This lecture will be presented by Megan Bonner on Monday, October 4, 1999
Textbook Assignment: Chapter 9, 277 - 285 (sections 9.6, 9.7, and 9.8). Also, the discussion of probes on pages 270 - 271 needs to be read (this was scheduled to be part of lecture 16, but since we have reversed the sequence, it needs to be read for this lecture).
Web pages: There is an interesting web page, entitled "Nobel Savage" written by Steven Shapin. It is an extended review of a book entitled "Dancing Naked in the Mind Field" written by Kary Mullis, the inventor of the polymerase chain reaction (PCR). This review, which is from the London Review of Books online, focuses on the unconventional personality and lifestyle of Kary Mullis. It also includes an account of how the idea for PCR first struck him as he was driving his car. If you are easily offended by profanity, you may want to skip this site. It is full of "colorful" quotations from Mullis. For a somewhat milder version of his lifestyle and the moment of discovery, try "The quirky genius who is changing our lives". The textbook web pages also have a number of interesting references about PCR, Southern blotting and other techniques described in this lecture that can be reached from Hypercontents for Chapter 9
Major concepts
Polymerase Chain Reaction
The polymerase chain reaction (PCR) is a clever technique for the amplification of a small sample of DNA to an amount large enough to work with. It is widely used, both in basic research and in forensic studies. In extreme cases, one can start with the haploid genome from a single sperm and amplify selected sequences sufficiently for detailed analysis. Some knowledge of the DNA sequence on each side of the segment that is to be studied is needed to perform PCR.
Primers: In order to perform PCR, it is necessary to have a pair of short single-stranded oligonucleotides that are capable of priming synthesis of the sequence that is to be amplified and its reverse complement. The sequences of the two oligonucleotide primers are identical to the 5'-ends of the sense and antisense strands that will be amplified. Thus, they will hybridize to the 3'-ends of the complementary strands that serve as templates for the double stranded DNA that is to be amplified. In each case, the primer must anneal with the 3'- portion of the template strand so that it can prime DNA synthesis that proceeds 5' to 3' across the region to be amplified. The synthesis itself is carried out by a heat-stable DNA polymerase derived from bacteria that live in near-boiling water either in hot springs or in deep-ocean vents.
Temperature cycling: The original DNA, the primers, the heat-resistant DNA polymerase, and a supply of dNTPs are mixed together and placed in a temperature cycling device. The temperature is raised enough to separate the strands of the parent DNA. The temperature is then lowered enough so that the primers, which are present in excess, will anneal onto the separated DNA strands. The DNA polymerase then synthesizes a new strand that is complementary to each of the parent strands (figure 9.20). The temperature is then raised to separate the newly synthesized strands from the original strands. When the temperature is lowered in the next cycle, all four strands are primed and serve as templates for synthesis of new complementary strands. In the next cycle, eight strands are primed and serve as templates, etc. (figure 9.21). By the time 30 cycles have been completed, the theoretical multiplication factor would be 230, which is roughly equivalent to 109. Even larger multiplications are possible, provided that the system does not run out of dNTPs or primers and the enzyme does not lose its activity.
Uniformity of PCR product: Each cycle is programmed with enough time at the lower temperature for synthesis to extend beyond the positions of the two primers. However, after a few cycles, most of the template strands will end at the position of the primer that initiated their synthesis (see figure 9.21, cycle 4). After the first few cycles of PCR, the lengths of nearly all of the strands will be the exactly the distance between the outer ends of the two primers. This high level of product uniformity makes it possible to detect the product as a sharp band in an electrophoretic gel. If desired, the PCR product can now be cloned into a vector and further amplified. It can also be used as a sample for procedures such as DNA fingerprinting, or it can be sequenced to detect any point mutations in the region that was amplified.
Danger of contamination: Because PCR is capable of amplifying a single DNA strand, great care must be taken to avoid even the slightest contamination of the starting template or the reagents with any foreign DNA. A tiny flake of dried skin or a drop of sweat is enough to produce an amplification product that may mask or confuse the desired result. Such considerations are particularly important when PCR is used in the analysis of forensic evidence.
Electrophoresis of nucleic acids: When suspended in a suitably porous support matrix, such as a polyacrylamide or agarose gel, nucleic acids, which are negatively charged, migrate in an electric field. In the absence of confounding effects such as double stranded vs. single stranded, linear vs. circular, and relaxed vs. supercoiled, the rate of migration is inversely proportional to the size of the nucleic acid fragment. Small pieces move rapidly and larger pieces more slowly (figures 9.24 and 9.25. By comparison with the migration rates of standards of known sizes, it is possible to estimate the size of an uncharacterized fragment of DNA (figure 9.25)
Electrophoretic separation of proteins by size: Electrophoresis of proteins is also possible, although it is made somewhat more complex by differences in electric charge from one protein to another. For separations based on size alone, the proteins are heated in a solution of sodium dodecyl sulfate (SDS), a detergent that both denatures the proteins and coats them with a uniform negative charge. Under these conditions, their electrophoretic mobilities are directly proportional to their molecular weights in much the same manner as the mobilities of nucleic acid fragments reflect their sizes (figure 9.26)
Separations of proteins based on charge: An alternative approach to electrophoresis of proteins places the untreated proteins in a special gel that generates a pH gradient as electric current flows through it. The pore size of the gel is usually loose enough so that mobility is not affected by protein size. The proteins begin to migrate in the pH gradient based on their charges. However, as a basic protein enters a more acidic part of the gradient, or vice versa, the ionizations of their weak basic or acidic groups tend to be neutralized. At some point in the pH gradient, each protein reaches its isoelectric point (the pH at which it has no net charge) and thus stops moving. Because each type of protein tends to have a different isoelectric point, it will stop moving at a different point in the pH gradient. This process of isoelectric focusing generates a spatial distribution in the gel based solely on the isoelectric points of the proteins (figure 9.27). A two dimensional array can be achieved by first separating by isoelectric focusing, and then by SDS electrophoresis (figure 9.29). Although not credited in our textbook, this type of two dimensional electrophoretic separation was invented by a graduate student in MCDB, Patrick O'Farrell, who is now a member of the faculty of the University of California, San Francisco. The two-dimensional gels are often referred to as "O'Farrell gels".
