Optimizing of the Conditions for the PCR Detection of mTOR, in DNA Derived from Human Cell Lines - Research Paper Example

Optimizing of the conditions for the PCR detection of mTOR, in DNA derived from human cell lines, which are associated with aging and senescence. By Presented to Due Date ABSTRACT PCR was developed by Kary Mullis,(Rainbow,1996) in 1983 and is now common and indispensable technique utilized in biological and medical research labs in a wide array of applications.(Saiki et al.,1985; Saiki et al., 1988) These applications include DNA-based phylogeny, DNA cloning used for sequencing, or genes functional analysis; hereditary diseases diagnosis; genetic fingerprints identification in paternity testing and forensic sciences; and detecting and diagnosing infectious diseases. In order for a successful PC procedure to be carried out, the optimal conditions need to be determined. In this paper, the optimizing of the conditions for the PCR detection of mTOR, in DNA derived from human cell lines, which are associated with aging and senescence was done. The results show that the optimal annealing temperature is 62.2 degrees, the optimal Mg Cl2 concentration required is 0.75ul and the optimal primer concentration is 0.4ul. Corresponding electrophoresed images clearly show that the variable and their values are precise for the PCR detection of mTOR Table of Contents ABSTRACT 2 1.0Introduction 4 1.1Protein Description 5 1.2Functions 6 1.3Significance of Study 8 2.0Methodology 9 2.1Brief Methodology 9 2.2Results 11 3.0Discussion 16 4.0Conclusion 21 1.0 Introduction The mechanistic target of rapamycin is also referred to as the mammalian target of rapamycin (mTOR) or the FK506-binding protein 12-rapamycin-associated protein 1. This protein is encoded in the human body by the MTOR gene.(Brown et al.,1994; Moore et al.,1996) mTOR gene is a protein kinase (serine/threonine) responsible for regulating cell growth, proliferation, motility, survival, as well as transcription and protein synthesis.(Hay et al., 2004) MTOR belongs to the protein family related to the phosphatidylinositol 3-kinase. The chromosomal location of mTOR is at 1p36.22 with the location base pair starting at 11166588 and ending at 11322608 bp from other as shown below where: EXOSC10 refers to exosome component 10, UBIAD1 refers to the domain UbiA prenyltransferase containing 1, ANGPTL7 is what encodes angiopoietin-like 7, PTCHD2 refers to the patched domain that contains 2, LOC100128221 is the same as hCG2041787, and SRM is what encodes spermidine synthase. The transcription length of mTOR is 8680bp. The gene is found on the minus strand and encompasses about 156 kb and has 58 exons. The reported NCB136 coordinates of human chromosomae1 location is between 11,089,179 and 11,245,151 bases and the ensemble49 coordinates are 11,089,180-11,245,176 bases. 1.1 Protein Description The FRAP amino terminus (mTOR) includes a number of repeat tandem HEAT ( i.e Huntingtin, EF2, A subunit of PP2A, TOR1) implicated in interactions of protein-protein (Bhaskar and Hay, 2007; Hay and Sonenberg, 2004; ). Every HEAT repeat includes two alpha helices that are close to 40 amino acids. The half that is a carboxy-terminal contains 2 FAT domains (i.e., FRAP, ATM, TRAP). The catalytic domain Upstream is the FRB domain (i.e., FKBP12-rapamycin binding). The sequence of the catalytic domain is similar to that of the phosphatidylinositol kinase (PIK) catalytic domain that is homologous to other protein kinase families known as PIKK (PIK-related kinase). mTOR has a domain that is putative negative regulator (NR) between FATC and the catalytic domain. The FATC domain (FRAP, ATM, TRAP C-terminal) is significant for kinase activity. The domains FAT and FATC interact such that they expose the catalytic domain. The protein is constituted by 2549 amino acids and a 288,891 da molecular weight. In humans, the inhibitor rapamycin, the ternary complex FKBP12 ( FK506-binding protein ), and FRB domain (the FKBP12-rapamycin-binding) of human FRAP crystallizes at a 2.7 angstroms resolution (Choi et al., 1996), and refines at 2.2 angstroms (Liang et al., 1999). 