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Custom Laboratory Polymerase Chain Reaction Technique essay paper sample

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Before commencing on the actual PCR process, it is eminent that one understands the initial processes applied in the production of the components and chemicals required in the technique. It is also crucial that the safety procedures and proper experiment conditions are ensured to come up with the required results, as the whole experiment is sensitive.

Some of the significant laboratory skills that one should be conversant with before carrying out the PCR experiment include:

  1. Establishment of uncontaminated culture systems
  2. Pupating small volumes of solutions of components
  3. Identification of molecular biology consumables
  4. Analysis of genomic DNA: The PCR process involves the manipulation of DNA molecules and thus acquiring of proper skills in the analysis of genomic DNA is not an option. The experiment involves the uses of a commercially available DNA safe rules and guidance on isolating genomic DNA from cells and tissues.

To isolate DNA from cells and tissues, first lyse the cells with an anionic detergent in the presence of a DNA stabilizer.  The stabilizer functions to limit the activity of the DNA enzymes contained on the body tissues. Treat the solution with RNA-digesting enzyme, to remove the contaminating RNA. The remaining contaminants, such as proteins, should be isolated by precipitating them with salt. The genomic DNA is then recovered by precipitation with alcohol. This is later dissolved in a solution containing DNA stabilizer. The extracted DNA requires is then quantified as PCR uses standard amounts.

  1. Quantification and evaluation of DNA

Two main approaches are used to determine the amount of nucleic acid in a preparation. The most simple and accurate process is the spectrophotometric measurement of the amount of ultra violet (UV) irradiation absorbed by nitrogenous bases. However, the process is suitable if the sample is devoid of any contaminants. If the quantity of RNA or DNA is small or if the sample harbors significant amounts of impurities, quantification can achieved through estimation of the intensity of fluorescence emitted by ethidium bromide. The readings denoting the amount of DNA in a sample should be take at wavelengths of 260nm and 280nm. The ratio between the readings at the specified wavelengths estimates the purity of the prepared nucleic acid.

  1. Preparation of DNA molecular weight marker by use of restriction enzymes: The ability to resize DNA into required fragments is an important aspect of molecular biology. Restriction enzymes target one palindrome and cuts at a restriction site with the selected palindrome. In the case, of repeated strands, the enzyme only cuts at the restriction sites. For instance, cut sites for the four Hind111 enzymes are shown in table 1 below.

TABLE 1: Illustration of enzyme cut sites in a palindrome sequence.

Restriction Enzyme 

Palindrome sequence 

Enzyme cut sites

Hind111 

 

AAGCTT

TTCGAA

A/AGCTT

 TTCGA/A

 

NB: The back slash represents the restriction site.

  1. Estimation of the size of the DNA fragments: this includes the Agarose Gel electrophoresis practice that includes the making and loading the gel.

This experiment is important because it allows visualization of DNA. The products of the DNA restriction enzyme digest or polymerase chain reaction are separated by gel electrophoresis for visualization. Gel electrophoresis works based on electric current that is used in the physical separation of mixtures in a matrix. The difference in the electric charges between nucleic acids and sugar phosphates provide the driving force that moves the molecules through the matrix. Having acquired the above skills, one can thus commence on the actual process.

The Polymerase Chain Reaction Process

The PCR process involves three main stages: Exponential amplification stage whereby at every cycle, the quantity is doubled. This reaction is very sensitive and only small amounts of DNA are required; leveling off stage whereby the reaction slows as the DNA polymerase ceases activity, lowering the amount of the reagents; and plateau phase whereby no more products  accumulates due to exhaustion of enzymes and reagents.

  1. Pipette the following chemicals and experimental components into two clean, sterile 200 micro liters PCR tubes that are placed in ice. Low temperatures curbs denaturation.                                    

TABLE 2: An illustration of the materials required in experiment.                                                                                                                                    

 

Tube 2

Tube 1

Reagent

µL

µL  

PCR Master Mix (contains 25mM MgCl2, reaction buffer, 2.5nmol/µL of each of 4 dNTPs dATP, dGTP, dCTP, dTTP), and polymerase (1U/µL)

12.5 

 

12.5

Forward primer (5pmol/µL) 

2.0 

 

2.0

Reverse primer (5pmol/µL)

2.0 

 

2.0

Nuclease-free water 

3.5 

 

3.5

DNA

5.0 

-

Nuclease-free water (in place of DNA) for negative control

 

-

5.0

Total

25.0 

25.0

 

  1. After dispensing the reagents, spin down the contents of the tube on ice and wait for the PCR to begin.
  2. Commence the PCR process by transferring the tubes into the thermal cycler at the same time. Amplify the target DNA, using the following conditions in all the cycles:

In the first cycle, denature at 95 OC for three minutes.

