PCR Today we will begin the long process of protein purification by performing PCR (polymerase chain reaction). PCR is a molecular biology technique for enzymatically replicating DNA without using a living organism, such as E. coli or yeast. PCR is commonly used in medical and biological research labs for a variety of tasks, such as the detection of hereditary diseases, the identification of genetic fingerprints, the diagnosis of infectious diseases, the cloning of genes, paternity testing, and DNA computing. PCR is used to amplify specific regions of a DNA strand. This can be a single gene, just a part of a gene, or a non-coding sequence. PCR, as currently practiced, requires several basic components. These components are: • DNA template, which contains the region of the DNA fragment to be amplified. In this case, we will use E. coli genomic DNA. • Two primers, which determine the beginning and end of the region to be amplified (see following section on primers) • Pfx polymerase (or another thermostable polymerase), a heat-stable DNA polymerase, which copies the region to be amplified • Deoxynucleotide triphosphates, (dNTPs) from which the DNA polymerase builds the new DNA • Buffer, which provides a suitable chemical environment for the DNA polymerase The PCR process is carried out in a thermal cycler. This is a machine that heats and cools the reaction tubes within it to the precise temperature required for each step of the reaction. The DNA fragment to be amplified is determined by selecting primers. Primers are short, artificial DNA strands, often not more than 50 and usually only 18 to 25 base pairs long, that are complementary to the beginning or the end of the DNA fragment to be amplified. They anneal by adhering to the DNA template at these starting and ending points, where the DNA polymerase binds and begins the synthesis of the new DNA strand. Designing primers for a PCR reaction is very important and the product yield depends on: • GC-content should be between 40-60%. • Calculated T m for both primers used in reaction should not differ >5°C and T m of the amplification product should not differ from primers by >10°C. • Annealing temperature usually is 5°C below the calculated lower T m . However it should be chosen empirically for individual conditions. • Inner self-complementary hairpins of >4 and of dimers >8 should be avoided. • Primer 3' terminus design is critical to PCR success since the primer extends from the 3' end. The 3' end should not be complementary over greater than 3-4 bases to any region of the other primer (or even the same primer) used in the reaction and must provide correct base matching to template. The PCR process usually consists of a series of twenty to thirty-five cycles. Each cycle consists of three steps (Fig. 1). 1. The double-stranded DNA has to be heated to 94-96°C (or 98°C if extremely thermostable polymerases are used) in order to separate the strands. This step is called denaturing; it breaks apart the hydrogen bonds that connect the two DNA strands. Prior to the first cycle, the DNA is often denatured for an extended time to ensure that both the template DNA and the primers have completely separated and are now single-strand only. Time: usually 1-2 minutes, but up to 5 minutes. Also, certain polymerases are activated at this step (see hot-start PCR). 2. After separating the DNA strands, the temperature is lowered so the primers can attach themselves to the single DNA strands. This step is called annealing. The temperature of this stage depends on the primers and is usually 5°C below their melting temperature (45-60°C). A wrong temperature during the annealing step can result in primers not binding to the template DNA at all, or binding at random. Time: 1-2 minutes. 3. Finally, the DNA polymerase has to copy the DNA strands. It starts at the annealed primer and works its way along the DNA strand. This step is called elongation. The elongation temperature depends on the DNA polymerase. The time for this step depends both on the DNA polymerase itself and on the length of the DNA fragment to be amplified. As a rule-of-thumb, this step takes 1 minute per thousand base pairs. A final elongation step is frequently used after the last cycle to ensure that any remaining pieces of single stranded DNA are completely copied. This differs from all other elongation steps only in that it is longer, typically 10-15 minutes. Figure 1: Schematic drawing of the PCR cycle. (1) Denaturing at 94-96°C. (2) Annealing at (eg) 68°C. (3) Elongation at 72°C (P=Polymerase). (4) The first cycle is complete. The two resulting DNA strands make up the template DNA for the next cycle, thus doubling the amount of DNA duplicated for each new cycle. The polymerase chain reaction is not perfect, and errors and mistakes can occur. These are some common errors and problems that may occur. Taq polymerase lacks a 3' to 5' exonuclease activity. This makes it impossible for it to check the base it has inserted and remove it if it is incorrect, a process common in higher organisms. This in turn results in a high error rate of approximately 1 in 10,000 bases, which, if an error occurs early, can alter large proportions of the final product. PCR works readily with DNA of lengths two to three thousand basepairs, but above this length the polymerase tends to fall off and the typical heating cycle does not leave enough time for polymerization to complete. It is possible to amplify larger pieces of up to 50,000 base pairs, with a slower heating cycle and special polymerases. These special polymerases are often polymerases fused to a DNA-binding protein, making them literally "stick" to the DNA longer. The non-specific binding of primers is always a possibility due to sequence duplications, non-specific binding and partial primer binding, leaving the 5' end unattached. This is increased by the use of degenerate sequences or bases in the primer. Manipulation of annealing temperature and magnesium ion (which stabilize DNA and RNA interactions) concentrations can increase specificity. Non-specific priming can be prevented during the low temperatures of reaction preparation by use of "hot-start" polymerase enzymes where the active site is blocked by an antibody or chemical that only dislodges once the reaction is heated to 95˚C during the denaturation step of the first cycle. Polymerase Chain Reaction Protocol: 1. Add the following reagents to a sterile PCR tube and keep it on ice: Component Volume Final Concentration 10X Pfx Amplification Buffer 5 μL 1X 10 mM dNTP mixture 1.5 μL 0.3 mM each 50 mM MgSO 4 1 μL 1 mM Primer mix (10 μM each) 1.5 μL 0.3 μM each Template DNA (10 pg - 200 ng) 1 μL As required Platinum Pfx DNA Polymerase 1 μL 1.0-2.5 units Autoclaved, distilled water to 50 μL Add water to the PCR tube first and then add the other components. As you add each component make sure the pipette tip enters the water and slowly pipette up and down two times to ensure efficient transfer. The DNA polymerase should be the last component added and can be obtained from the instructor. 2. Mix tube contents carefully. 3. Cap the tube and centrifuge briefly to collect the contents. 4. Denature the template for 2 min at 94°C. Perform 35 cycles of PCR amplification as follows: Three-step cycling Denature: 94°C for 1 min Anneal: 55°C for 30 s Extend: 68°C for 1 min 5. Maintain the reaction at 4°C after cycling. Samples can be stored at -20°C until use. 6. Analyze the products by agarose gel electrophoresis. 7. Put your sample in the freezer. . PCR Today we will begin the long process of protein purification by performing PCR (polymerase chain reaction). PCR is a molecular biology. computing. PCR is used to amplify specific regions of a DNA strand. This can be a single gene, just a part of a gene, or a non-coding sequence. PCR, as currently