Essay: The Polymerase Chain Reaction

History
A revolutionary method in amplifying many copies of a specific small sequence of DNA from a longer DNA molecule has greatly influenced the molecular biology world. By 1980, many factors that were needed for the polymerase chain reaction were already discovered, but it was not until in 1983 that Kary B. Mullis perceived the idea of this breakthrough technique. With knowledge about primers annealing to a complementary sequence of DNA, Mullis’ initial experiments of PCR involved him having to self-adjust the temperatures during the processes of DNA denaturation, annealing, and elongation, whilst also adding in new DNA polymerases for every cycle. This proved to be very tedious and time consuming, until the purification of Taq polymerase in 1985. This heat stable DNA polymerase was able to sustain the high temperatures of denaturation, and could efficiently elongate DNA strands through numerous cycles. Then, in 1987, the first PCR thermal cycler machine allowed for the regulation of temperature and timing, which significantly reduced the costs and hours of manually adjusting the different processes of PCR that Mullis originally had endured.
Equipment
In order to perform PCR, several ingredients need to be included, which are: a template DNA strand, forward and reverse primers, dNTPs, a thermostable DNA polymerase enzyme, a reaction buffer, MgCl2, water, and a thermal cycler. The template DNA strand contains the target DNA sequence that is going to be amplified. The forward and reverse primers are synthetic DNA molecules that have sequences that will anneal complementary to the template DNA strand, binding to the two ends that surround the DNA sequence of interest. For PCR, only a specific region is going to be amplified, which will be from the 5′ end of the forward primer, to the 5′ end of the reverse primer, thus it is vital to have well designed primers. When designing primers, a few factors come into play, in order for both forward and reverse primers to function effectively during PCR. They must be complementary to the template DNA, their length are designed to be between 20 and 30 nucleotides long, which allows them to be sufficiently specific to the region of DNA. In addition, both primers should have similar melting point temperatures, which can be calculated based on their length and number of A and T nucleotides relative to the number of C and G nucleotides in the sequence. A thermostable DNA polymerase enzyme, such as Taq polymerase, will function at high temperatures, and not degrade during DNA denaturation; it will read the DNA template and assemble the dNTPs, which are the nucleotides used to make up the new DNA. A reaction buffer, such as TBE buffer, will allow for maintaining an ideal pH balance, while MgCl2 will reduce charge repulsion between the primer and the template strand and is a required cofactor for the Taq polymerase; adding too little cation may result in difficult annealing and reduce the amount of product, while adding too much cation may reduce specificity of annealing and produce non-specific DNA products. Lastly, a thermal cycler is an instrument that is used in order to control the temperature and time changes to perform PCR efficiently.
Experimental Protocol
Once the necessary ingredients are added in as the master mix with the template DNA strand into a tube, the sample is loaded into the Thermal cycler machine. The three vital steps in order to carry out PCR are: denaturation, annealing, and extension, which are then repeated continuously. In the first step, denaturation, the thermal cycler raises the temperature to around 95?? C, which is sufficiently high enough in order to break the hydrogen bonds that are present in between the two strands of the double-stranded template DNA. Now with the single-stranded template DNA, annealing of the primers can occur. The temperature and length time during primer annealing will vary, as it depends upon the base composition, length, and concentration of the primers. If the annealing temperature is set higher than the melting temperature of the primers, they would not be able to anneal to the target DNA, and if the annealing temperature is too low, there will be mismatched hybrid and not all of the correct base pairing will be formed. Thus, an ideal annealing temperature will usually be 5?? C below the melting temperature of the primers. Once the temperature is lowered, it allows for new hydrogen bonds to form; the forward and reverse primers that are in the mix bind to their complementary sequences on the single-stranded template DNA. With the primers flanking the DNA sequence wanting to be amplified, extension of new DNA can now be performed. The temperature is raised to a favorable temperature in order for the DNA polymerase to begin adding in dNTPs; this is around 72??C to 74?? C for Taq polymerase. The DNA polymerase will read the template DNA strand and add in complementary dNTPs quickly, resulting in the DNA synthesis of two new double-stranded DNA, each composed of one of the original template DNA strand and the newly assembled complementary strand. This process of denaturation, annealing, and extension is repeated, and the amount of DNA after one cycle will be doubled each generation. Each cycle takes relatively little time, which allows for the production of millions of copies of a specific DNA sequence.
