Group 1 Project - Fluorescent-PCR

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Introduction

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 gel electrophoresis. Gene amplification accounts for the high sensitivity of PCR where single copies of genes could be analyzed. 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

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, however it would be relevant to understand the underlying mechanism of the conventional PCR. The basic principles of amplification in PCR evolved from the knowledge about DNA replication and denaturation.

Polymerase Chain Reaction

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.

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.

The processes involved in fluorescent analysis were fluorescent labelling, gel electrophoresis and detection of fluorescence for quantification.

1) Fluorescent-labelling

Fluorescence probes are added to the amplified genetic molecules after PCR. Examples of fluorescent probes include MB-Green and MB-Red (Vogelstein, 1999). 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.

In Situ Fluorescent PCR PMCID: PMC17763
  • 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.


2) Gel Electrophoresis

Gel electrophoresis is used for separation of the amplified genetic product according to its molecular size.

3) Quantification by fluorescent intensity

For fluorescence analysis, the fluorescence intensity of MB-Green was read at excitation wavelength of 485/530 nm while MB-Red was read at 530/590 nm. The fluorescence intensity was the difference in the initial fluorescence and level after the amplification.


Comparison against Conventional PCR

Applications of Fluorescent-PCR in Research

References

Websites

  • http://en.wikipedia.org/wiki/PCR - under the "variations" heading there is an explanation of the different types of PCR, and i'm pretty sure ours falls under "quantitative PCR"

Papers

  1. P W Chiang, W J Song, K Y Wu, J R Korenberg, E J Fogel, M L Van Keuren, D Lashkari, and D M Kurnit.Use of a fluorescent-PCR reaction to detect genomic sequence copy number and transcriptional abundance. Genome Res. 1996. 6: 1013-1026 [Paper 1 | http://genome.cshlp.org/content/6/10/1013]
  2. 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.[Paper 2 | http://www.ncbi.nlm.nih.gov/pubmed/19950004]
  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 [Paper 3 | http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0007205]
  4. Vogelstein B, Kinzler KW. Digital PCR. Proc Natl Acad Sci U S A. 1999 August 3; 96(16): 9236–9241. PMCID: PMC17763 [Paper 4 | http://www.ncbi.nlm.nih.gov/pmc/articles/PMC17763/]


Images

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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