In the second cycle, denature at 95 OC for 30 seconds, anneal at 54 OC for 30 seconds, and extend at 72 OC for 45 minutes. Then, complete the cycle by synthesizing at 72 OC for approximately 5 minutes.  

  1. After completion of the process, add the appropriate amount of the loading dye to another tube and electrophorase the samples. Electrophoresis helps in visualization of the DNA products amplified in the reaction.

Gel Electrophoresis

The procedure is used to check whether PCR has generated the required DNA fragment. Gel is a buffer solution that ensures that the DNA will be negatively charged. It is made up of 32 wells, which are formed with combs inserted into the gel before hardening. The PCR product and the dye is pipette into these wells. Once the PCR products are put ion the well, the electric current of 100v is turned on for 40 to 60 minutes.

After undertaking the PCR process, visualization is carried out by use of a suitable stain after separation by use of gel electrophoresis. Ethidium bromide and Sybr Green are the most common dyes that are used to detect amplified products. However, other visible dyes may be used provided sufficient DNA is available. The size of the dye PCR products is determined by comparison with a molecular weight maker that contains fragments of known size.

The gel is then illuminated with an ultra violet lamp after electrophoresis. This is usually done by placing the gel in a light box and exposing it to ultraviolet light to limit the exposure to radiation. The Ethidium Bromide fluoresces in the presence the DNA.

In molecular biology, the polymerase chain reaction involves making g the multiple copies of a specific deoxyribonucleic acids (DNA) sequence. These copies are subjected to analysis processes such as cloning and sequencing to compare the types and numbers of PCR products amplified in standard conditions. Ideally, a PCR technique should be sensitive enough to locate and amplify a mono copy of DNA. The three main steps involved in the process are denaturation (heating), annealing (hybridization), and extending (synthesis). These three steps are repeated for 30 to 40 times in a cycle to amplify the double stranded DNA models located in the primers. They take place at a different temperature:

  1. Denaturation: the template DNA is heated at temperatures above 92 C (200F). This makes the double-stranded DNA to melt and open into two pieces of single stranded DNA. This forms the target DNA that is to be simplified.
  2. Annealing: this is the pairing up of the single stranded template with the oligonucleotide primers that occurs at relatively medium temperatures (around 54 oC). The polymerase attaches and starts copying the template after the double stranded DNA are formed by annealing.
  3. Synthesis: at a higher temperature (approximately 72 oC), the polymerase reaction progresses, and couples the DNA building blocks complementary to the template. This leads to the formation of a double stranded DNA molecule. Synthesis is only possible under the following conditions: Presence of the template DNA, primers, stable DNA polymerase molecule, the four deoxyribonucleotide triphosphate molecules (dNTPs), a buffer, magnesium chloride and water.

IMAGE 1: Summarized illustration of the PCR process.

Image adopted from educational science online science and nature store, 1998.

For gel electrophoresis method, the direction of migration, from the negative and positive charged electrodes, is due to the presence of negatively charged sugar phosphate. Double stranded DNA fragments behave as long rods, so their migration is determined by their size. Mono stranded DNA tend to form molecules with complex shapes as they fold up together and migrate through the gel in a complicated manner. The role of the formamide as used in the experiment is to denature these nucleic acids and make them behave as long strands.

Image adopted from educational science online science and nature store, (1998).

By looking at the DNA molecular standards, it shows migration though the gel is not linear. This indicates that smaller fragments of DNA travel much faster than larger fragments. The cut and uncut plasmids run differently in a gel. This is because the plasmid occurs in many coiled forms in the bacteria. When the super coiled forms are isolated, each form will migrate differently on the gel forming three major bands and other minor bands. If they are cut with restriction enzymes, they unwind and linearize; thus, becoming identical, and they run at the same rate; only one band is visible on the gel.

The molecular size of an unknown linear DNA is then estimated by the distance it travels to that of the standard molecular weights. However, this is not applicable in the super coiled forms. This implies that linearizing is required to find the molecular weight of a plasmid. In the example above, the molecular weight of the plasmid is approximately 3.0 kb.