Examples
PCR is used in a variety of ways in daily life. In the journal article, ‘Bite Injuries of Grey Seals (Halichoerus grypus) on Harbour Porpoises (Phocoena phocoena)’, there were speculations as to grey seals being the culprit for the bite-like skin abrasions that were being found on harbour porpoises, found along the Belgian and northern France coastline. Initially, visual observations for the predation of the harbour porpoises by grey seals were reported, but these visual observations were seen from a far distance and were not efficient enough to pinpoint the bite marks to grey seals. Thus, genetic evidence was sought out and gathered in order to obtain a definitive answer. From the study, the skin lesions from five harbour porpoises were swabbed. In addition, as a positive control, the head of a dead grey seal was used to imitate a bite-like injury on a corpse of another harbour porpoise, and DNA samples were swabbed from these two animals. Once the DNA was extracted from the seven mammals, primers were designed to flank and amplify a region of the mitochondrial 16S DNA sequence, which differ amongst the grey seal and harbour porpoises. PCR reactions were conducted to obtain many copies of the DNA; electrophoresis, sequencing, and the use of GenBank Blast was used in order to compare the sequences of the amplified DNA fragments. On the PCR gel electrophoresis, it was revealed that the grey seal mitochondrial 16s DNA was present in the swabs from the grey seal and the imitated bite mark on the porpoise carcass, which was used as the positive control. For the five harbour porpoises with the questioning skin lesions that were swabbed, two of them showed a positive result in having the grey seal mitochondrial DNA. For the three porpoises that were negative for grey seal mitochondrial DNA, it was hypothesized that the grey seal DNA may have been washed out of the swabbed region by seawater.
Aside from using PCR for animal DNA, this application is widely seen amongst the food industry. With the increasing rise of having informed choices for food consumption, individuals in Saudi Arabia have expressed their voices in wanting to know whether or not the foods they purchase from their markets have been genetically modified. Described in ‘Monitoring of genetically modified food in Saudi Arabia’, the production of genetically modified crops have been increasing in Saudi Arabia, in order to maintain their growing demand of produce. Many countries regulate the labeling of foods, in order to inform whether or not the product has been genetically modified. In order to do this, PCR is used to obtain information about the presence or absence of a genetically modified food. Today, a vast majority of genetically modified plants are transformed with the Cauliflower mosaic virus 35S promoter and the Agrobacterium tumefaciens nos terminator, and by utilizing this known information, PCR is used in order to screen for the presence of the 35S promoter and the nos-terminator sequence within foods. In the study, 202 samples of foods from Saudi Arabian markets had their DNA extracted, and PCR was used in order amplify these specific DNA regions, using primers to flank the 35S promoter and a different set of primers to surround the nos-terminator sequence. From the 202 samples tested, 20 samples were positive in producing PCR products, resulting in these 20 samples as having some form of genetic modification: 16 of the samples were meats with genetically modified soybeans, two corn products had the 35S promoter, one corn product had both the 35S promoter and the nos terminator sequence, and one potato sample was positive for both the 35S promoter and the nos terminator sequence.
Future Directions
As of now, PCR works well in amplifying small fragments of DNA; amplification efficiency greatly decreases if the region wanting to be amplified is significantly large. Improvements are being made by adjusting the variables in PCR, such as the chemical reagents used and the pH of the buffers, in order to have successful copying of larger pieces of DNA. As technology advances, enhancements of thermal cyclers are posing for increasing the speed of PCR in order for DNA generation to be done in a much quicker fashion; this is highly anticipated because although this technique can generate large amounts of DNA, it is still too slow to be used in clinical settings. In addition, smaller devices that can regulate the temperatures and timing of a PCR reaction are in the developmental stages, which would replace the need for a large thermal cycler; this would be efficient in providing quick and more mobile results of DNA, while simplifying the procedure and reducing costs.
Northern Blotting
History
The origin of northern blotting was built upon the discovery of southern blotting. In 1975, Edward Southern popularized the technique of separating DNA fragments by using gel electrophoresis, transferring, or blotting, the DNA fragments onto a solid membrane, and then incubating the membrane with a radioactively labeled probe that was specific for the DNA fragments of interest. This process then allowed for the visualization of the gene of interest with the usage of an instrument that could detect the labeled probe. Not too long afterwards, in 1977, George Stark, Ames Alwine, and David Kemp built upon Southern’s technique. However, rather than analyzing DNA, they worked with RNA, and named the technique Northern blotting. By studying RNA, rather than DNA, northern blotting allows for the measurement of a messenger RNA, which is useful in determining which cells express a particular gene, or determining the factors that regulate the expression of a certain gene.