1.2 Functions The protein kinase Serine/threonine is a central regulator for growth factors, cellular metabolism, nutrients, hormone response growth and survival, stress signals, and energy. mTOR regulates the direct or indirect phosphorylation of approximately 800 proteins. It operates as part of two functionally and structurally separate signaling complexes namely: mTOR complex 1 and 2 (mTORC1 and mTORC2). The activated mTORC1 regulates protein synthesis upwards by phosphorylating m RNA translation and synthesis of ribosomes key regulators. This is inclusive of EIF4EBP1 phosphorylation of and its inhibition release toward the factor 4E elongation initiation (eiF4E). In addition it activates and phosphorylates RPS6KB1 and also RPS6KB2 which promote synthesis of proteins by activity modulation of their targets downstream including S6 ribosomal protein, translation of eukaryotic initiation factor EIF4B, as well as inhibits PDCD4 translation. It stimulates the biosynthesis pathway of pyrimidine through the acute regulation via phosphorylation that is RPS6KB1-mediated of the CAD biosynthetic enzyme, as well as delayed regulation, through pentose phosphate pathway transcriptional enhancement producing CAD allosteric activator: 5-phosphoribosyl-1-pyrophosphate (PRPP), at a later synthesis step, and is a mTORC1 complex dependent function. The mTOR signaling pathway Further, mTOR regulates the synthesis of the ribosome by RNA polymerase III -dependent transcription activation through the RNA polymerase III-repressor MAF1 phosphorylation and inhibition. It also regulates the synthesis of lipids through LPIN1 and SREBF1/SREBP1. In order for Mtorc1 to maintain energy homeostasis, it regulates biogenesis of mitochondria through PPARGC1A regulation. mTORC1 is also responsible for negative autophagy through ULK1phosphorylation. ULK1 phosphorylates at Ser-758, disrupt the AMPK interaction and prevent ULK1 activation. It also prevents autophagy by autophagy inhibitor DAP phosphorylation. A feedback control is exerted by mTOR on the signaling of upstream growth factor that includes GRB10 phosphorylation and activation, a signaling INSR-dependent suppressor. Other potential spots mTORC1 may include CLIP1 phosphorylation and microtubules regulation. MTOR may also be involved in the regulation of other cellular functions such as organization and survival of the cytoskeleton. It plays a vital role in AKT1 phosphorylation at Ser-473, a phosphoinositide 3-kinase pro-survival effector and facilitates its PDK1 activation. mTORC2 may also facilitate in cytoskeleton regulation, through PXN. PRKCA phosphorylation and Rho-type guanine nucleotide activation RAC1B, RAC1A and RHOA exchange factors. mTORC2 also plays a role in the phosphorylation regulation of SGK1 at Ser-422 1.3 Significance of Study mTOR signaling modification modulates longevity in laboratory models of fruit flies, nematodes, yeast, and also in mammals (Vellai et al., 2003; Kaphali et al., 2004). The significance of this pathway in aging and longevity determination in these systems is in the demonstration observed when the mTORC1 pathway is targeting immunosuppressant. Rapamycin is the single pharmacological intervention that is shown to increase rodents’ lifespan (Cao, 2001; Harrison et al., 2009). The calorific restriction that is known in the regulation of mTOR signaling is also linked to animal model increased lifespan (Masoro, 2005; Colman, 2009). Accordingly, a number of cellular processes that are influenced by mTOR pathway are linked to age advancement in animals and humans, changes that are age related such as autophagy, protein synthesis, vascular plasticity, lipid metabolism and inflammation are characterized well (Kennedy et al, 2009; Kolovou et al, 2011; AbouRjaili et al, 2010; Chung et al, 2011) The mTOR pathway is also being increasingly implicated in several diseases that are age related such as type 2 diabetes, cancer, obesity and neurodegeneration (Laplante et al., 2012) Microarray analysis utilizing a bespoke mTOR-related genes transcript set demonstrated that individuals that were older from study population showed changes in expression that were consistent with the overall mTORC1 and mTORC2 components down-regulation in the mTOR pathway, results that were consistent with animal model findings (Harries, 2012). The data from the study reinforced the mTOR signaling potential importance in aging. The study demonstrated the relevance of the further study of the mTOR pathway status amongst the human population. In order to detect the mTOR genes, it is critical that the condition for PCR detection is at an optimum. This study seeks to determine the optimum conditions for the PCR detection of specific genes, in DNA derived from human cell lines, which are associated with aging and senescence. 2.0 Methodology A number of experts in the field of biology of ageing hold the belief that pharmacological interventions that can slow ageing are an issue of ‘when’ as opposed to ‘if’. A forefront target for such pharmacological interventions is the response pathway that is defined by the rapamycin mechanistic target. This pathway inhibition extends model organism lifespan as well as confers protection from an increasing list of pathologies that are age-related. Characterized pathway inhibitors are already approved clinically while others are still under development. It is, therefore, critical to understanding the optimum conditions for the PCR detection of specific genes, in DNA, derived from human cell lines, which are associated with aging and senescence. 