Group 1 Project - Fluorescent-PCR

From CellBiology

Introduction

PCR tubes. Each tube contains a 100ul reaction.

Genetic expression has established the basis of clinical diagnosis and molecular analysis. Any alterations of genetic expression may result in the development of a disease state or abnormal cellular process. Fluorescent polymerase chain reaction (abbreviated as fluorescent-PCR) has been an efficient analytical method that could detect genetic material in an organism with high precision. When genetic material is present in limited amounts, DNA or RNA could be amplified exponentially to a substantial level for detection in a simple enzymatic reaction. Gene amplification accounts for the high sensitivity of PCR where single copies of genes are analyzed. PCR is also characterized by its high selectivity and speed. Therefore, fluorescent-PCR is a diagnostic test that provides simplicity, accuracy, reliability and performance. These characteristics justify the extensive use of fluorescent PCR for genetic screening and analysis in medical research.


Basic Concept of DNA

Amplification of genetic material by PCR is developed on the basis of understanding the DNA biology. Comprehending the basic concept of DNA structure will enhance our appreciation of the PCR technique for current molecular biological research.

Complementary base pairings. In DNA double-helix, hydrogen bonds were formed between the nitrogenous bases of the nucleotides known as complementary base pairs.

DNA has a double-helical structure that constitutes two anti-parallel strands intertwined with one another. Each DNA template strand is a polymer of subunits called the nucleotides. A nucleotide subunit is composed by a phosphate molecule, deoxyribose sugar and a nitrogenous base. The deoxyribose sugar molecules are linked together by the phosphate groups that formed the backbone of the DNA strand. The linkage is known as 3'-5' phosphodiester linkage that connects 3'C of sugar molecule to the 5'C of the adjacent nucleotide subunit.

Deoxyribose sugar molecule is covalently bonded to one of the nitrogenous bases (Adenine, Guanine, Cytosine and Thymine). Adenine (A) and Guanine (G) are double-ringed bases known as purines; while Cytosine (C) and Thymine (T) are the single-ringed bases known as pyrimidines.

The double-helical DNA structure is constructed by two DNA strands oriented in opposite directions and held together by hydrogen bonds. Adenine and Thymine can form a base pair with two hydrogen bonds; and Guanine and Cytosine form a base pair by three hydrogen bonds. These base pairings occur exclusively either between A-T or G-C; therefore, they are called complementary base pairs. The complementary base-pairings contribute to selectivity and specificity of the PCR technique, where a targeted sequence is amplified by initiating DNA extension with primers of complementary gene sequences. In the double-stranded DNA, interaction energies between the complementary bases arise from the hydrogen bondings and the hydrophobic interactions between the neighbouring stacks of base pairs[1] . In the denaturation phase of thermal cycling, these hydrogen bonds are broken to generate single-stranded DNA by high temperatures. However, renaturation can occur as the two single polynucleotide chains unite to form a double-helix where the original hydrogen bonds can reform [2]. This accounts for the annealing of the primers to the complementary sequences of the template strand when the temperature is lowered.


Development of PCR

Anchor PCR. Anchor primer binds to the artificial tail of the unknown sequence before amplification.
Kary Mullis. Founder of the PCR technique
Inverse PCR. Circular DNA is cleaved by the restriction enzymes before amplification of each circular DNA strand.

Since the early 1990s, PCR has predominantly become the basic tool for application in molecular biology. As it progressed to mid 1990s, PCR was used as a diagnostic and screening tool for genetic diseases.

Before PCR was introduced, molecular cloning allowed molecular biological research and studying of genetic structures. The efficiency of this method relied on the DNA replication of plasmids and vectors in cellular division. Researchers recognised that molecular cloning was laborious and possessed low selectivity. Thus, it was difficult to isolate specific DNA from cells in biological specimens.

Development of PCR has revolutionized the procedures of studying molecular biology. This DNA amplification procedure was initiated by Kary B. Mullis and his team from Cetus Corp in 1984[3]. As compared to molecular cloning, PCR amplified DNA via a in-vitro setting instead of in-vivo. Since then, PCR has accelerated the analysis of genetic data. Since its discovery in 1983, Basic PCR has generated large quantities of DNA sequences if the DNA sequences of the primer molecules were known. Primer's DNA sequences would bind to complementary DNA sequences on template strand for amplification. The limitation of this method is the need to know the DNA sequence at both ends of targeted template strand to synthesize the primers.