Equipment
For preparation of the Formaldehyde Agarose gel, the solutions needed are: agarose, 10X MOPS-EDTA-Sodium Acetate buffer, formaldehyde, and an electrophoresis chamber with its casting trays and combs.
For preparation of the RNA sample: RNA sample buffer, isolated RNA sample from cells by RNA preparation methods which include: H2O, EtOH, RNAzol, chloroform, isopropanol, 5 ml Falcon tubes, 8ml Starstedt tubes, centrifuge machine
For gel transfer: UV trans illuminator for visualization of the gel, denaturing solution (NaCl, NaOH), water, neutralizing solution (NaCl, TRIS HCl), 20X SSPE (EDTA, NaCl, phosphate buffer), Whatman filter paper, solid membrane, 10X SSPE buffer, paper towels
For prehybridization: prehybridization solution (100X Denhardt’s solution, 10% SDS, 20X SSPE, water), salmon sperm DNA, UV crosslinker
For preparation of the probe: 1X probe mix (oligonucleotide probe, probe buffer, water, 32P-ATP, T4 phosphonucleotide kinase), hybridization solution (10% SDS, water, 20X SSPE), wash solution (10% SSDS, 20X SSPE, water)
For detection of the probe: autoradiograph using x-ray film
Experimental Protocol
Northern blotting begins with isolating and purifying the RNA from the cell of interest. First, the cells are disrupted by the addition of guanidium thiocyanate and subjected to vortexing. Once the cells have been lysed, the RNA sample is separated from the solution by the addition of phenol and chloroform-isoamyl alcohol. With the RNA present in the aqueous solution, it is separated and the addition of isopropanol, followed by centrifugation, precipitates the total RNA. Then, to remove any impurities that still may be present, the precipitate is subjected to ethanol, giving the final RNA product.
The purified RNA sample contains a vast variety of types of RNA molecules, so separation is done in order to determine the size and how much RNA is present. Thus, the RNA samples are subjected to gel electrophoresis, which allows the RNA sample to separate into fragments relative to their sequence size through the usage of the electric field; small RNA fragments will run fast and further along the agarose gel, while large RNA fragments will run slow and not as far on the agarose gel.
Once the RNA molecules are separated, they are transferred to a solid membrane, which can be done electrically or by capillary action.
Capillary transferring of the RNA involves transfer of the RNA molecule onto a filter paper through the usage of capillary force. First, the agarose gel is washed with the denaturing solution, water, the neutralizing solution, and 10X SSPE. The nylon membrane is soaked in water, while the Whatman paper is soaked in 10X SSPE buffer. The gel is placed faced down on top of the soaked Whatman paper. Then, the nylon membrane is placed on top of the gel, subsequently with three pieces of Whatman paper and a stack of paper towels. The buffer solution will soak and pass through the gel and filter paper, thus transferring the RNA molecules onto the solid membrane. If the gel were subjected to electrical transferring, it involves the transfer of the RNA molecules to a solid membrane by the usage of an electric current. Here, the solid membrane, usually a filter paper, will be sobbed with the transfer buffer. The agarose gel is then placed in between the filter paper and a wet sponge, and inserted into a blotting chamber. Within the chamber an electric current allows for the transfer of the RNA molecules onto the filter paper.
After blotting, RNA is immobilized on the membrane, by exposure of ultraviolet light, in order to covalently link the RNA to the membrane so that is prevents the RNA from being washed away after hybridization.
Now with the RNA molecule on a solid membrane, hybridization is done in order to identify the RNA of interest. This is done by using a detection probe, which is the labeled nucleotide sequence that will complementary base pair to the RNA of interest. When labeling the complementary probe, it can be done radioactively or chemically, which will aid in the process of visually being able to quantify the RNA of interest. Thus, with the labeled detection probe and the blotted membrane, hybridizing is done by incubating the membrane in a solution that contains the probe.
During the incubation process, the probes will complementary base pair to the appropriate RNA sequence; any extra detection probes that did not hybridize are then washed off, in order to yield a clean film without unnecessary high background radioactivity. The hybridized membrane is then analyzed by using a detector that will detect the labeled probe, such as an autoradiography. The results observed on the autoradiograph will present bands that the probe has hybridized to on the RNA of the filter paper, representing where the gene is being expressed.
