Lecture Four. Molecular Approaches I: Nucleic Acids I. Recombinant DNA and Gene Cloning Recombinant DNA is DNA that has been created artificially. DNA from two or more sources is incorporated into a single recombinant molecule. In essence, in order to amplify any DNA sequence in vivo, the sequence in question must be linked to primary sequence elements capable of directing the replication and propagation of themselves and the linked sequence in the desired target host. The required sequence elements differ according to host, but invariably include an origin of replication, and a selectable marker. In practice, however, a number of other features are desired and a variety of specialized cloning vectors exist that allow protein expression, tagging, single stranded RNA and DNA production and a host of other manipulations that are useful in downstream applications. In the classical restriction and ligation cloning protocols, cloning of any DNA fragment essentially involves four steps: fragmentation, ligation, transformation, and screening/selection. Plasmids Plasmids are molecules of DNA that are found in bacteria separate from the bacterial chromosome. They are small (a few thousand base pairs), usually carry only one or a few genes, are circular, and have a single origin of replication. The same machinery that replicates the bacterial chromosome replicates plasmids. Some plasmids are copied at about the same rate as the chromosome, so a single cell is apt to have only a single copy of the plasmid. Other plasmids are copied at a high rate and a single cell may have 50 or more of them. Genes on plasmids with high numbers of copies are usually expressed at high levels. In nature, these genes often encode proteins (e.g., enzymes) that protect the bacterium from one or more antibiotics. Plasmids enter the bacterial cell with relative ease. This occurs in nature and may account for the rapid spread of antibiotic resistance in hospitals and elsewhere. Plasmids can be deliberately introduced into bacteria in the laboratory transforming the cell with the incoming genes. Restriction Mapping Restriction mapping: Digestion to completion with a restriction enzyme generates a series of fragments of discrete sizes from genomic DNA. In many cases, a single probe, such as a cdna is long enough to hybridize with more than one such fragment in a Southern blot. Alternatively, a large cloned sequence can be cut into a series of smaller fragments that can be identified by size without the need for a specific hybridization probe. This can be done through the use of a fluorescent dye such as ethidium bromide, which intercalates into the DNA and causes all bands to fluoresce.
Partial digests: Partial digestion will yield a series of fragments of various lengths, some of which are the sum of two or three or more shorter fragments. It is possible to end label the shorter fragments and use them as probes to determine which of the longer fragments contain the same sequences. This allows nested sets of fragments that contain the same sequences to be identified, and when overlapping fragments are obtained, it is often possible to fit them all together to reconstruct the original larger fragment, or the entire area that hybridizes with a probe. Double digests: Digestion with two different restriction endonucleases separately and with the two together yields a larger total number of fragments that can be fitted together to generate a more detailed restriction map of the area of interest. Restriction mapping with two or more enzymes yields relatively short segments of DNA of known position that provide a physical map of the genomic region upon which known mutations and other markers of interest can be localized. It also provides an ordered series of relatively small fragments for sequencing, which collectively can be used to sequence a relatively long stretch of DNA. Isolation of insert Initially, the DNA fragment to be cloned needs to be isolated. Preparation of DNA fragments for cloning can be accomplished in a number of alternative ways. Insert preparation is frequently achieved by means of PCR, but it may also be accomplished by restriction enzyme digestion, DNA sonication and fractionation by agarose gel electrophoresis. Chemically synthesized oligonucleotides can also be used if the target sequence size does not exceed the limit of chemical synthesis. When using restriction endonucleases, the ends of the cut have an overhanging piece of single-stranded DNA and are called "sticky ends" because they are able to base pair with any DNA molecule containing the complementary sticky end. DNA ligase covalently links the two into a molecule of recombinant DNA. The ends may also be blunt, but ligation efficiency for blunt ends is not as efficient. Ligation Subsequently, a ligation procedure is employed whereby the amplified fragment is inserted into a vector. The vector (which is frequently circular) is linearized by means of restriction enzymes, and incubated with the fragment of interest under appropriate conditions with an enzyme exhibiting DNA ligase activity. Ligation procedures usually employ sticky ends, single stranded DNA overhangs which allow annealing of the insert with the vector sequence. Sticky ends can be incorporated into inserts either by chemical modification and attachment of adapter molecules or by incorporation of restriction enzyme recognition sequences into PCR primers and digestion of PCR products with the appropriate restriction enzyme prior to ligation.
Alternatively 3' A overhangs produced by non-proofreading DNA polymerases utilized in PCR can be used. Sticky ends allow for both higher efficiency transformations and can be used directional insertion of the insert into the vector, thus minimizing the need for subsequent screening.