Crystal Structure of Taq DNA polymerase

In the early 1990s, anchored PCR was developed by Gail Martin and Mark Davis of Stanford. This method overcame the limitation in the basic PCR. Anchored PCR employed the use of one primer and another "anchor" primer binding to a sequence artificially linked by unknown an sequence of target. Another strategy developed by Washington University was inverse PCR. DNA template strands were cleaved by restriction enzyme and annealed at each ends to form circular DNA. Synthesis of primers were based on the ends of the known sequence. At each template strand, DNA is transcribed from one primer site to another. This method generates linear DNA molecules where DNA sequences are anti-parallel to the template strand in the first round of amplification. Subsequently, ordinary PCR will proceed.

DNA samples in early PCR experiment were amplified by the Escherichia coli DNA polymerase 1 at the temperature of 37°C. The outcome was incomplete pure target products determined by gel electrophoresis. Isolated heat-resistant DNA polymerase from Thermus aquaticus permits the annealing and extension of DNA at higher temperatures. This DNA polymerase is not denatured at 95°C and it works optimally at 72°C. Non-complementary annealing of the primer and template strand is reduced to negligible level.

Basic PCR Timeline

1971 – Gobind Khorana described a basic principle of replicating a piece of DNA using two primers. Progress was then limited by primer synthesis and polymerase purification issues.

1976 – Taq polymerase was discovered (from Thermus aquaticus) which paved the way for PCR concept. Taq is stable at high temperatures and remains active after DNA denaturation, eliminating the need to replace the DNA polymerase after each cycle of DNA replication.

1983 – Kary Mullis at Cetus Corporation conceived a way to start and stop a polymerase’s action at specific points along a single DNA strand. Mullis also realised that by harnessing this component of molecular reproduction technology, the target DNA could be exponentially amplified.

1985 – Science publishes the first paper on PCR.

1989 – PCR “explosion” can be seen as a result of a combination of the improvements and optimising of the methodology and the introduction of new deviations on the basic PCR concept.

1993 – Kary B Mullis receives a noble prize in chemistry for inventing the concept of PCR.

Present – Many variations have built upon the fundamental PCR method, including that of Fluorescent PCR[4].


Principles of Fluorescent-PCR Procedures

Multiple copies of a desired DNA sequence could be amplified through the polymerase chain reaction technique. The sensitivity of this technique is enhanced by performing hybridization of a fluorescent probe to the PCR products. With a fluorescent detector, the PCR products are analyzed based on the fluorescence intensity.

Polymerase Chain Reaction

PCR thermal cycler.

Although fluorescent-PCR may be the topic of interest, it is relevant to understand the underlying mechanism of the conventional PCR, as fluorescent-PCR is an extension of this fundamental process. The basic principles of amplification in PCR evolved from the knowledge about DNA replication, denaturation and renaturation.

Polymerase Chain Reaction. Three processes in thermal cycling are denaturation, annealing and extension of the DNA.
Principle of Fluorescent Probe. Labelling illustrated on one DNA template strand, 5' to 3' end.
Structure of Fluorescent Probe PMCID: PMC17763

Amplification of the genetic material requires DNA polymerase, two oligonucleotides primer (where each primer is complementary to one strand of parenteral DNA template) and repetitive cycles at three different temperatures known as thermal cycling[5].

The three processes in thermal cycling are: denaturation, annealing and extension.

  • Denaturation is the process of heating the DNA duplex to a temperature of 90-95°C. At a high temperature of 90°C, the hydrogen bonds between the complementary strands of DNA helix are broken. Subsequently, two single-stranded of DNA are generated.
  • Annealing occurs as the temperature of the process is reduced to 50-65°C. As the reaction is cooled down to 50°C, hydrogen bonds form between the bases of oligonucleotide primers with the DNA template strand. The primers anneal to the complementary DNA sequence on the single-stranded DNA template that begins at the 5' end.
  • Extension of the new DNA strand require the temperature to be raised to 72°C. The new DNA strand is extended by Taq DNA polymerase, an enzyme that polymerizes the additional deoxy-nucleotides (dNTP) in a DNA sequences that are complementary to the DNA template. Taq DNA polymerase functions optimally at 72°C, with magnesium in the PCR buffer facilitating the reaction.

