Biochemical Techniques Biochemistry 24 Polymerase Chain Reaction Description of Module Subject Name Biochemistry Paper Name 12 Biochemical Techniques Module Name/Title 24 Polymerase Chain Reaction Biochemical Techniques Biochemistry 24 Polymerase Chain Reaction 1. Objectives 1.1 To understand principle of Polymerase chain reaction (PCR) technique. 1.2 To explain how this techniques is performed. 1.3 What are helpful hints which help in successful performance of PCR technique? 1.4 What are potential applications of PCR? 1.5 What are different variants of PCR? 2.0 Introduction and principle Polymerase chain reaction (PCR) can be recognized as a revolutionary technology for which the inventor Kary B. Mullis was awarded with the prestigious Nobel Prize in 1993 for chemistry along with Michael Smith. The secret story of any living organism which gives it unique characteristics is coded within the large and complexed DNA of that individual. To understand the story, it was needed to bring the DNA outside the living cell into the laboratory, this gave birth to the concept of PCR. Kary Mullis conceptualized of a machine through which from a single molecule of DNA we can get multiples of its copy after the end, like a Xerox machine does. With this in 1983 Kary Mullis developed PCR which is now a most generalized technique used in medical or biological science research labs for a range of applications. Examples are its use in DNA cloning, phylogeny, functional analysis of genes, diagnosis of diseases, genetic fingerprints for forensic sciences as paternity test where the aim is to amplify a single or a few copies of a specific DNA segment in generating thousands to millions of copies of that specific DNA sequence. The PCR techniques currently available can amplify DNA from a wide range of samples as small as hair, blood spot, and ancient biological samples. 3.0 Principle The principle behind PCR is based on using the ability of DNA to replicate into a new strand of DNA complementary to the previous template strand. PCR differs from the replication in the sense that here only a fragment of DNA is multiplied several time to get a multiple copy of the fragment. Biochemical Techniques Biochemistry 24 Polymerase Chain Reaction The PCR is a chain reaction of thermal cycling, which consists of repeated heating and cooling steps of reaction involving 3 basic steps 1. Denaturation 2. Annealing 3. Elongation 3.1 Denaturation: Denaturation step is the first regular step of PCR and is consists of heating the reaction mixture containing template DNA to a temperature around 94–98 °C for 20–30 seconds. This results in breakage of the hydrogen bonds between complementary bases A-T and G-C and leads to denaturation of. Consequently, DNA is unzipped to open and single- stranded DNA molecules are obtained. The time of denaturation should be kept optimum such that the complete portion of the DNA strand to be amplified would be unzipped. 3.2 Annealing: Biochemical Techniques Biochemistry 24 Polymerase Chain Reaction In the next step gradually the temperature of reaction mixture is lowered down to around 50–65 °C for 15–60 seconds which allows the DNA molecules to reanneal but during this step the short stretches of ss oligonucleotides (primers) added in the reaction mixture incorporates and get annealed to the single-stranded DNA template. The temperature selected for this step must be low enough to allow for hybridization of the primer to the strand, but should be higher enough to let the binding be sequence specific between template and primer. In other words, primer can only bind to a perfectly complementary sequence of the template. If the temperature is selected very low, the primer would bind nonspecifically. When annealing temperature is kept too high, primer will fail to anneal as H-bonds cannot form. In general, the annealing temperature is adjusted about 3–5 °C below the Tm of the primers. Tm is the temperature at which 50% of the oligonucleotide and its perfect complement are in duplex. It is dependent on the length of the DNA molecule and its specific nucleotide sequence. Theoretically, Tm = {4(G+C) + 2(A+T)}. Now the polymerase can bind to the primer-template hybrid and further starts formation of new strand complimentary to the template. 