with ribonuclease enzymes
    5. Proteins can be denatured and removed by extraction with phenol
  2. Density gradient ultracentrifugation separates nucleic acids by their density. The nucleic acids are centrifuged in a solution of cesium chloride (CsCl), resulting in the formation of a CsCl gradient in which the nucleic acid forms a band at a density of CsCl equal to its own density. Ethidium bromide is used to detect the nucleic acids. The density of DNA is determined by its G/C content - the more G/C content, the denser the DNA.

III. GEL ELECTROPHORESIS

  1. DNA will migrate in an electric field due to its net negative charge.
  2. Gels consist of agarose or polyacrylamide; these substances form complex fibrous networks through which DNA can travel. The pore size of the matrix depends on the concentration of gel materials and the degree of crosslinking. Since DNA has the same charge/mass ratio regardless of its size, migration of DNA fragments in electric fields depends only on their size - smaller fragments travel further than larger fragments.
  3. The resolution of acrylamide gels is excellent - DNA fragments that differ in size by just one nucleotide an be separated. Agarose gels are ususally used to resolve larger fragments.
  4. Detection of nucleic acids in gels is typically accomplished with UV or autoradiography.

IV. DNA SEQUENCING (p.141-142)

F. Sanger's (1975) method of DNA sequencing involves the synthesis of radiolabeled DNA in the presence of dideoxynucleotide triphosphates (ddNTPs). These nucleotide derivatives do not have 3' hydroxyl groups, having instead a 3' hydrogen. Since the mechanism of DNA replication involves the attack of the 3' hydroxyl group on dNTPs, when ddNTPs are incorporated into DNA, no more DNA synthesis may occur.

    A. The procedure requires:

V. CUTTING AND SPLICING OF DNA

A. Restriction enzymes recognize and cut specific sequences of DNA. They provide protection against foreign DNA for the organisms that produce them.

  1. Restriction sites are typically palindromes: e.g. EcoR I recognition site.
    5'GAATTC3'
    3'CTTAAG5'
    Restriction enzymes are usually dimeric and each subunit "reads" and cuts a strand of DNA accounting for the palindromic nature of restriction sites and resulting in both strands of the DNA being cut
  2. The frequency of the occurrence of a restriction site can be calculated as (1/4)^n where n is the length of the recognition sequence.
  3. Restriction enzymes are named in reference to the organism that produces them: e.g. EcoR I comes from Escherichia coli Hind III comes from Haemophilus influenzae
  4. Restriction enzyme cleavage can result in the formation of "sticky" or "blunt ends". Sticky ends of DNA have protruding single stranded regions while blunt ends are flush, entirely double-stranded.
  5. Uses for restriction enzymes: a. Mapping DNA b. Cloning

B. Ligase can connect DNA having "compatible" sticky ends (i.e. those that have complementary sequences) or blunt ended DNA.

VI. PCR = POLYMERASE CHAIN REACTION

PCR involves the exponential amplification of a target DNA. Click here for an animated illustration of this process.

A. PCR requires:

  1. A DNA template to be amplified
  2. Knowledge of the boundaries (i.e. one must know the sequence of the regions flanking the region to be amplified)
  3. Two primers - short, single-stranded DNA complementary to each end of the DNA to be amplified and present in vast excess relative to the target DNA
  4. Substrate dNTPs
  5. A heat-stable DNA polymerase to construct the DNA

B. A PCR cycle is repeated 20-30 times to synthesize copies of the target DNA exponentially. Each cycle consists of three steps:

  1. Heat denaturation of DNA to separate the target DNA into its component strands
  2. Cooling to hybridize primers to the target DNA
  3. DNA polymerzation (extension) to make copies of the DNA

C. Uses

  1. Amplification of DNA allows it to be detected easily
  2. DNA fingerprinting - forensics investigations for identification of individuals (e.g. O.J. Simpson) e.g. VNTRs = Variable Number of Tandem Repeats - eukaryotic DNA contains short, non-coding sequences that vary in length from organism to organism. These regions can be amplified by PCR and their lengths determined - a "bar code" for an individual

VII. GENE CLONING

Why clone? The average length of a gene is 1 kb while the E. coli genome is 4700 kb and the human genome is three million kb in length. In other words, each gene is a very small fraction of the entire genome. Cloning a gene allows one to amplify it and thus facilitate its analysis.

Gene cloning requires a cloning vector, such as a plasmid (e.g. plasmid pBR322).

  1. The plasmid replicates independently of the chromosome by having its own replication orgin. pBR322 has the replication orgin ori and is maintained at 20-30 copies per cell.
  2. The plasmid must contain a selectable marker such as an antibiotic resistance gene. pBR322 has genes encoding resistance to ampicillin and tetracycline.

A classic strategy for gene cloning using pBR322 involves the inactivation of one the antibiotic resistance genes by the insertion of foreign DNA into it. Cells are screened for sensitivity to the antibiotic whose resistance gene has been disrupted in order to locate plasmids carrying foreign DNA (Figure 8.2).

VIII. TRANSFORMATION/TRANSFECTION

Transformation is a process by which DNA is inserted into bacteria. Transfection is the analogous term for eukaryotes. Several procedures are used:

A. Calcium chloride method

  1. Used to transform E. coli and some other Gram negative bacteria
  2. DNA is added to CaCl2-treated cells on ice; cells are transferred to 42 C briefly (heat shock)

B. Electroporation - exposure to pulsed electric fields

  1. Used to transform Gram positive bacteria, Gram negative bacteria and eukaryotes
  2. Currently supplanting other methods

C. Microprojectile gun - nylon particles are coated with nucleic acids and fired at the cells 1. Used to transform yeast, algae, plant cells, mitochondria, and chloroplasts