One cycle comprises of denaturation, annealing and extension. For substantial genetic material to be analyzed, 25-35 cycles are performed in PCR. DNA strands are amplified exponentially, where the number of DNA strands can be quantified by this formula, 2n, where n denotes the number of thermal cycles.


Fluorescent Analysis

When the amplification of genetic material is completed, the quantity and molecular size (in bps) of amplified products can be determined by gel electrophoresis and fluorescent analysis. When fluorescent probes are used, they sensitize the analytical technique allowing visualization through the emission of a fluorescent signal. The processes involved in fluorescent analysis are fluorescent labelling, gel electrophoresis and detection of fluorescence for quantification.

1) Fluorescent-labelling

Fluorescent probes are added to the amplified genetic molecules after PCR. Examples of fluorescent probes include MB-Green and MB-Red [6]. A fluorescent-labelled primer is an alternative for detection in quantification. Fluorescent probes are used to identify the presence of a specific genetic sequence in PCR products. If the fluorescent probe has a complementary DNA sequence to the wild-type PCR product, mutations present in the DNA sequencing will disrupt the hybridization between the probe and amplified product. Different fluorophores in the fluorescent probes serve as markers for multiple target genes by variation in colour coding. Presence of mutations and multiple PCR products can be detected simultaneously after PCR is complete.

  • In the labelling process, fluorescent probes or primers are added as one of the components in the PCR solution.
  • Before the amplification process, the probes are added to the solution. In addition, other essential constituents are added to the PCR solution including oligonucleotides (dGTP, dTTP, dATP, dATP), Taq ploymerase, magnesium chloride and Tris solution.
  • PCR is performed in a thermal cycler for amplification at three different temperatures, then incubated at room temperature.
  • Fluorescent probes provide the benefits of in-situ hybridization where the amplification and labelling are performed in the same well.
  • The principle of the fluorescent labelling is dependant on the structure of the fluorescent probes and the intrinsic nuclease activity of Taq DNA polymerase. These probes will initially be hybridized to the target DNA sequence before cleavage by Taq DNA polymerase during amplification.
  • Fluorescent probes are oligonucleotides that have a fluorescent dye at the 5' end and a quencher (E.g. Dabcyl or rhodamine) at the 3' end. These oligonucleotides exist in stem-loop structures where hydrogen bonds are formed between the complementary bases within the same oligonucleotide strand. Upon excitation by irradiation at certain wavelengths, the fluorescent dye emits fluorescence through the resonance energy transfer[7]. If the quencher is in close proximity to the fluorescent dye, the fluorescence emission will be quenched. During extension phase, the hybridized probe will be cleaved by Taq DNA polymerase by its intrinsic nuclease activity from 5' to 3' end. Both fluorescent dye and quencher group are released from the probe after cleavage, and the fluorescent dye is no longer quenched. Subsequently, an increase in fluorescence intensity can be observed, where the degree of quenching is inversely proportional to the distance between the fluorescent dye and quencher. Therefore, amplifications of PCR products are quantified by the elevation of fluorescence signal.

2) Gel Electrophoresis

Procedures of Gel Electrophoresis.
Gel Electrophoresis. Image captured by the UV transilluminator after gel electrophoresis of PCR products and DNA ladder.

Gel electrophoresis is used for separation of the amplified genetic product according to its molecular size for quantification in a genescanner. Molecular size is expressed as the number of nucleotide base pairs. In addition, gel electrophoresis can be used to distinguish the intended amplicon from the false amplicon based on the molecular size[8]. Unintended amplicons may occur by the hybridization between the primers that generate a false signal. The separation of PCR products is performed on an agarose or polyacrylamide gel immersed in an electrophoretic buffer that maintains the pH at a consistent value. The degree of DNA fragment separation can be adjusted by varying the concentration of the agarose or polyacrylamide gels. PCR products separate once they are subjected to the electrical field generated from the polarities by the electrodes.