Transformation Following ligation, a portion of the ligation reaction is transformed into cells. A number of alternative techniques are available, such as chemical sensitization of cells, electroporation and biolistics. Chemical sensitization of cells is frequently employed since this does not require specialized equipment and provides relatively high transformation efficiencies (usually involves preparing the cells in a calcium environment). Electroporation is used when extremely high transformation efficiencies are required, as in very inefficient cloning strategies. Biolistics are mainly utilized in plant cell transformations, where the cell wall is a major obstacle in DNA uptake by cells. Selection As ligation and transformation tend to be low efficiency, there is a need to identify the cells that contain the desired insert at the appropriate orientation and isolate these from those not successfully transformed. Modern cloning vectors include selectable markers (most frequently antibiotic resistance
markers) that allow only cells in which the vector, but not necessarily the insert, has been transfected to grow. Additionally, the cloning vectors may contain color selection markers that provide blue/white screening (via α- factor complementation) on X-gal medium. Nevertheless, these selection steps do not absolutely guarantee that the DNA insert is present in the cells. Further investigation of the resulting colonies is required to confirm that cloning was successful. This may be accomplished by means of PCR, restriction fragment analysis and/or DNA sequencing.
II. DNA Gel electrophoresis DNA electrophoresis is an analytical technique used to separate DNA fragments by size. An electric field forces the fragments to migrate through a gel. DNA molecules normally migrate from negative to positive potential due to the net negative charge of the phosphate backbone of the DNA chain. Longer molecules migrate more slowly because they are more easily 'trapped' in the network. After the separation is completed, the fractions of DNA fragments of different length are often visualized using a fluorescent dye specific for DNA, such as ethidium bromide. The gel shows bands corresponding to different DNA molecules populations with different molecular weight. Fragment size is usually reported in "nucleotides", "base pairs" or "kb" (for 1000's of base pairs) depending upon whether single- or double-stranded DNA has been separated. Fragment size determination is typically done by comparison to commercially available DNA ladders containing linear DNA fragments of known length. Gels have conventionally been run in a "slab" format such as that shown in the figure, but capillary electrophoresis has become important for applications such as high-throughput DNA sequencing. Electrophoresis techniques used in the assessment of DNA damage include alkaline gel electrophoresis and pulsed field gel electrophoresis. The measurement and analysis are mostly done with specialized gel analysis software. Capillary electrophoresis results are typically displayed in a trace view called an electropherogram. The DNA strand is cut into smaller fragments using restriction enzymes, then samples of the DNA solution (DNA sample and buffer) are placed in the wells of the gel, and allowed to run through the gel.
III. Southern analysis A technique developed by E. M. Southern in 1975 for the detection of a specific DNA sequence (gene or other) in a large, complex sample of DNA (e.g. cellular DNA). It is also used to determine the molecular weight of a restriction fragment and to measure relative amounts in different samples. Under optimal conditions, Southern blotting detects ~ 0.1 pg of the DNA of interest. Southern blots are used in gene discovery and mapping, evolution and development studies, diagnostics and forensics. In regards to genetically modified organisms, Southern blotting is used as a definitive test to ensure that a particular section of DNA of known genetic sequence has been successfully incorporated into the genome of the host organism. Genomic DNA or DNA from a specific source, such as a lambda phage or cosmid clone, is digested, usually to completion, with a restriction endonuclease. Electrophoresis is then used to separate the fragments by size. The fragments are then blotted from the electrophoretic gel onto a sheet of nitrocellulose or similar support material, and fixed onto it by heating or other treatments. The attached DNA fragments are denatured to separate the strands and annealed with a radioactive probe that is single stranded or also denatured. The nitrocellulose sheet is then washed, removing all unbound probe, and leaving radioactivity only where the probe has hybridized to the original DNA bound to the membrane. A sheet of X-ray film is then laid over the nitrocellulose for a time period long enough for the radioactivity to "expose" the film. When the film is developed, dark bands appear wherever there were DNA fragments capable of hybridizing with the radioactive probe. Size standards run on the same electrophoretic gel allow the sizes of the fragments identified by the probe to be determined.
A Northern analysis involves assessment of RNA samples, run through a formaldehyde slab gel, using essentially the same approach as a Southern. IV. Polymerase Chain reaction (PCR) The purpose of a PCR (Polymerase Chain Reaction) is to make a huge number of copies of a gene. There are three major steps in a PCR, which are repeated for 30 or 40 cycles. This is done on an automated cycler, which can heat and cool the tubes with the reaction mixture in a very short time.
Denaturation at 94 C: During the denaturation, the double strand melts open to single stranded DNA, all enzymatic reactions stop (for example: the extension from a previous cycle). Annealing at 54 C: The primers are jiggling around, caused by the Brownian motion. Ionic bonds are constantly formed and broken between the single stranded primer and the single stranded template. The more stable bonds last a longer (primers that match exactly); and the polymerase can attach on the region of double stranded DNA (template and primer), and start copying the template. Extension at 72 C: This is the ideal working temperature for the polymerase. The bases (complementary to the template) are coupled to the primer on the 3' side (the polymerase adds dntp's from 5' to 3', reading the template from 3' to 5' side, bases are added complementary to the template.
V. DNA sequencing