Native protein electrophoresis: In many cases, it is desirable to locate an enzyme protein in an electrophoretic gel. For this purpose, the gel is usually buffered at a fixed pH, such that the separation of proteins is influenced both by net charge at that pH and by size of the protein. After electrophoresis has been completed, the gel can be tested for the presence of enzymatic activity. The most convenient assays are those that generate a color change when the enzymatic activity is present (figure 9.29).
Hybridization probes: Any DNA (or RNA) that can be prepared as a single uniform sequence can be used as a probe. Because complementary strands of DNA, RNA, or DNA plus RNA readily form stable double stranded helical structures when placed under suitable annealing conditions, labeled probes are widely used to detect the presence of complementary sequences. Radioactive labels, which are easy to incorporate into the nucleic acids and easy to detect, have traditionally been used. However, because of the hazards associated with handling radioactivity, alternative probes, often based on the use of fluorescent derivatives of nucleic acid bases or attached ligands that can be detected as staining reactions, are becoming increasingly popular. Cloned DNA is particularly useful as a probe, because it consists of multiple copies of a single sequence, and is generally carried in a vector that is sufficiently "foreign" so that it will not react with any of the DNA that is being analyzed.
Procedures for probing: The DNA that is to be probed is immobilized and denatured on a support, such as a nitrocellulose membrane and exposed to the probe under conditions that will promote hybridization, typically at a temperature about 25°C below the melting temperature for the expected double-stranded structure. Probe sequences that do not hybridize because there is no immobilized complementary strand for them are washed off. The sites that contain sequences capable of hybridization retain the probe and can be detected by autoradiography, fluorescence, or other techniques that are appropriate for the type of label on the probe. When combined with electrophoretic separation of DNA (or RNA) fragments by size, hybridization probes play major roles in many different molecular biology procedures, including Northern and Southern blotting (discussed below), DNA fingerprinting, etc. Probes are also widely used to identify bacterial colonies or viral plaques that contain specific cloned gene sequences (figure 9.15).
Southern blotting: The Southern blot (named for its inventor) uses gel electrophoresis together with hybridization probes to characterize DNA restriction fragments. 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 (or sometimes with two or more restriction endonucleases). 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 (figure 9.31).
Interpreting Southern blots: Matching the positions of the radioactive spots with those of the size standards identifies the sizes of the digestion fragments that hybridize with the probe. For example, a cDNA probe for a gene that contains two internal cut sites for the restriction enzyme will generate three fragments (which will usually have enough size difference so that all three can be detected). More complex patterns generated by repetitive sequences form the basis for DNA fingerprinting, which will be discussed in a future lecture. Note that it is not necessary for the entire length of the probe to hybridize with the entire length of the DNA fragment. A relatively short complementary sequence (less than 100 bp) is usually enough to obtain a strong hybridization signal. In addition, modification of the annealing conditions can alter the stringency of hybridization (the precision of base-pair matching needed for hybridization). By using reduced stringency, it is often possible to obtain hybridization between the coding sequences for the same protein from different species.
Alternative sources of multiple bands: If a probe hybridizes with only a single band, one can conclude that only one size class of fragments contains the probe sequence. However, if two or more bands hybridize, two very different interpretations are possible: 1) that there is a restriction endonuclease cut site within the sequence that hybridizes to the probe, causing the hybridizing sequence to be cleaved into two different restriction fragments that can both hybridize with parts of the probe; or 2) that more than one copy of the target sequence was present in the original DNA sample, with each copy emerging in a different sized restriction fragment. It is thus often desirable to use a relatively small probe to minimize the chance of a single target sequence being cleaved into two halves during digestion of the original sample. On the other hand, there are times when a much larger probe is more effective, for example to be certain that all of the genomic restriction fragments that contain any part of a protein coding sequence have been identified.
Dot blotting: In cases where the goal is simply to determine whether a particular gene has been cloned, size separation can be bypassed altogether and a bit of DNA from each putative clone can be transferred to a nitrocellulose membrane as a "dot", followed by hybridization to a probe and autoradiography. Only those dots that contain the desired sequence will hybridize and become radioactive. This procedure is similar to the colony and plaque hybridization techniques discussed in lecture 16.
Northern blotting: In a Northern blot (named because it is the opposite of a Southern blot), RNA molecules of varying lengths (often naturally occurring mRNAs) are separated by size and blotted onto nitrocellulose. A DNA probe (often a cDNA) is then used to identify bands that contain particular sequences. Northern blots are particularly useful for determining the conditions under which specific genes are being expressed, including which tissues in a complex organism express which of its genes at the mRNA level.
Western blotting: In a Western blot, proteins are separated by electrophoresis and blotted onto an appropriate support matrix. The matrix is then exposed to an antibody to the desired protein and all unbound antibody is washed off. The bands (or spots in a dot blot) where the antibody has bound are then detected by various means, such as binding of a second antibody that is radioactively labeled and specific for the first antibody.
Non-radioactive techniques: Although we do not have time to discuss them in detail, a variety of alternative techniques are beginning to replace the use of radioactive labels. These include fluorescent labels and a variety of reactions that produce colored end products.