  • For conducting gel electrophoresis, the PCR products or DNA sample are initially diluted in Tris solution and EDTA. The diluted PCR products is mixed with the loading buffer that may be constituted by a density gradient agent (E.g. glycerol, sucrose or Ficoll), EDTA and tracking dye (E.g. xylene cyanol, bromophenol blue or orange G). In gel electrophoresis for conventional PCR, ethidium bromide as a fluorescent dye is used for staining nucleic acids for visualisation with a UV transilluminator. In fluorescent- PCR, a fluorescent signal is detected upon excitation which obliterates the use of ethidium bromide. Amplified DNA samples are loaded into the sample wells of agarose gel by a pipette. Beside the PCR products, a DNA ladder is also electrophoresed on the agarose gel at a designated voltage.
  • The main principle of gel electrophoresis is utilising both electric field and the porosity of the electrophoresis gel to separate DNA molecules. Nucleic acids possess negative charges from their phosphate backbone. The electrodes (cathode and anode) generate an electric field that separate the molecules based on their charges. For a negatively-charged DNA molecule, it will migrate towards the positively-charged cathode. Electrophoresis gel is a matrix composed by cross-linked polymers from polysaccharides (agarose gel) or acrylamide (polyacrylamide) that form mesh networks for separating the nucleic acid molecules. By varying the concentration of agarose or polyacrylamide, the porosity of the gel is adjusted according to the molecular size of DNA molecules. When subjected to an electric field, the DNA molecules will migrate through the matrix at different rates according to its molecular size and charge. Smaller molecules (E.g. DNA) have a greater migration distance than large molecules (E.g. RNA molecules).
  1. Density gradient agent: Glycerol or sucrose will increase the density of the DNA material so that it will be layered at the base of the agarose gel's sample well for separation.
  2. Tracking dye: Allows visualisation of the DNA sample and monitoring the progress of the electrophoresis.
  3. DNA ladder: Contains DNA molecules with different molecular sizes (in base pairs) to approximate the size of the unknown DNA molecules.

With the advancement of current technology, the genescanner has the ability to quantify the amplicons based on their fluorescent intensity. Gel electrophoresis is no longer used after PCR for quantification. The main use of gel electrophoresis is to identify any false postitives that may arise from non-complementary hybridization between the primers and the DNA template strand.

3) Quantification by fluorescent intensity

Quantification may be performed during amplification process or gel electrophoresis. If PCR was performed in a spectrofluorometric thermal cycler, fluorescent intensity can be monitored at the annealing phase of the thermal cycle. Alternatively, if quantification occurs in gel electrophoresis, the DNA samples have to be substantially separated before excitation by irradiation for quantification. The fluorescent signal can be enhanced by a photomultiplier and subsequently analyzed by a computer. The fluorescent intensity is the difference in the initial fluorescence and the level of fluorescence after the amplification. The genescanner detects the level of fluorescence emitted from the fluorescent probes after cleavage by the Taq DNA polymerase in PCR. Fluorescence intensity is used to quantify the PCR products where incremental amounts of DNA materials result in increased fluorescence.

  • The principle of this quantification process arises from the irradiation (e.g. Laser) emitted from the genescanner. Depending on the fluorescent probe, the fluorescent dye will be absorbing light at a particular wavelength (e.g. MB-Green 485/530 nm, MB-Red 530/590 nm), thus exciting it. Subsequent emission of fluorescence is derived from the dye through resonance energy transfer. This energy transfer will be increased by distancing the fluorescent dye and the quencher group in the amplification. Quantity of the PCR products are determined by the relative intensities of fluorescence[9].
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To enhance your understanding of fluorescent PCR, the video will feature the three phases of thermal cycling, process of fluorescent-labelling and fluorescence signaling.

Video of PCR and Real-time PCR Process (Scanelis)





Comparison against Conventional PCR

To recap, in order to obtain substantial quantity and molecular size of genetic material, PCR cycles have to be completed. In conventional-PCR, agarose or acrylamide gels are separated by an electric field according to their molecular size. Smaller amplicons migrate at a greater rate through the gel. Conventional-PCR can be made more accurate by labelling primers with fluorescent markers this procedure is referred to as fluorescent-PCR. A sensitive system fluorescent DNA sequencer, also known as a genescanner can be used to separate and analyze the fluorescent-PCR products. As a result fluorescent-PCR allows detection of product without agarose or acrylamide gel electrophoresis.[10]

Advantages of Fluorescent-PCR

These are the advantages provided by fluorescent-PCR when compared to conventional-PCR [11]:

Table comparing Conventional and Fluorescent PCR
Parameters Conventional PCR Fluorescent PCR
Sensitivity Less sensitive More sensitive by about 1000-fold
Accuracy Lower accuracy High accuracy (97-98%) from 1-2 base pairs difference
Number of PCR cycles More PCR cycles required Less PCR cycles required for the same level of detection
Loading of product More loading of products Less loading of products; more efficient because of more repeated sampling
Detection reagents Radioactive labelled nucleotides used Fluorescent-tagged nucleotides used; less toxic
Quantitative analysis Gel electrophoresis Detection of fluorescence signal intensity
Detection of multiple products Separation of products by molecular size in gel electrophoresis Different fluorescent dye to detect multiple products


Disadvantages of Fluorescent-PCR

The potential disadvantages that may arise from this technique include:

  • Cross contamination can easily produce false positive results.
  • Specificity: Amplified PCR products can only be obtained when conditions are specific.
    • Slight differences can reduce yield dramatically.
    • There are different requirements for each primer set; thus all conditions are optimized to maximize the yield of PCR products.
    • Optimization of many procedural conditions is essential for multiple reaction where several different primer sets are used.
    • Conditions will include temperatures for different phases of thermal cycle, magnesium concentration etc.
  • The technique is costly and requires specialized equipments.


Clinical Applications of Fluorescent-PCR for Diagnosis

Identification of Retroviruses

Fluorescence of Retroviruses. Intensity of the fluorescence increases as the thermal cycling progresses that indicates the exponential multiplication of genetic material.

A multiplex nucleic acid assay was developed by Vet et al., 1999, that both identified and quantified the abundance of retroviruses including the HIV-1, HIV-2 and human T-lymphotropic virus type I and II. Amplification of the retroviral DNA sequences is performed through PCR assays in a spectrofluorometric thermal cycler. The amplified retroviral DNA is hybridized to specific fluorescent probes which include fluorescein for HIV-1, tetracholoro-6-carboxyfluorescein (TET) for HIV-2, tetramethylrhodamine (TMR) for HTLV-I and carboxyrhodamine (RHD) for HTLV-II. The fluorescence colour is crucial for identification of the specific retroviruses. Fluorescence spectrum at 500-650 nm is detected from the assay sample during the annealing phase of the thermal cycle. Quantification of the retro-viral DNA abundance is conducted in real-time, where the intensity of the fluorescence signal is increased significantly with the number of thermal cycles. The reliability of the assay is demonstrated with clinical samples. The retroviruses are identified with false positives eliminated. Therefore, the use of fluorescent-PCR in the assay has enhanced the efficiency and reliability of screening donated blood and transplanted tissue for retroviruses.

Results are plotted with fluorescence intensity of fluorescent probes against the number of completed thermal cycles. The colour of the fluorescence acts as the indicator of the retroviral DNA that has been amplified. In the laboratory samples, the results show that there is a positive correlation between the fluorescence intensity and the number of thermal cycles. Increases in fluorescence intensity are observed for all four nucleotide sequences of HIV-1, HIV-2, HTLV-I and HTLV-II. Elevation of fluorescence signal results from the higher target copy number of the amplified retrovirus DNA.

In the clinical samples, retroviruses are correctly identified by the assay indicated by the colour of the fluorescence. In samples of healthy individuals, the assay gives a negative result due to the absence of retrovirus DNA. The PCR assays allow fast and sensitive detection of the amplified retrovirus DNA, which is essential for rapid screening of diagnostic samples. [12]

Analysis of Insulin-like Growth Factor Receptor 1 Gene Copy Number in Survival of Small-cell Lung Cancer

Quantitative fluorescent-PCR technique has also been used in cancer research. One study focused on the correlation between two growth factors, insulin-like growth factor-1 receptor (IGF1R) and epidermal growth factor receptor (EGFR), being expressed in non-small-cell lung cancers (NSCLC). IGF1R gene expression was evaluated using quantitative reverse transcription polymerase chain reaction. Fluorescent in situ hybridization was subsequently used to assess IGF1R gene expression using customized probes. IGF1R and EGFR protein expression showed significant correlation and presence within cancers. Prognosis of the cancer is dependent on the gene copy number. Amplification of HER2 or high EGFR gene copy number is related to the malignancy and poor survival of patients. It was concluded that IGF1R gene expression does not associate with survival, however high IGF1R gene harbors positive prognostic value. By utilising fluorescent-PCR, prognosis of patients can be determined by high gene copy number of HER2, EGFR and IGF1R.[13]

