Group 1 Project - Fluorescent-PCR

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Gel Electrophoresis doi:10.1371/journal.pone.0007205

Genetic expression has established the basis of clinical diagnosis and molecular analysis. Any alteration of genetic expression may result in developing 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 the 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 could be analyzed. PCR is also characterised by its high selectivity and speed. Therefore, fluorescent-PCR is a diagnostic test that provides simplicity, accuracy, reliability and performance. These characteristics justified the extensive use of fluorescent PCR for genetic screening and analysis in medical research.

Development of PCR

Anchor PCR. Anchor primer binds to the artificial tail of the unknown sequence before amplification.
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 proceed to mid 1990s, PCR was used as a diagnostic and screening tool for genetic diseases.

Before PCR was introduced, molecular cloning has allowed molecular biology 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 is labourious and possesses low selectivity. Thus, it is difficult to isolate specific DNA from cells in biological specimen.

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[1]. As compared to molecular cloning, PCR is amplifying DNA via the in-vitro instead of in-vivo. Since then, PCR has accelerated the analysis of genetic data. Basic PCR, invented in 1984, generated large quantities of DNA sequence if the DNA sequences of the primer molecules are known. Primer's DNA sequence would bind to complementary DNA sequence 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 Tap DNA polymerase

In the early 1990s, anchored PCR was developed by Gail Martin and Mark Davis of Stanford. This method overcomes the limitation in the basic PCR. Anchored PCR employed the use of one primer and another "anchor" primer binds to sequence artificially linked by unknown sequence of target. Another strategy developed by Washington University is inverse PCR. DNA template strands were cleaved by restriction enzyme and annealed at each ends to form circular DNA. Synthesis of primers is based on the ends of the known sequence. It allows DNA transcription from one primer site to another at each template strand. This method generate linear DNA molecules where DNA sequences are anti-parallel to 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 I at temperature of 37°C. The outcome was incomplete pure target product determined by gel electrophoresis. Isolated heat-resistant DNA polymerase from Thermus aquaticus permits the annealing and extension of DNA at higher temperatures; as this DNA polymerase is not denatured at 95°C but works optimally at 72°C. Non-complementary annealing of the primer and template strand is reduced to negligible level.

===Basic PCR Timeline===[2]

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 is discovered (from Thermus aquaticus) which pathed 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 publishers 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.

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 and through a fluorescent detector, to analyze the PCR products based on the fluorescence intensity.

Polymerase Chain Reaction

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

Polymerase Chain Reaction. Three processes in thermal cycling are denaturation, annealing and extension of the DNA.

Amplification of the genetic material requires DNA polymerase, two oligonucleotides primer (where each primer is complementary to one parental of DNA template) and repetitive cycles at three different temperatures known as thermal cycling[3].

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 would be broken. Subsequently, two single-stranded of DNA are generated.
  • Annealing occurs when the temperature of the process is reduced to 50-65°C. As the reaction is cooling down to 50°C, hydrogen bonds are formed between the bases of oligonucleotide primers with the DNA template strand. The primers would anneal to the complementary DNA sequence on the single-stranded DNA template that begins at the 5' end.
  • Extension of the new DNA strand would require the temperature to be raise till 72°C. The new DNA strand is extended by Taq DNA polymerase, an enzyme that polymerizes the additional deoxy-nucleotides (dNTP) in a DNA sequence that is complementary to the DNA template. Taq DNA polymerase functions optimally at 72°C; and with magnesium in the PCR buffer, it would facilitate 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 could 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 could be determined by gel electrophoresis and fluorescent analysis. When fluorescent probes are used, it would sensitize the analytical technique and allowing visualization through the emission of fluorescence signal.The processes involved in fluorescent analysis were fluorescent labelling, gel electrophoresis and detection of fluorescence for quantification.

Structure of Fluorescent Probe PMCID: PMC17763

1) Fluorescent-labelling

Fluorescence probes are added to the amplified genetic molecules after PCR. Examples of fluorescent probes include MB-Green and MB-Red [4]. Fluorescent-labelled primer is an alternative for detection in quantification. Fluorescent probes are used to identify the presence of specific genetic sequence in PCR products. If the fluorescent probe has a complementary DNA sequence to the wild-type PCR product, any mutations present in the DNA sequencing disrupts the hybridization between the probe and amplified product. With different fluorophores in the fluorescent probes, mutation and PCR product could be detected simultaneously after PCR is completed.