2.1 Brief Methodology DNA extraction was done from six cell lines and a super mix obtained by adding 2.5ul 10x ionic buffer, 0.75ul of MgCl2, 0.5ul of dNTPs, 0.5ul of forward primer (GAPDH), 0.5ul of reverse primer (GAPDH), 0.1ul of Taq and 18.15ul of molecular water and finally adding 2ul of DNA template (THP1) to have a total of 25ul in each 0.2ml PCR tube. After preparing the samples, the samples were placed in the thermal cycler (C1000 Thermal cycler-BioRad, Oxford, UK) to amplify the DNA fragment. For experiments 2, 3, 4 and 5 the same method was used but the recipe of the supermix was altered in experiment 4 and 5 to obtain the optimal condition for PCR to detect mTOR gene using HACAT cell line. 9 8 7 6 5 4 3 2 1 samples 10 9 8 7 6 5 4 3 2 Well 2.25 ul(4.50Mm) 2.00ul (4.5Mm) 1.75ul (3.5Mm) 1.50ul (3.0mM) 1.25ul (2.5mM) 1.00ul (2.0mM) 0.75ul (1.5Mm) 0.50ul (1.5Mm) 0.25ul (0.5Mm) MgCl2 The following table shows the amount of primers added to have 25ul in total in each sample tube to compensate amount with molecular water. 8 7 6 5 4 3 2 1 samples 9 8 7 6 5 4 3 2 well 0.70ul 65ul 60ul 55ul 0.50ul 0.45 0.40ul 0.35ul forward 0.70ul 65ul 60ul 55ul 0.50ul 0.45 0.40ul 0.35ul reverse To run the gel electrophoresis, the gel was made by mixing 0.6g of 2% agarose and 30ml of TBE buffer in the flask. The initial experiment was undertaken to ensure that it was possible to conduct a PCR experiment using the university laboratory facilities before starting to optimize the condition of mTOR in HACAT. The primer that was used was GAPDH, and the cell line was THP1. 2.2 Results An initial experiment was undertaken to confirm the possibility of conducting a PCR experiment using the university laboratory facilities prior to optimizing the condition of mTOR in HACAT. The primer that was used was GAPDH and the cell line wass THP1. The following photo was obtained The picture confirms that indeed a PCR process can be undertaken in the university laboratory facilities. In addition, the DNA concentration is high with samples 1, 2, 4, 5, 6, 7 and 8 showing multiple bands but sample 3 shows one clear band. The single band on lane three is indicative of the purity of the sample used and also shows that the annealing temperature used was sufficient. The multiple bands observed in other lanes could indicate a number of reasons such as the annealing temperature was too low or impurity of the DNA samples used. The second procedure was also a control to ensure that the university facilities were able to work with a mTOR primer under general conditions. The following picture was obtained. From the picture it is seen that the It can be clearly seen from the picture that the mTOR primer worked under general condition. It is also observed that clear bands are present in temperatures that are 64oC. This acted as a guide in knowing the temperature range for mTOR annealing in the experiment that followed. The third experiment was done to get the optimum anealing temperature for PCR detection of mTOR gene in HACAT. The temperature gradient was between 58oC - 63oC. The following picture was obtained. It can be clearly seen from the picture that the optimal annealing temperature for PCR detection of mTOR gene in HACAT is 62.2oC. A clear band is observed in the third lane from the left which corresponds to this temperature. The reaction temperature in a PCR process is normally lowered to temperatures of 50–65 °C for a period of 20–40 seconds in order to allow the primers to anneal to a single-stranded DNA template. The temperature is made low enough to allow the primers to hybridize to the primer strand. The temperatures are also made high enough to ensure that the hybridization is very specific where the primers bind perfectly to the template complementary part. Too low temperatures could result in imperfect binding while too high temperature could cause no binding at all. The optimal temperature is therefore critical for the perfect annealing of primers. The fourth experiment was undertaken to get the optimum MgCl2 concentration for PCR detection of mTOR gene in HACAT. The following results were obtained The optimal value of MgCl2 as seen from the picture is 0.75ul (1.5mM) which is the same amount as was used in the general condition. The role of the Magnesium is that of a cofactor to improve the efficiency of the Taq polymerase and