Presentation on theme: "Research Techniques Made Simple: Polymerase Chain Reaction Lilit Garibyan 1 and Nidhi Avashia 2 1. Department of Dermatology, Massachusetts General Hospital."— Presentation transcript:
Research Techniques Made Simple: Polymerase Chain Reaction Lilit Garibyan 1 and Nidhi Avashia 2 1. Department of Dermatology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, USA 2. Department of Dermatology, Boston University and Boston Medical Center, Boston, Massachusetts, USA
Introduction Widely used for diagnostic and research purposes Allows millions to billions copies of a specific DNA product to be made in vitro Invented in 1983 by Dr. Kary Mullis, for which he received the Nobel Prize The key enzyme in the reaction is DNA Polymerase; therefore it was named Polymerase Chain Reaction=PCR
Application of PCR in Basic and Clinical Research Gene cloning Pathogen identification and diagnosis Drug discovery Gene expression studies Gene sequencing Disease identification Genotyping
Components of the PCR Reaction 1.Target/Template DNA: contains the sequence to be amplified 2.Pair of Primers: oligonucleotides that define the segment of DNA to be amplified 3.Nucleotides: which make up the DNA building block and are composed of adenosine, thymine, cytosine and guanine 4.DNA polymerase: the enzyme which catalyzes the reaction 5.Buffer solution: maintains pH, ionic strength of the reaction solution suitable for the activity of the enzyme
The PCR Reaction PCR MachinePCR Reaction in a Test Tube
PCR: 3 Basic Steps 1.Denaturation: Heated above the melting point of the two complementary strands of the template DNA, which allows the strands to separate. 2.Annealing: The temperature is lowered which allows the primers to bind to the specific and complementary DNA sequence to be amplified 3.Extension: The DNA polymerase is able to extend the primers by adding nucleotides to the developing DNA strand.
Exponential PCR Amplification With each repeat of these three steps, the number of copied DNA molecules is doubled.
Analysis of the PCR Product Two main methods of visualizing the PCR products: 1.Staining of the amplified DNA product using a chemical dye such as ethidium bromide, which intercalates between the two strands of the duplex; or 2.Labeling the PCR primers or nucleotides with fluorescent dyes (fluorophores) prior to PCR amplification. The most widely used method of analysis of the PCR product is via the use of simple agarose gel electorophoresis as shown here From Riedl E, Tada AY, Udey MC. Identification and characterization of an alternatively spliced isoform of mouse Langerin/CD207. J Invest Dermatol 2004. Jul;123(1):78-86.
Qualitative/Quantitative PCR When PCR is used to detect the presence or absence of a specific DNA product, it is referred to as qualitative PCR. Quantitative real-time PCR (qRT-PCR) is able to indicate how much of a specific DNA or gene is present in the sample. –qPCR allows both detection and quantification of the PCR product in real-time as it is being synthesized.
Advantages/Limitations of PCR ADVANTAGES PCR is a very sensitive technique that allows rapid amplification of a specific segment of DNA. Simple to understand and carry out. qPCR has the advantage of quantification of the synthesized product. Thus, it can be used to analyze the alterations of the gene expression levels in tumors, microbes or other disease states. LIMITATIONS The DNA polymerase used in the PCR reaction is error prone and can lead to mutations in the fragment that is generated. The specificity of the generated PCR product might be altered by the nonspecific binding of the primers to other similar sequences on the template DNA. In order to design primers to generate a PCR product, some prior sequence information is usually necessary.