Performing Preimplantation Genetic Diagnosis for myotonic dystrophy or Steiner’s disease

Comparison of conventional PCR with Fluorescent PCR. Allelic dropouts and amplification of myotonic dystrophy embryonic DNA sample in patients using conventional PCR and fluorescent PCR

Myotonic dystrophy also known as Steiner’s disease is described as a disease that causes muscular weakness, atrophy and myotonia. It is also an autosomal dominant disease. The disease affects the distal muscles of the limbs; frontal baldness; atrophy of the sternomastoids and gonads; lens opacity; mild endocrine and bone changes as wells as cardiomyopathy. In 1992 the gene on loci 19q was located and cloned and it was observed to contain CTG triplet repeat. Normal individuals were known to contain 5-30 repeats, mildly affected patients have 50-80 repeats and the severely affected have over 200 repeats. This repeat was used to diagnose and develop a preimplantation genetic diagnosis (PGD) for myotonic dystrophy. When conventional-PCR was used, it was decided that a setback of this assay was, the loss of healthy embryos that were misdiagnosed as affected. Consequently, leading to a high allelic dropout (ADO) rate of more than 20%. In comparison, fluorescent-PCR ADO rate was found to be much lower because it is more sensitive to conventional-PCR. Therefore, their study concluded that fluorescent-PCR for PGD is superior to conventional-PCR because of its superior accuracy, thus a lower ADO rate resulting in less embryos being lost due to misdiagnosis.[14]

Detection of Mycobacterium tuberculosis Genome DNA

Probe diffusion time against the cycle number of experiment. Exponential increase in diffusion time of probe as the number of thermal cycles was increased.

Fluorescence correlation is utilized to detect specific in-vitro amplification of genetic sequences. The fluorescent signal is detected by fluorescence correlation spectroscopy (FCS) after probe extension during PCR amplification of a specific sequence for Mycobacterium tuberculosis. The pathogenic genome from Mycobacterium tuberculosis is detected after subjecting it to PCR amplification with Stoffel fragment of Thermus aquaticus DNA polymerase. This amplification is initiated in the presence of rhodamine-labelled probe binding to the targeted DNA sequences between the amplification primers. In the presence of the template, the probe is necessary for the formation of specific amplicons. Without the template DNA strand, the primers can initiate non-specific amplification that consequently yields non specific products during PCR. This does not interfere with the diffusion time of the low concentration fluorescent probe.

Gel electrophoresis performed after PCR amplification. Gel electrophoresis is a confirmatory test for specificity of target amplification by molecular size of the DNA.

Results show that the correlation between the fluorescent signal and diffusion time of the probe increases progressively with increasing PCR thermal cycles, this observation demonstrates the specificity of PCR amplification. Therefore, low input of genome can be quantified without the need of manipulation after PCR. However, it is necessary to avoid false-positives in diagnostic test by ensuring the targeting amplification is specific. Confirmation is accomplished by gel electrophoresis by processing the DNA sample in a gel. In this technique, low concentrations of probe are used. Therefore, it provides more stringent conditions for a probe binding to its specific target. Thermus aquaticus DNA polymerase is used for DNA extension in this study. As compared to Taq DNA polymerase, Stoffel fragment of Thermus aquaticus DNA polymerase lacks the intrinsic exonuclease activity that explained the low concentration of probes utilized in the study.

Mycobacterium species are slow growing pathogens, identification of these pathogens is reliant on long and laborious culturing techniques. This PCR technique has improved the analysis techniques for diagnosis. Rapid diagnosis is critical for treatment and preventing the resurgence of tuberculosis worldwide [15].

Fluorescent-PCR Applications in Cell Biology Research

Evaluating the effects of kinase inhibitors on epithelial to mesenchymal transition

Treatment with a TRβI inhibitor reverses PAI-1 RNA level in TGF-β1-induced mesenchymal renal tubular epithelial cells to levels present in epithelial cells.

Epithelial to Mesenchymal Transition (EMT) is the cellular event, which contributes to organogenesis, cancer and organ fibrosis. This event is triggered by the transforming growth factor (TGF). Kinases are implicated in the signaling pathways that induce mesenchymal state. Inhibitors of the kinases in EMT were evaluated in this study for the reversal of the induced mesenchymal state.