The same data can also be analyzed graphically by use of Ferguson plots. This employs the separation of the DNA mixture and standardized sample to determine the charge and mass. The equation governing mobility as stipulated:

log (Rf)= log Yo - KRT
Rf – is the relative mobility, that is normalized by the dye front.
Yo – is the relative mobility of the protein in the absence of any sieving matrix.
KR – is the extent to which the gel matrix affects mobility.
T - % represents the monomer of the gel matrix.

The graph is plotted as log Rf against %T. Following the illustrated equation, the has a slope of KR and a Y-intercept (ar %T=0) of Yo. The comparison with the standards known size and charge yields the molecular weight and charge of the samples.

IMAGE 3: Ferguson plots showing the effect of the variation of charge and mass of DNA on mobility

   

Ferguson plot representing three proteins with different mass of the same charge.

Ferguson plot representing three proteins of different charge but same mass.

Images adopted from JA Negron’s PCR workshop, (2007)

In the quantification, and evaluation of DNA, An optical density (OD) of 1.0 corresponds to approximately 50µgm/L, for double-stranded DNA, 40%uF020µgm/L for RNA.  The ratio between  the  readings  at  260nm  and  280nm  provides  an  estimate  of  the  purity  of  the  nucleic  acid preparation. Pure  preparations  of  DNA  have  an  OD260/OD280 ratio  of  1.8,  pure  preparations  of  RNA  have  an OD260/OD 280 ratio  of  2.0.    If  there  is  significant  contamination  of  the  preparation  with  proteins  or phenols, the ratio will be significantly less than these values.  In general, higher ratio values indicate contamination with carbohydrates.  In these cases, accurate quantification of the nucleic acid will not be realized.

Yield and Kinetics of PCR Reactions

PCR Reactions produce products in a nonlinear pattern. Amplification follows a non-linear (exponential) curve until saturation is reached. Amplification normally generates a maximum of 1-5 micrograms of products. Saturation by one product cannot prevent the amplification of other unwanted products. Thus, over cycling decreases the quality of an otherwise standard reaction. Samples are normally taken after 5 to 10m cycles to verify the number of cycles needed.

An example of an exponential curve formed by PCR amplification is seen in figure:

Image 4: Exponential curve formed by PCR amplification

Adopted from promega web page (2003).

Application of the PCR in the Identification of Human Transposable Element

The ‘Alu’ can be detected by use of polymerase chain reaction technique. The technique allows one to amplify a targeted region of DNA in the genome. Molecular primers are used to target the sequence of interest. In the reaction, multiple copies of the target sequence are synthesized exponentially, so that downstream analysis and manipulation can be effective. In the experiment, PCR is use to amplify a nucleotide sequence from a human chromosome to look for the insertion of ‘Alu’ (short DNA sequence). The chromosome is extracted from the genome DNA contained in the hair follicles. Alu sequences are transposable elements that are DNA sequences of approximately 300bp in length, and interspersed within the human genome, at many different sites. They occur within the human Tissue Plasminogen Activator (TPA) gene. Oligonucleotide primers, flanking the insertion site, are used to amplify a 400-bp fragment, when the Alu sequence (TPA-25) is present and a 100-bp fragment, when it is absent. Each of the three possible genotypes--homozygous for the presence of TPA-25 (400-bp fragment only), homozygous for absence of TPA-25 (100-bp fragment only), and heterozygous (400-bp and 100-bp fragments), are distinguished following electrophoresis in Agarose gels.

To perform this analysis, you must first extract genomic DNA from your hair follicles, then use the PCR technique to target the flanking regions of the ‘Alu’ sequence within your genome, and finally, analyze your result using gel electrophoresis. Polymorphic ‘Alu’ insertions represent a significant source of genetic material required in the studies of human population genetics. Alu elements seem stable integrations into the genome that seldom delete from a location. Even when rare deletion occurs, the template of the original insertion event remains as the occurrence of exact excision has a low probability. ‘Alu’ insertion helps in dissecting the evolutionary history of individual populations.

The results from the experiment should help you to understand and value the importance of working in a clean, aseptic culture environment. They will reinforce the need to understand the source environment that the cell or tissue was taken from and its potential effect on the establishment of a successful culture and sample system.

The polymerase chain reaction is a scientific technique used in amplification of single or less copies of a piece of DNA to yield many copies of a certain DNA sequence. Kary Mullis invented PCR in 1983. In the PCR procedure, small amounts of DNA are rapidly copied to produce a reasonable quantity for investigation by use of simple but sensitive laboratory methods. In this way, it is possible to produce a DNA sequence. For the process to succeed, the original DNA that is being copied does not necessary need to be pure or abundant. Due to this, Mullis method has ushered the new age of genomics. Almost all areas of the current genetic research depend on this procedure.