its subsequent amplification. The total amount of concentration of magnesium ion should exceed that of the total dNTP concentration hence in a PCR a range of between 1.5-4.0mM is used to ensure maximum reaction is catalyzed. The optimum amount in this experiment shows that the Mg Cl2 catalyzes the Taq polymerase optimally at a concentration of 0.75ul. The final experiment as to determine the optimum concentration of the mtor primers and the following picture was produced It can be seen from the picture that the optimal amount for both (F & R) is 0.40ul. In the general condition 0.50ul of each primer was used but this amount is excluded because the sample containing this amount shows primer dimers. The optimal primers concentration is 1.60mM. When the primer concentrations are too high they increase the chances of mispriming resulting in PCR products that are nonspecific. Limiting the primer concentrations may also result in PCR reactions that are extremely inefficient. The primer dimmers observed in the sample amount is an indication that the sample had excessive amounts of both the forward and reverse primers which resulted in false annealing where primers anneal to regions that are not the target regions. It is therefore important to know the suitable range of primer concentration to ensure accuracy in the annealing process. 3.0 Discussion PCR was developed by Kary Mullis,(Rainbow,1996) in 1983 and is now common and indispensable technique utilized in biological and medical research labs in a wide array of applications.(Saiki et al.,1985; Saiki et al., 1988) These applications include DNA-based phylogeny, DNA cloning used for sequencing, or genes functional analysis; hereditary diseases diagnosis; genetic fingerprints identification in paternity testing and forensic sciences; and detecting and diagnosing infectious diseases. In 1993, Mullis received the Nobel Prize in Chemistry for the work on PCR. (Mullis,1993) PCR can be modified extensively to perform a broad range of manipulations and thus provides many advantages when compared to traditional techniques. Several PCR tests can be performed rapidly and given a same day interpretation upon submission. Large sample numbers can be completed simultaneously. A major PCR advantage is in its ability to identify organisms rapidly that are challenging to culture, for example, Lawsonia. PCR differential assays can be used in determining if an isolate has properties that are toxigenic or nonpathogenic (genes) necessary for inducement of disease. In addition, PCR can be used in amplifying very small genetic material amounts and hence able to detect very low organism numbers in a sample. The major PCR drawbacks are in its lack of data on antimicrobial sensitivity, assay complexity and the high price of the kits and PCR equipment. The PCR sensitivity is also its major disadvantage as minute amounts of contaminant DNA from different samples can be amplified in the processes. QPCR is a technique used for the amplification of DNA trace amounts (and at times, RNA) that is found on or in almost any surface or liquid where strands of DNA may be deposited. The key to PCR understanding is in the knowledge that all animals, humans, parasites, virus, bacterium, plant contain genetic material either DNA or RNA nucleotide sequences unique to every species as well as the individual species members. Hence a sample containing DNA or RNA segments can be amplified using PCR to make several copies that are identical that are used in the determination of the high probability of the source identity (i.e. specific pathogen animal or person) of the sample trace DNA or RNA in close to all and any type of material sample. PCR amplification forms a part of the test for identification with other parts following this first step. After the amplification is completed, the segments that are amplified are compared to other segments of nucleotides from known sources (such as a particular pathogen, animal or person). This unique segments comparison is done by the placement of the nucleotide sequences that are PCR-generated next to the nucleotide sequences of known pathogens, animal or humans in a separating gel. The gel is then subjected to an electrical current, and the different nucleotide sequences result in the formation of bands resembling a "ladder" that are arranged according to their molecular size and electrical charge. This process is termed as gel electrophoresis. The bands that migrate to levels similar to the ones in the gel indicate the nucleotide sequences identity. This is the traditional and most popular PCR ways used and is the same that was used in this analysis. Recent advances in technology have