Restoration of epithelial gene expression patterns requires a combination of kinase inhibitors.

Quantitative or fluorescent PCR are used to quantify the total RNA isolated from the cells by the UV spectrophotometer. Results have demonstrated that the treatment with a TGF-βI inhibitor will reverse the PA-1 RNA levels in TGF-βI induced mesenchymal renal tubular cells to baseline levels present in epithelial cells.

In addition, the kinase inhibitors in mono-therapy or combined therapy are evaluated on the expression of several altered genes in EMT. These genetic expression are examined by fluorescent-PCR. It begins with the induction to a mesenchymal state by the incubation with TGF-βI; then the cells are treated with the kinase inhibitors. RNA level of Ksp-cadherin, SM22 and MMP-9 are determined with fluorescent-PCR. Significant differences can be observed between the untreated cells and the cells treated with kinase inhibitors. RNA levels drastically alter by the administration of kinase inhibitors. Kinase inhibitors exert their therapeutic effects by inducing the accumulation of transcripts specific to epithelial cells or certain additional specific transcripts such as Ksp-cadherin associated with the reversal of EMT. Through the fluorescent-PCR technique, the kinase inhibitors can be evaluated on their efficacy in reversing EMT. This concludes that the combination of the small molecule therapy that target several kinases may be necessary to reverse the disease conditions[16].

Studying the modulation of E2-stimulated expression of ERα target genes by Keratin 18

K18 modulates E2-stimulated expression of ERα target genes and the recruitment of ERα to its target DNA in MCF-7 cells.doi:10.1186/1471-2121-10-96

The excessive activation of estrogen receptor (ERα) and over-expression of its co-activators are attributable to the oncogenesis in breast cancer. LRP 16 is acting both as an ERα target gene and co-activator; therefore, it is involved in the proliferation of MCF-7 breast cancer cells. Intermediate filament protein keratin 18 (K18) is a LRP16 interacting protein. Expression of K18 in MCF-7 cells has significantly lowered the association of LRP16 with ERα. Consequently, it attenuated ERα-activated reporter gene activity and decreasing gene expression by inhibiting ERα recruitment to the DNA.

To study if K18 affects E2 induction of ERα target genes in MCF-7 cells, fluorescent- or quantitative PCR is used to measure the mRNA expression levels of cyclin D1 that is known to be E-2 regulated in MCF-7 cells. Over-expression of the K18 has attenuated this induction. While the over-expression of LRP 16 antagonizes the K18 inhibition of E2-induced expression of target genes. The MC7 cells are cultured and transfected with the indicated vectors. The RNA is extracted from the cells for analysis and the expression of the indicated RNA abundance is analyzed by fluorescent-PCR.

From the observations, it has been concluded that K18 has the role of sequestering LRP 16 in the cytoplasm. Therefore, it prevents the nuclear action of LRP 16 and attenuates ERα signalling. As a result, it will blunt estrogen-stimulated cell-cycle progression of ERα-positive breast cancer cells. The level of K18 gene can be used as a marker for the diagnosis of breast cancer, where the level of K18 gene expression correlates inversely with the progression of breast cancer[17].


Conclusion and future directions

The Fluorescent PCR technique has been a very important tool in the area of DNA identification and genetic analysis. It has been shown to be an advantageous method of research allowing a higher accuracy of detection of base pairs, a lower number of cycles required for the same level of detection, with multiple products detected and less loading of products needed compared to that of the conventional PCR method. As mentioned above, Fluorescent PCR is being used in and primarily supports a great number of clinical applications whether that is the identification and analysis of specific genes within retroviruses, the marking of growth factor receptors in small cell lung cancers or the genetic detection and diagnosis of the disease myotonic dystrophy. Future projections in regards to the use of the Fluorescent PCR technique suggests the continued application in the areas of cancer research and other common diseases within the population, as well as the furthering of our knowledge and understanding in the realm of stem cell research[18].