Examples
Northern blotting can be utilized in a multitude of ways in order to visualize a certain gene’s expression. In ‘Type 2C protein phosphatase ABI1 is a negative regulator of strawberry fruit ripening’, researchers sought out for the FaABI1 gene found in strawberries to determine whether it had a role in fruit ripening. Seven different samples of strawberries were obtained during their developmental stages: small green, large green, degreening, white, initial red, partial red, and full red. To determine the expression of the FaABI1 gene, northern blotting was administered. The RNA from these different variants were isolated and purified, separated by gel electrophoresis, transferred onto nylon membranes, and hybridized using a FaABI1 probe, washed, and analyzed. The results from real-time PCR and northern blotting for the seven stages showed high expression levels of FaABI1 at the beginning of the strawberry developing stages, with decline as it ripened; this potentially meant that this gene was negatively regulating the ripening of strawberries. Another experiment was done in order to see the effects of silencing the FaABI1 gene in strawberries. Comparison between a control strawberry and a strawberry with the FaABI1 gene silenced, showed that silencing the FaABI1 gene promoted ripening; real-time PCR and northern blotting was administered to ensure that the FaABI1 gene was down regulated in the RNAi strawberry, in comparison to the control strawberry. To further confirm the negative regulation of the FaABI1 gene in the strawberry ripening, overexpression of the gene was done in a separate experiment. Here, it showed that the strawberries who had upregulated expression of the gene remained white, while the control strawberry fully turned red, concluding that overexpression of the FaABI1 gene would lead to inhibition of development.
In ‘Identification of UV-B-induced microRNAs in wheat’, the concern of ultraviolet-B radiation, which have wavelengths between 280 to 320 nm, as having a negative effect on plants is addressed. As plants are constantly adapting to their changing environment, they do this by adjusting the expression of their various genes. Specifically, microRNAs have been studied, as being an important factor in regulating post-transcription of plant genes during their adaptation to the environment because microRNAs can change their levels of expression in response to stress. In particular, wheat was studied in order to see the effects that the UV-B radiation had on its miRNAs. Northern blotting was done in order to see the expression levels of six miRNAs after the wheat samples were treated with the UV-B radiation at six different time points. Total RNA from the wheat leaf cells underwent gel electrophoresis, blotted onto a solid membrane, radioactively labeled using probes with [??-32P]-ATP, washed, and analyzed by using a Typhoon scanner. By analyzing the northern blot of the six different miRNAs at the six progressive times of 0 hours, 1 hour, 2 hours, 6 hours, 12 hours, and 24 hours of exposure of UV-B radiation, it was apparent that three miRNAs (miR164, miR39, and miR16) were down regulated, while three miRNAs (miR159, miR167, and miR171) were upregulated. These results demonstrated that the varying expressions of the miRNA genes may be an adaptation that wheat portrays, due to the constantly changing environment. Further research showed that in particular, miR159, is responsive for dehydration and hormone signaling for wheat development, in order to aid it from damage from the ultraviolet-B radiation and for adaptations to the physical activities of the wheat plants after long exposure to UV-B radiation. With the varying up regulation and down regulation of the miRNAs in the study, it demonstrates that miRNAs respond and interact with each other when the plant undergoes stressful circumstances, in order to readjust to the diversifying environment.
Future Directions
The vast advancements in technology has proved to be a vital key in being able to conduct molecular biology techniques in a more efficient manner. Second generation sequencing technologies has provided some advantages of studying gene expression of RNA, in comparison to Northern blotting. In particular, RNA-seq is a methodology for profiling RNA through the usage of next-generation sequencing. This allows for high resolution exploration of all the RNAs in a given sample, such that RNA-seq can identify the RNA sequence and quantify the relative amounts, simultaneously. Northern blotting relies on knowledge about the genome in order to design the probe and allow for its hybridization onto RNA, while RNA-seq uses matching of data to genes by sequencing alignments. The advantages of this is that the entire transcription product can be studied, rather than studying only the specific transcript region where the probe hybridized to. RNA-seq measures gene expression through analyzing the amount of data that matches the sequence, giving a dynamic range of what can be studied, and thus providing higher levels of reproducibility. Also, this method is more accurate, allowing for higher precision for sequencing. With many innovative ideas and access to larger resources, the field of molecular biology techniques is continually improving, which entails for exciting new possibilities and profound discoveries.