brought about a new, innovation in PCR termed as Real-Time PCR or QPCR. This method has gained importance in research laboratories and clinical diagnostics due to its generating capacity for quantitative results. The technique allows for the accompanying reaction as well as results presentation in an accurate and faster fashion than the conventional PCR; that displays qualitative results only. (Kubista et al., 2006; Morillo et al., 2003; Novais et al., 2004). Real-Time PCR (QPCR) monitoring has changed the DNA and RNA fragments quantification process. Real-Time PCR allows for quantification that is of the nucleic acids and also with reproducibility that is greater. This technique is a sensitive and accurate quantification method of individual species, whose relevance is applicable in the diagnosis of genetic diseases and pathogens. The advantages of the Real-Time PCR include: quantification ease, rapid analysis, sensitivity is greater, as well as the precision and reproducibility, better process quality control and a lower contamination risk. (Morillo et al., 2003; Novais et al., 2004). Real-Time PCR utilizes a thermocycler coupled with an optical system for capturing fluorescence as well as computer software that captures data and performs the final reaction analysis. The software programs are supplied by diverse manufacturers show differences concerning excitation method, sample capacity, total sensitivity. Differences are also with regard the processing data. The fluorescence emission generates a signal that increases proportionally to the PCR products amounts. The fluorescence values are noted for each cycle and are representative of the amplified product the amount. The fluorescent composites commonly used are TaqMan® and SYBR® Green (Kubista et al., 2006; Novais et al., 2004). QPCR is a more suitable method than the traditional method for measuring repair and damage in the functional units in the subgenre level such as the intron, exons and promotor regions, (Grimaldi,1994). Most of the Real-Time applications include mRNA expression levels measurement, allelic discrimination, copy number of DNA, copy number of transgene and expression analysis, and viral titers measurement (Ginzinger, 2002).The method is also suitable for the determination of damaged DNA and repair originating from oxidative stress and genotoxic agents (Van Houten et al., 2000; Ayala-Torres, 2000; Yakes, 1997). The most crucial step in QPCR is in the PCR optimization. The thermal conditions and specifically the annealing temperature should be optimized hence the results from the analysis carried out in this paper are relevant discussed in this paper The most critical optimization points include: 1. Determination of the temperature for annealing 2. Extension temperature optimization (this temperature could be lower for amplifications of long PCR) 3. Adjuvants where necessary 4. Hot start PCR (nontarget amplification as well as primer-dimer formation minimization) 5. cycling number determination running of PCR 50% of template and nontemplate controls ( template control of 50% is given a signal amplification reduction of 50% with 40-60% reduction values being acceptable. Nontemplate control detects PCR products or spurious DNA contamination). Studies on mice have been shown to detect mtDNA damage originating from genotoxic agents, age, and oxidative stress (Mutlu et al., 2009a; Mutlu et al., 2009b; Mutlu, 2011). In addition, QPCR is a suitable method for cancer as well as nutritional studies. The cellular and molecular aging process basis is not well understood, especially in humans as predictive biomarkers set is yet to be created which can be utilized for the quantification of particular senescence aspects within cell populations and/or discrete regions. A given species longevity is dependent on several factors that include senescence (frailty change with time) and frailty ( the natural vulnerability to death) (De Magalhaes, 2004; McElwee,2004). Actuarial rates, as well as the survival curves, do not offer much in the prediction of brain aging rates of mammals. The general consensus is in the use of surrogate cellular and molecular markers in evaluating senescence throughout the animal model lifespan a plausible approach to gaining greater perspective into underlying aging mechanisms in humans, aiming at the development of pharmacotherapeutic rational interventions to avoid progressive late-onset scourge of neurodegenerative disorders as well as all related phenomena occurring in a brain that is aging. There is little likelihood that a single gene causes aging. A complex genes pattern probably influences a broad range of critical parameters which includes