Glossary

  • Amplicon: The amplified sequence of DNA in the PCR process.
  • Atrophy: A wasting or decrease in size of a body organ, tissue, or part owing to disease, injury, or lack of use.
  • Autosomal disease: A disease caused by a gene located on a chromosome other than a sex chromosome (autosomal chromosome).
  • DNA duplex: Known as DNA double helix; it is a typical conformation of a double-stranded DNA molecule in which two polynucleotide strands are wounded around each other with base pairing between the double strands.
  • DNA template strand: Also known as parenteral DNA strand is the DNA or RNA sequence that is going to be amplified.
  • Dominant disease: A disease caused by an affected individual who has inherited one copy of a mutant gene and one normal gene on a pair of autosomal chromosomes. Individuals with autosomal dominant diseases have a 50-50 chance of passing the mutant gene and therefore the disorder onto each of their children.
  • EDTA: Ethylenediaminetetraacetic acid; a complex molecule used to chelate magnesium ion (as a co-factor for DNA polymerase) that subsequently inhibits the action of DNA polymerase to extend the DNA strand.
  • Fluorophore: The fluorescent dye used to monitor dye accumulation.
  • Fluorescent probe: A probe which is labelled with a fluorescent dye, so that the signal emitted can be captured by photometric methods.
  • Gene copy number: A copy number variation (CNV) is a segment of DNA in which copy-number differences have been found by comparison of two or more genomes.
  • Hybridization: Experimental process in which two complementary nucleic acid strands form a double helix; a powerful technique for detecting specific nucleotide sequences.
  • In-situ hybridization: Technique in which a single-stranded RNA or DNA probe is used to locate a gene or an mRNA molecule in an entire cell or tissue.
  • Intrinsic nuclease activity: Property of the Taq polymerase to catalyze the hydrolysis of terminal nucleotide from the nucleic acid chains.
  • in vitro: Term used by the biochemist to describe a process taking place in an isolated cell-free extract. Also used by cell biologists to refer to cells growing in culture (in vitro), as opposed to in an organism (in vivo).
  • in vivo: In an intact cell or organism.
  • Molecular beacons: These hairpin probes consist of a sequence-specific loop region flanked by two inverted repeats. Reporter and quencher dyes are attached to each end of the molecule.
  • Myotonia: Tonic spasm or temporary rigidity of one or more muscles, often characteristic of various muscular disorders.
  • Oligonucleotides: A short polymer composed of nucleosides with a series of one or more phosphate groups joined by an ester linkage to the sugar moiety.
  • Organogenesis: The formation and development of the organs of living things.
  • Plasmids: Small circular DNA molecule that replicates independently of the genome. Used extensively as a vector for DNA cloning.
  • Primers: A short length of RNA made at the beginning of a DNA synthesis event catalyzed by DNA polymerase; these RNA primers are subsequently removed and filled in with DNA.
  • Quencher: The molecule that absorbs the emission of fluorescent reporter dye when in close vicinity (6 to 10 nucleotides). It is also called as the acceptor dye.
  • Restriction enzymes: Nuclease that can cleave a DNA molecule at any site where a specific short sequence of nucleotides occurs. Different restriction enzymes cut at different sequences.
  • Resonance energy transfer: The interaction between the electronic and excited state of two dye molecules. The excitation is transferred from the donor dye molecule to the acceptor dye molecule.
  • Radioactive-labelled nucleotides: A defined segment of nucleic acid that is tagged with a radioactive marker. It is necessary to identify the specific segments of DNA that carries the complementary sequence.
  • Retrovirus: RNA-containing virus that replicates in a cell by first making a double-stranded DNA intermediate. This DNA is inserted into the cell's chromosome, where it can be maintained for a long time and is transcribed to produce new viral genomes and mRNAs that encode viral proteins.
  • Stoffel fragment: An unique enzyme is a 61 kDa modified form of recombinant Taq DNA Polymerase from which the N-terminal 289 amino acids have been deleted to increase stringency at lower ionic strength and reduce misextension.
  • Thermal cycling: A process of temperature modulation that consists of cycles of repeated heating and cooling of DNA for denaturation and enzymatic extension of the DNA template.
  • Vectors: Genetic element, usually a bacteriophage or plasmid, that is used to carry a fragment of DNA into a recipient cell for the purpose of gene cloning.
  • Wild-type: Normal, non mutant form of a species resulting from breeding under natural conditions.


References

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2010 Projects

Fluorescent-PCR | RNA Interference | Immunohistochemistry | Cell Culture | Electron Microsopy | Confocal Microscopy | Monoclonal Antibodies | Microarray | Fluorescent Proteins | Somatic Cell Nuclear Transfer