3.4 Elongation: This is the step where the polymerase attached to primer-template hybrid starts extending the new strand of DNA by adding specific nucleotides dNTPs which are complementary to the template strand into the developing strand. The temperature is different for different DNA polymerase. Most commonly used polymerase is Taq polymerase which is derived from a thermostable bacterium (Thermus aquaticus) and has its optimum activity at temperature 75–80 °C. However, chain elongation is carried out at 72 °C. The time required for this step depends both on the length of the target DNA to be amplified and on the efficiency of DNA polymerase used and. In general, DNA polymerase can add a thousand bases per minute. At each extension step, the amount of DNA target is doubled. These three steps are repeated in a cyclic process which gives this technique the name Polymerase Chain Reaction which means a chain of reaction involving polymerase. PCR are carried out for 25 to 30 cycles in an automated thermal cycler, which can heat and cool the reaction mixture speedily. It is theoretically calculated that a single molecule of DNA can give 1 billion copies of the template after 30 cycles of PCR. Biochemical Techniques Biochemistry 24 Polymerase Chain Reaction 4.0 Requirements for PCR Any PCR reaction requires the following components for reaction: (i) DNA Template (ii) DNA polymerase (iii) Buffer (iv) dNTPs (v) Primers 4.1 DNA Template: DNA template is the sample DNA that contains the target sequence to be amplified by PCR. At beginning of reaction a high temperature is applied to this template DNA to separate the strand, called as initial denaturation. For PCR a very small quantity of DNA is required as more amount of DNA template will increase the amount of contaminants and reduce the efficiency of PCR. 4.2 DNA polymerase: DNA polymerase is the enzyme required for amplification of template DNA. These enzymes should have: 1) Capability to generate new strands of DNA by using the DNA template and primers, 2) The enzyme should be resistant to high temperature. In these parameters the most common enzyme used for PCR is Taq DNA polymerase (isolated from Thermus aquaticus) as it is better than other polymerase giving good results whereas it is having no 3’ to 5’ proof reading activity and results to low replication fidelity showing an error in 9,000 nucleotides. Other polymerase Pfu DNA polymerase (obtained from Pyrococcus furiosus) is widely used due to its higher fidelity when copying DNA. In addition, Taq also adds an extra A at the 3’ end (it is found useful for TA cloning). 4.3 Buffer: For any enzymatic reaction an optimal reaction condition is needed which is provided by the Buffer solution. For example, buffer used for PCR reaction provides a suitable Biochemical Techniques Biochemistry 24 Polymerase Chain Reaction chemical environment for optimum activity and stability of the DNA polymerase. PCR buffer contains tris-HCl, KCl or MgCl2 and glycerol or Triton X-100 and water. In most cases the PCR reaction buffer is provided along with the DNA polymerase and in 10X concentration, which is diluted to 1X in final reaction volume. 4.4 dNTPs: dNTP (Deoxynucleoside triphosphates) is the basic substrate for PCR reaction which is a mixture of dATP, dTTP, dGTP and dCTP. It acts as the building block for polymerization of new DNA strand. Excess of dNTP can increase rate of error and can also inhibit the Taq. Lower amount of dNTP may reduce rate of reaction. Thus an optimum concentration of dNTP is required to be taken for PCR . Usually dNTP are used at 10-50 µM concentration. If we need to amplify large size of DNA, higher concentration of dNTP is required. 4.5 Primers: Primers are the last but the most important in PCR. They are short pieces of single- stranded DNA about 10 base pair that are complementary to the target sequence. DNA polymerase starts synthesizing new DNA from the end of the primer. Up to 3µM of primers is sufficient. High template to primer ratio may result to non-specific amplification and formation of dimers of primer. In most of PCR a set of 2 primers (forward and reverse) is used for amplifying a specific region of gene. But a number of PCR have been designed which can run with a single primer as RAPD (Rapid Amplification of Polymorphic DNA) where the primer binds to several polymorphic sites in genome and amplifies all of these regions resulting into PCR product of variable sizes. RAPD is successfully used for differentiating plant varieties, animal breeds, and microbial strains. While designing any primer few points are to be considered: i. It should be of a size around 20-30 nucleotide. ii. It should avoid formation of primer-dimer. iii. GC content should be between 40-60%. iv. Tm should be between 55-65 oC. Biochemical Techniques Biochemistry 24 Polymerase Chain Reaction Increasing number of information about DNA sequences over various public database has made it easy to design specific primer sets. A number of free online bioinformatics tools can be used for designing primers and PCR conditions as Primer3, Primer-BLAST etc. 5.0 Instrument: Any PCR reaction is most commonly carried out in a reaction tube (volume 0.2–0.5 ml) covering reaction volume of 10-200 μl in a thermal cycler.