PolymeraseChain Reaction is a method in the field of molecular genetics. Thereaction involves the amplification of single deoxyribonucleic acid(DNA) into a big number of similar fragments. The method isapplicable medical and scientific processes like molecularidentification, genetic engineering, and DNA sequencing. Scientistsuse three stages in which they employ about thirty cycles in eachstage (Verkuil et al., 2008).
Ithas an imperative role in biochemistry, and other modified processesare crucial in the genetic identification and manipulation. Themodified techniques include DNA fingerprinting, genetical problemsdiagnosis, screening of the fetus during prenatal stage and groupingof organisms. Polymerase chain reaction was the idea of Karry Mullisin the 80s. The millions of similar DNA cells in over 30 cycles takeplace in an automated machine referred to as thermal recycler. Themachine regulates temperature in the various stages through its timedincrease or lowering depending on the stage of the reaction (Verkuilet al., 2008). There are various materials required for the PCRprocess to be effective. They include DNA templates that harbor thetargeted sequence, DNA polymerase, primers and deoxynucleotideTriphosphates.
Somepolymerases can give the desired results in the PCR process. Thefirst one comes from Pyrococcusfuriosusand scientists refer to it as Pfu polymerase. The second is Taqpolymerase and scientists extract them from Thermisaquaticus.These are the two commonly used polymerases. Unlike another DNApolymerase like Escherichia coli that cannot withstand hightemperature, these two can give results even in high temperatures,and it explains why scientists prefer them. Before the discovery ofthe tolerant temperature polymerase, scientists used the Escherichiacoli polymerase, and since it cannot withstand high temperature, theyhad to add more of it in every cycle. Its optimal temperature standsat 37 degrees Celsius. The introduction of Pfu and Taq simplified theprocess and made it more efficient. The specificity of theamplification led to the achievement of specific DNA targets (Cohn &Russel, 2012).
Theprocess occurs in various stages, and each stage requires a differentamount temperature to give the bet results. The temperature variousoccurs on the basis of the quantity of divalent ions, the type ofpolymerase in action and the type of divalent ions used. Some of thepolymerase used requires high temperatures that may go up to 98degrees Celsius for them to become active. Also, the primers used inthe process melt at different temperatures and, therefore, theprocess would not be effective with the application of a constanttemperature. The heat tolerance of primers depends on the bonding oftheir building units. That is guanine, cytosine, and thymine. Thecytosine-guanine bonds are very strong since they have three hydrogenbonds unlike adenine-thymine bonds have two hydrogen molecules. Theadenine-thymine bonds, therefore, require low temperatures for themto melt (Cohn & Russel, 2012).
Theprocess involves the following stages
Inthis stage, the individuals carrying out the process identify the DNApolymerase they are going to use. It involves the activation ofhot-start that requires high temperature.The hot-start PCR is an advanced method that makes the DNA polymeraseinactive at low temperatures. The approach helps in avoiding theundesired non-particular amplification of the targeted DNA sequence.It is effective when performed in a temperature range of 94 and 98degrees Celsius. The optimum time for the process is between one andnine minutes (Shafique, 2012).
Thepurpose of denaturing is to obtain a DNA that has a single strandthat becomes the genesis of amplification to give the desiredfragments. Denaturing takes place through the introduction of hightemperature ranging between 94 and 95 degrees Celsius. The optimumtime for the best results is about 30seconds. The purpose of the hightemperature in this stage is to break the bonds of the particularnucleotide bases that join two strands and therefore remaining withsingle DNA strands (Shafique, 2012).
Inthis step, temperatures reduce to become favorable for the primer.The low temperature facilitates the binding of y primer to thecorresponding target sequence of the DNA. However, the temperatureshould not go to low to ensure a specific annealing of the primer.The optimum temperature range for this stage is about 55 to 60degrees Celsius. The temperature is about 50 degrees below themelting point of the primer. When the primers bind to specificnucleotide bases, they form very strong hydrogen bonds. The optimumtime for this stage is about 20 seconds. After the binding, thepolymerase begins elongating the DNA sequence through the applicationof Deoxynucleotide Triphosphates (Shafique, 2012).
4.The elongation of the targeted DNA nucleotide sequence
Theelongation process entirely relies on the polymerase. The optimumapplicable at this stage depends on the polymerase used. Taqpolymerase is the widely used, and it functions optimally at 76 to 80degrees Celsius. For an elongation involving this polymerase, thetemperature falls between this range and the optimum happens to be atthe average level. That is 73 degrees Celsius. The elongationinvolves the addition of Deoxynucleoetide Triphosphates thatcorresponds well with the nucleotide bases in the DNA template. Orthe process to be complete, the condensation of the 5-phosphate grouptakes place together with the 3-hydro group found in the DNA templatenucleotide bases. The process leads to the formation of hydrogenbonds that joins the template and the elongated sequence strand (Cohn& Russel, 2012). The elongation depends on the targeted size ofthe DNA sequence and the efficiency of the involved enzymes. Undernormal circumstances, the elongation leads to the doubling of thetemplates, and this creates an exponential increase in the sequence.
