Why is it necessary to have a primer on each side of the DNA segment to be ampli
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Why is it necessary to have a primer on each side of the DNA segment to be amplified? How did taq DNA polymerase acquire its name? Why are there nucleotides (A, T, G and C) in the master mix? What are the other components of the master mix, and what are their functions? Describe the three main steps of each cycle of PCR amplification and what reactions occur at each temperature. Explain why the precise length target DNA sequence doesn't get amplified until the third cycle. You may need to use additional paper and a drawing to explain your answer.Explanation / Answer
6) Ans: The sides of a DNA molecule are antiparallel, that is, they run in opposite directions. the sugar molecule of one base pair compared to the other is flipped over--so that the two sides of a DNA chain are replicated from different directions. Since DNA polymerase operates from the 5' (phosphate) to the 3' (sugar) end, this means that two different DNA primers are needed--one for each 5' end of side of the molecule.A second primer with sequences that are complementary to the end of the targeted segment is added to the PCR brew. In this way it is possible to create short segments of "target DNA" that include only the marker of interest and a bit of its flanking region on either end.
7)Ans:The Taq name is a shortened for Thermophilus aquaticus, a thermophilic bacteria that is the source of the particular DNA polymerase enzyme.The primers are two short single-stranded DNA molecules (23 bases long), one that is complementary to a portion of one strand of the template, and another that is complementary to a portion of the opposite strand. These primers anneal to the separated template strands and serve as starting points for DNA Taq replication by DNA polymerase. Taq DNA polymerase extends the annealed primers by “reading” the template strand and synthesizing the complementary sequence. In this way, Taq polymerase replicates the two template DNA strands. This polymerase was isolated from a heat-stable bacterium (Thermus aquaticus) which in nature lives within high temperature steam vents such as those found in Yellowstone National Park.6 For this reason these enzymes have evolved to withstand high temperatures (94°C) and can be used in the PCR reaction.
8)Ans: Because master mix containing enough of the reagents to perform PCR on all of your samples. The master mix is then aliquoted into separate PCR tubes, DNA is added and the tubes are placed into a thermalcycler to perform the DNA replication. Following the reaction, the PCR products will be visualized on an agarose gel.
Template DNA(target gene) , PCR Buffer , PCR primers, are the other components of master mix .
master mix is made in which enough of each reagent to satisfy all reactions is combined into one tube, and then dispensed among the individual reaction tubes.Template DNA is then added to each tube. This limits pipetting errors as well as the potential for contamination, as the reagent tubes are opened and dispensed from only one time per reaction set-up.
9)Ans:Amplification of a DNA sequence typically involves 20-30 cycles of PCR and each cycle of PCR requires three temperature shifts. These repeated cycles of temperature shifts are usually done automatically in a thermocycler, a machine that sequentially shifts between the desired temperatures and remains at each temperature for a specified length of time.
1)Denaturation:Denaturation for 45 seconds at 94-95°C is sufficient to completely denature the DNA synthesized in the first amplification cycle. If the amplified DNA has a veryhigh GC content, denaturation time may be increased up to 3-4 minutes.
2.)Primer annealing: The annealing step lasts for approximately 45 seconds, which allows enough time for the primers to locate their complement and anneal to the template DNA strand. The optimal annealing temperature (TA) is commonly 5°C lower than themelting temperature of primer-template DNA duplex. In most cases the TA is between 50 and 65o C. However, if nonspecific PCR products are obtained in addition to the expected product, the annealing temperature should be optimized by increasing it stepwise by 1-2o C.
3.Extension: The processivity of Taq DNA polymerase is approximately 150 nucleotides sec-1. Based on this estimate, the extension time necessary to synthesize copies of the 16S rRNA gene (1500 bp) is 10 seconds. However, reaction conditions, template quality, and the presence of inhibitors require that the extension time be increased, usually to between 45-60 seconds for 1500 bp. When larger DNA fragments are amplified, the extending time is usually increased by 1min for each 1000 bp. The extending step is commonly performed at 70-75°C, as the rate of DNA synthesis by Taq DNA polymerase is highest at this temperature.
10.)Ans:The enzyme DNA polymerase then adds nucleotide bases to the end of each primer, using the template DNA as a guide to extend the primer thereby producing new double stranded DNA. This process is repeated for a number of cycles to enrich the DNA sample for the desired genes targeted by the oligonucleotide primers. Since each cycle of PCR involves creating two new double stranded DNAs from each DNA molecule present, the amount of DNA theoretically doubles with every cycle of PCR. Therefore, after two cycles the concentration of DNA increases by 22-fold, after 3 cycles a 23-fold increase, etc. After N cycles, PCR generates a 2N-fold increase in the target DNA.PCR allows the production of more than 10 million copies of a target DNA sequence from only a few molecules. Therefore, PCR is very sensitive to contamination from non-target DNA.
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