Principle of Fluorescent Probe. Labelling illustrated on one DNA template strand, 5' to 3' end.
  • In the labelling process, fluorescent probes or primers constitute as one of the components in the PCR solution. Addition of probes was carried out before the amplification process; where oligonucleotides (dGTP, dTTP, dATP, dATP), Taq ploymerase, magnesium chloride and Tris solution are other essential constituents in PCR solution. PCR was performed in a thermal cycling for amplification at three different temperatures and incubated at room temperature. With the use of fluorescent probes, it provides the benefit of in-situ hybridization where the amplification and labelling were performed in the same well.
  • The principle of the fluorescent labelling is based on the structural formation of the fluorescent probes and 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 has 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 structure where hydrogen bonds are formed between the complementary bases within the oligonucleotide strand. Upon excitation by irradiation at certain wavelength, the fluorescent dye would emit fluorescence through the resonance energy transfer[5]. 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 will be released from the probe after cleavage. Fluorescent dye is not quenched that leads to a subsequent increase in fluorescence intensity, where the degree of quenching is inversely proportional to the distance between the fluorescent dye and quencher. Therefore, amplification of PCR products could be 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 gene scanner. In addition, gel electrophoresis could be used to distinguish the intended amplicon (i.e. amplified PCR product) from the false amplicon based on the molecular size[6]. Unintended amplicons may occur by the hybridization between the primers that generates a false signal. The separation of PCR products is performed on a agarose or polyacrylamide gel immersed within electrophoretic buffer that maintain the pH at a consistent value. Degree of separating the DNA fragments could be adjusted by varying the concentration of the agarose or polyacrylamide gels. PCR products will be separated 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 will be initially diluted in Tris solution and EDTA. The diluted PCR products is mixed with the loading buffer that may be constitute 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 would be used for staining nucleic acids for visualisation with a UV transilluminator. In fluorescent PCR, fluorescence signal is detected upon excitation that obliterates the use of ethidium bromide. Amplified DNA sample are loaded into the sample wells of agarose gel by a pipette. Beside the PCR products, a DNA ladder would also be 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 separation of 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 electric field, the DNA molecules will migrate through the matrix at different rates accounted by its molecular size and charge. Smaller molecules (E.g. DNA) have a greater migration distance than large molecules (E.g. RNA or DNA molecules).
  1. Density gradient agent: Glycerol or sucrose will increase the density of a DNA material so that it will be layered at the base of the agarose gel's sample well for separation.
  2. Tracking dye: Allow visualisation of the DNA sample and monitoring the progress of the electrophoresis.
  3. DNA ladder: Contain DNA molecules with different molecular sizes (in base pairs) to approximate the size of the unknown DNA molecules.

3) Quantification by fluorescent intensity

Quantification may be performed during amplification process or gel electrophoresis. If PCR was performed in the spectrofluorometric thermal cycler, fluorescence intensity could 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 the irradiation for quantification. The fluorescent signal could be enhanced by a photomultiplier and subsequent analyzed by the computer. The fluorescence intensity was the difference in the initial fluorescence and level after the amplification. The gene scanner 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 the incremental amounts of DNA materials result in increased fluorescence.

  • The principle of this quantification process arise from the irradiation (E.g. Laser) emitted from the gene scanner. 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) and excited. Subsequent emission of fluorescence is derived from the dye through resonance energy transfer. This energy transfer will be increased by the distancing the fluorescent dye and the quencher group in the amplification. Quantity of the PCR products is determined by the relative intensities of fluorescence.

Comparison against Conventional PCR

Before we begin to compare fluorescent PCR (F-PCR) against conventional PCR (C-PCR) it is important to recap what we mean by F-PCR and C-PCR. In order to establish quantity and molecular size, PCR cycles have to be completed. In C-PCR agarose or acrylamide gels are used with an electric current to separate products according to size with smaller products moving faster down the gel. However, C-PCR can be made more accurate by labelling primers with fluorescent markers. A sensitive system fluorescent DNA sequencer, also known as a gene scanner can be used to separate, and analyse the F-PCR products, therefore F-PCR allows detection of product without agarose or acrylamide gel electrophoresis.[7]

These are the advantages provided by F-PCR when compared to C-PCR [8]:

  • FPCR is more sensitive to C-PCR by about 1000-fold, therefore F-PCR is more sensitive, accurate (1-2 bp) and reliable than C-PCR as a detection system
  • Results for single cell defects are highly accurate (97-98%) and highly reliable (97%), this allows for multiple diagnosis to be simulataneously within a few hours
  • Less PCR cycles are required in F-PCR for the same level of detection in CPCR, hence allowing diagnosis to be quicker than C-PCR
  • Less loading of product is required for loading (1-1.5 ul), thus it is more efficient than CPCR as it allows more repeated sampling
  • Toxic reagents are reduced as radioactive labelled nucleotides are not needed
  • C-PCR is difficult to use in a developing country in paticular by mobile teams under field conditions as it requires a stable electricity.

Applications of Fluorescent-PCR in Research

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 identifies and quantifies 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 was performed through PCR assays in spectrofluorometric thermal cycler. The amplified retroviral DNA were hybridized to specific fluorescent probes that includes 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 would be crucial for identification of the specific retroviruses. Fluorescence spectrum at 500-650 nm was detected from the assay sample during the annealing phase of the thermal cycle. Quantification of the retro-viral DNA abundance was conducted in real-time, where the intensity of the fluorescence signal was increased significantly with the number of thermal cycles. The reliability of the assay was demonstrated with clinical samples. The retroviruses was identified and false positives were eliminated. Therefore, through the use of fluorescent-PCR in the assay, it enhanced the efficiency and reliability of screening donated blood and transplanted tissue for retroviruses.