Afterthe elongation, some of the strands may remain un-elongated. Thisprocess takes place to ensure that no single strand remains withoutextension. It happens after the polymerase chain reaction cycle. Theoptimum time for this reaction is about 10-12 minutes. It takes placeat around 73 degrees and 74 degree Celsius (Cohn & Russel, 2012).
Thisstage occurs as a temporary storage of the reaction. The duration ofthis process is not specific. The optimum temperature for thisreaction is between 5 degrees and 14 degrees Celsius (Cohn &Russel, 2012).
Occasionally,the PCR process may not be successful due to various factors. It maybe due to increased sensitivity of the reaction. The occurrence makesthe process delicate to impurities that may get introduced in thevarious stages. To increase the effectiveness of the process,scientists apply various methods.
1.Modification of the buffer concentration
Themajority of the buffers contain potassium chloride. The process ofbinding becomes effective with the introduction of the potassiumchloride. However, an increased concentration of the chemical tolevels more than 50mm affects the functioning of the polymerase. Theuse of buffers in optimal concentration increases the effectivenessof the process.
2.Regulation of the cycling requirement
Taqpolymerase requires activation through the use hot-start PCR. Tofacilitate this, a temperature of 94 degrees Celsius becomesnecessary. The temperature initiates the activity. The temperatureinvolved in the annealing stage is about 50 degrees Celsius. Thetemperature is the melting pint of primer and this avoids itsdestruction. An increase in temperature during the annealing stagereduces the yields, and a temperature increase reduces specificity.
3.The number of cycles
Theduplication of the DNA occurs exponentially. That is, there is doubleduplication for every DNA sequence. The number of cycles in everyprocess is between 20 and 30. Taq polymerase has a half-life of about30 minutes when subjected to 95 degrees Celsius. Every processemploys 94 degrees Celsius in a span of one minute. It translates toabout 30 minutes of exposure at 94 degree Celsius (Shafique, 2012).At the 30th cycle, Taq reduces by half, and it is thereforeimperative to maintain the number of cycles at 30.
Therehave been progressive efforts to improve the process of PCR to makeit effective. The giant leaps have enabled its use in variousprocedures such as the analysis of gene mutation. The modificationsinclude
Theform of modification determines the amount of target DNA or RNAsequence applied in the process (Cohn & Russel, 2012). Thescientists use a thermal recycler tat measures the quantity of theproducts resulting from the amplification in the duplication phase.
Thetechnique applies many primers that have different target DNAs. Itmakes it possible for the amplification of more than one target in asingle reaction. The genetic analysis takes place through thismodification to detect gene mutation and variations among individuals(Yu et al., 2012).
Thepurpose of this technique is to optimize the specificity during theannealing stage and the bidding of the primer. The process takesplace by the use of two pairs of primers in two stages. The firstpair results in the amplification of the DNA. The products of theprocess proceed to the subsequent step ad utilizes one or two primersthat base their binding sites in the initial set (Watson, 2012).
Theaim of this technique is to intensify the efficiency of thereplication process. In this technique, there is an elevatedtemperature of about 30 degrees Celsius. The temperature reduces inthe final stages. In the annealing stage, it leads to an increasedspecificity of the primers.
Inthe occurrence of a crime, the culprits always leave behind traces ofobtainable DNA that manifests as evidence. Forensic scientists employthe technique to amplify the small fragments into thousands, and theycan therefore, link suspects to particular crimes (Watson, 2012).
Thetechnique has been very instrumental in the diagnosis of infectiousdiseases. It clearly identifies viruses, and mycobacteria through theapplication of tissue culture. It makes it possible to distinguishbetween pathogenic and non-pathogenic bacteria. The PCR techniquesalso identify malignant cells, and this makes it possible to diagnosecancer at an early stage. It is also a primary process in theidentification of Alzheimer disease by identifying the 84 genesresponsible for the progression disease.
3.Analysis of drug metabolism enzyme carriers
Theprocess is crucial during the metabolism of hormones, drugs,nutrients and toxins (Bustin & Nolan, 2013). When there is analteration of the genes involved in this process, some complicationsmay result. The application PCR helps to identify the genes withabnormalities (Watson, 2012).
4.DNA cloning and hybrid formation
Thewhole process requires a lot of DNA nucleotides. The amplificationprocess gives millions of fragments that facilitate cloning andhybridization.
Thefuture of PCR
The has improved over time since its innovationby Karry Mullis. It role in genetic screening and molecular biologyhas been of importance in the scientific process. The intensifyingtechnology involved in the process will reduce the cost of carryingit out, and this will attract a lot of investors to scale up its useand diversify its uses. The conventional methods of the process willbecome outdated with time, and the technology has to move with thewave (Verma et al., 2012)
Conclusively,PCR techniques have been primary inputs in the field of molecularbiology. Its application has brought to light various aspects ofmolecular genetics, and this has led in the solving of differentmedical issues. The improving technology is a promise to productivefuture for the PCR, and there are prospects of increased efficiencyand results. Scientists are still doing procedures and test toimprove the quality and specificity of the results, and this will bea primary input in the field of forensic science and criminology.
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