and generalized homeostasis, disease resistance, and cellular defense. There is no known exact gene products mosaic responsible for aging, and current research has begun identifying candidate markers drawn from a broad range of transcript classes. Another obstacle linked to aging research by using animal models has been in the uniform genetic backgrounds divergence in model systems leading to phenotypic expression variable complexity. Further, there lacks effective experimental means for measuring potentially significant but small gene expression changes (Galvin et al., 2005) PCR can be used in the quantification of amplicon product formation in every amplification cycle thus eliminating many concerns plaguing conventional methods of PCR. Other advantages linked to qPCR include simultaneous multiplex reactions, high throughput capabilities, increased sensitivity, inter-assay variation is reduced, post-PCR manipulations are lacking. Currently, a number of dye chemistries are being exploited in for use in the qPCR systems and include molecular beacons, hydrolysis probes, and binding dyes of double-stranded (ds) DNA (Bustin, 2002). As mentioned earlier, TaqMan assay is used as a hydrolysis probe. In this procedure, the enzyme Taq polymerase cleaves to specific TaqMan probe in the PCR extension phase. The probe is then dual-labeled having both a quenching dye and reporter dye at the two separate ends. The probe needs to remain intact, that is in its free form, for there to be fluorescence emission when the quenching dye absorbs the reporter dye via energy transfer of fluorescence resonance energy transfer (FRET) (Giulietti A, et al. 2001; Shintani-Ishida et al ,2005) There is an increase in emission of fluorescence emission when reporter and quencher dyes separate during PCR reaction nuclease degradation (Cardullo RA, et al. 1988). This is an occurring process in each PCR cycle and does not interrupt the amplified product exponential accumulation. Another qPCR-based detection methodology involves molecular beacons use. The molecular beacons are probes forming a structure that is stem-looped from DNA single-strand molecule (Bonnet G, et al. 1999; Tan L, et al. 2005). They are significant in the identification of point mutations where different targets o so by a single nucleotide that can be delineated. SYBR green DNA binding dye incorporates into ds DNA selectively. The SYBR green then emits fluorescence levels that are undetectable when in free form after that a robust signal hat is fluorescent is emitted upon binding (Kricka, 2002). The advantage of utilizing the chemistry of DNA binding dye is in the implementation of assays for any target sequence together with any primer set making it a flexible process that is less expensive in comparison to the dye chemistries that are probe-based (Providenti,2005). The downside is that the sensitivity of the assay can be diminished in the utilization of the system of ds DNA binding dye because of the increased amplification risk of PCR products that are nonspecific. A careful design of primer set and assay optimization that is rigorous can alleviate most of the nonspecific issues linked to the ds DNA binding dyes. 4.0 Conclusion The mechanism that underlie the aging process is quite complex with a high probability of CNS expression profiles revealing several mosaics at cellular, nuclear, laminar, and regional levels. There is exists no current accepted aging biomarker hence the molecular neuroscience goal is the development of reliable age-related molecular fingerprints (Galvin, 2005; McClain KL, et al., 2005. This protein is encoded in the human body by the MTOR gene. mTOR signaling modification modulates longevity in laboratory models of fruit flies, nematodes, yeast, and also in mammals (Vellai et al., 2003; Kaphali et al., 2004). The significance of this pathway in aging and longevity determination in these systems is in the demonstration observed when the mTORC1 pathway targeting immunosuppressant, rapamycin is the single pharmacological intervention that is shown to increase rodents’ lifespan (Cao, 2001; Harrison et al., 2009). There is no known exact gene products mosaic responsible for aging, and current research has begun identifying candidate markers drawn from a broad range of transcript classes. Currently, a number of dye chemistries are being exploited in for use in the qPCR systems and include molecular beacons, hydrolysis probes, and binding dyes of double-stranded (ds) DNA. A careful design of primer set and assay optimization that is rigorous can alleviate most of the nonspecific issues linked to the ds DNA binding dyes. 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