The basic PCR procedure is simple. It is a chain reaction as the name stipulates that is, one DNA molecule produces two molecules and the doubling progresses. The doubling is accomplished polymerases, which are enzymes that strong together individual DNA building blocks into long molecular strands.  Polymerases require a supply of DNA building blocks known as nucleotides and the primer that attaches the building blocks to longer DNA molecule. PCR is also applicable in the manufacture of m-RNA, which are the working copies of genes. The RNA first undergoes reverse Transcription PCR to transcribe it into DNA. This process requires a small segment fragment of DNA that serves as a template to produce the primers that initiate the reaction. It is thus possible to clone the DNA whose sequence is unknown. PCR is thus used whenever the sequence of the DNA needs to be determined, for example, in gene research, genome-sequencing projects, investigation of genomic changes, and in the search for targets.

Quantitative PCR can be applied in the world of molecular diagnosis of diseases. To follow the progress of a disease, for example, AIDS, doctors need to know the concentration f pathogens present in the body. For instance, in viral diseases, the viral load in the blood stream provides an insight on the progress of a disease. Quantitative determinations also monitor the success of a treatment. PCR can also be applied in cancer therapy. The procedure is applied in the identification of that have are associated in the development of cancer.

The technique has proved exceptionally valuable in law due to its ability to identify and copy the tiniest amount of even damaged and old DNA. It is an indispensable adjunct to DNA fingerprinting as applied in forensic science. The blood found on a murder victim is enough to identify the likelihood of the suspect. Archaeologists to identify evolutionary relationships among dead creatures are also currently applying it. A relationship among extinct human groups and tracing human migrations has been made possible by this procedure.

The analysis of the PCR procedure is risky due to the use of the ultra violet radiation that is dangerous to both the human beings and the nucleic acids. The UV damage to the sample reduces the efficiency of the subsequent manipulation of the sample. In cases where a large amount of genes are required, the technique cannot act as a substitute for gene cloning in cells. The occasional errors with the process limit the number of relevant copies that can be made.

Electrophoresis in Genotyping

It applies the use of single stranded target that is thermal stable. In the heterozygote, the presence of second alternative sequence is normally verified by a second oligonucleotide that is complementary to the variant. At a certain percentage, the polyacrilamide migrates rapidly. The full profile of the oligonucleated dissociation occurs, and a single oligonucleotide is enough to confirm heterozygosity.

During the procedure, it is eminent that one remembers the following precautions:

  • Always keep the PCR reagents on ice.
  • The centrifuge tubes should be well covered to avoid contamination.
  • Use gloves o avoid contamination of the centrifuge tubes
  • Do not flame the centrifuge tubes when making the cell suspension as they may melt.
  • Make sure you are using the correct pipette as they come in different sizes.
  • Be careful not to contaminate the pipettes by slowly releasing the plunger
  • Clean the tip of the pipette even in the cou4rse of the experiment to prevent contamination.
  • Label everything you are using or else you will have to start again.
  • Dirty pipette tips should be autoclaved so that they are not disposed in the tin cans with red bags.
  • Balance the micro-centrifuge machine.

PCR is a sensitive process and can fail due to various reasons, in part because of the contamination that may lead to amplification of the contaminants instead of the DNA itself. Because of this, optimization procedures and techniques have bee developed. Contamination in the laboratory can be addressed by formulation of procedures and protocols that separate other mixtures from potential DNA products.  This involves spatial separation of PCR designated areas from areas of other experiments. The different stages of the process should also be carried in specified areas as the DNA to avoid contamination from pre-PCR mixtures. Pre-PCR contamination can also be curbed through the use of disposable plastic ware and thorough clean up of the surfaces in between the reaction set-ups. Primer design techniques are crucial in promoting the PCR product yield. Amplification of the problematic regions of the DNA is achieved through usage of alternate buffer components. Its specificity can be improved by use of reagents such as formamide. In electronic PCR, computer simulations may be performed to assist in primer design.

The practical bit of the above procedure helps in the clarification of a process that has a wide scope of applications in the modern world. This process is, especially important in the identification of human transposable elements. If proper precautions are followed when undertaking the technique in the laboratory, required result are accrued. Every step in the procedure is influential to the result; thus, should be performed scientifically. 

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