Results were plotted with fluorescence intensity of molecular beacons against the number of completed thermal cycles. The colour of the fluorescence acts as the indicator of the of the retroviral DNA that was amplified. In the laboratory samples, the results have shown that there is a positive correlation between the fluorescence intensity and the number of thermal cycles. Increase in fluorescence intensity were observed for all four nucleotide sequences of HIV-1, HIV-2, HTLV-I and HTLV-II. Elevation of fluorescence signal resulted from the higher target copy number of the amplified retrovirus DNA.

In the clinical samples, the retroviruses were correctly identified by the assay indicated by the colour of the fluorescence. In samples of healthy individuals, the assay correctly gave a negative result due to the absence of retrovirus DNA. The PCR assays allow fast and sensitive detection of the amplified retrovirus DNA that is essential for screening of diagnostic samples. [9]

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 looked into the correlation between that of 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, while IGF1R gene expression was assessed by fluorescent in situ hybridization 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, whereas 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.[10]

Clinical application of Fluorescent-PCR (F-PCR) 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 weakness; atrophy and myotonia, it is also and 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 contain 5-30 repeats; mildly affected patients have 50-80 repeats; severely affected have over 200. This repeat was used to diagnose, thus developing a preimplantation genetic diagnosis (PGD) for myotonic dystrophy. When conventional-PCR (C-PCR) was used it was decided that a setback of this assay was the loss of healthy embryos that were misdiagnosed as affected leading to a high allelic dropout (ADO) rate (more than 20%). In comparison,F-PCR ADO rate was found to be much lower because it is more sensitive to C-PCR. Therefore, their study concluded that F-PCR for PGD is superior to conventional PCR due to its superior accuracy leading to a lower ADO rate and subsequently there will be less embryos lost due to misdiagnosis.[11]



  1. Arnheim N, Erlich H. Polymerase Chain Reaction Strategy. Annu. Rev. Biochem. 1992. 61:131-156.
  2. Paul Rabinow. Introduction. Making PCR. The University of Chicago Press, 1996. pp: 1-19
  3. Hauge B, Oggero C, Nguyen N, Fu C, Dong F, 2009 Single Tube, High Throughput Cloning of Inverted Repeat Constructs for Double-Stranded RNA Expression. PLoS ONE 4(9): e7205. doi:10.1371/journal.pone.0007205 Public Library of Science (PLoS One)
  4. Vogelstein B, Kinzler KW. Digital PCR. Proc Natl Acad Sci U S A. 1999 August 3; 96(16): 9236–9241. PMCID: PMC17763 PNAS
  5. Morrison LE. Basic principles of fluorescence and energy transfer applied to real-time PCR. Mol Biotechnol. 2010 Feb;44(2):168-76. Review. PubMed PMID:19950004.PubMed
  6. Vet AM, Majithia AR, Marras AE, Tyagi S, Syamalima D, Poiesz BJ, Kramer FR. Multiplex detection of four pathogenic retroviruses using molecular beacons. Proc. Natl . Acad. Sci . USAVol. 96, pp. 6394 – 6399 Proceedings of the National Academy of Sciences (PNAS)
  7. A J Woolford and J W Dale. Simplified procedures for detection of amplified DNA using fluorescent label incorporation and reverse probing.FEMS Microbiol Lett. 1992 December 1; 78(2-3): 311–316.PubMed
  8. I Findlay and P Quirke.Fluorescent polymerase chain reaction: Part I. A new method allowing genetic diagnosis and DNA fingerprinting of single cells.Hum Reprod Update. 1996 Mar–Apr; 2(2): 137–152.PubMed
  9. Vet AM, Majithia AR, Marras AE, Tyagi S, Syamalima D, Poiesz BJ, Kramer FR. Multiplex detection of four pathogenic retroviruses using molecular beacons. Proc. Natl . Acad. Sci . USAVol. 96, pp. 6394 – 6399 Proceedings of the National Academy of Sciences (PNAS)
  10. Dziadziuszko R, Merrick DT, Witta SE, Mendoza AD, Szostakiewicz B, Szymanowska A, Rzyman W, Dziadziuszko K, Jassem J, Bunn PA Jr, Varella-Garcia M, Hirsch FR.Insulin-like growth factor receptor 1 (IGF1R) gene copy number is associated with survival in operable non-small-cell lung cancer: a comparison between IGF1R fluorescent in situ hybridization, protein expression, and mRNA expression.J Clin Oncol. 2010 May 1;28(13):2174-80.
  11. K Sermon, A De Vos, H Van de Velde, S Seneca, W Lissens, H Joris, M Vandervorst, A Van Steirteghem, and I Liebaers. Fluorescent PCR and automated fragment analysis for the clinical application of preimplantation genetic diagnosis of myotonic dystrophy (Steinert's disease). Mol Hum Reprod. 1998 August; 4(8): 791–796.

2010 Projects

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