Briefly explain the rationales of adding chemicals that can affect DNA stability in a polymerase chain reaction (PCR). Why DNA melting is required in PCR? Briefly explain how PCR can be used to detect DNA mutation.
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- Briefly explain the rationales of adding chemicals that can affect DNA stability in a polymerase chain reaction (PCR). Why DNA melting is required in PCR? Briefly explain how PCR can be used to detect DNA mutation.
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- Explain why DNA ladders are usually included during gel electrophoresis. One aspect of PCR that can be modified is the annealing temperature. In general, higher annealing temperatures show more specificity towards a single template, whereas lower annealing temperatures show less specificity and may bind to multiple regions throughout the genome. Discuss how using an annealing temperature that is too high or too low might influence the results of a PCR assay (and gel electrophoresis results) such as the one used in this study.Why DNA melting is required in PCR? Briefly explain how PCR can be used to detect DNA mutation.Briefly explain the rationales of adding chemicals which can affect DNA stability in polymerase chain reaction (PCR).
- The process of PCR essentially revolves around three phases. Briefly describe these phases and the events that occur in them. Take note the temperature on which these phases take place.Briefly describe what happens during each of the phases of PCR (denaturation, annealing, and extension), including when you need each of the major components of the reaction (template, primers, nucleotides, DNA polymerase).Examine the DNA fragment sequence below. Your job is to design primers for PCR that would be able to amplify this DNA fragment. Design the primers so that they are 7 bases in length. Don’t forget to indicate direction (polarity) of the primers. Also describe where the primer would bind (i.e. top or bottom strand, left or right side of the DNA strand). Please organize your response so that each primer, and associated information, is separated by at least one blank line 5’ - TCCACTTGCTGTGTAGCTAAATCATATAACAG3’ - AGGTGAACGACACATCGATTTAGTATATTGAC
- Explain how the percentage efficiency of a real-time PCR reaction is determined using a theoretical experiment and why this is essential in any real-time PCR analysis.The exponential nature of PCR allows spectacular increases in the abundance of a DNA sequence being amplified. Consider a 10-kbp DNA sequence in a genome of 1010 base pairs. What fraction of the genome is represented by this sequence; i.e., what is the fractional abundance of this sequence in this genome? Calculate the fractional abundance of this target sequence after 10, 15, and 20 cycles of PCR, starting with DNA representing the whole genome and assuming that no other sequences in the genome undergo amplification in the process.PCR is a powerful technique to screen and amplify segments of DNA for use in recombinant protein technologies. Describe in detail, the components of a PCR reaction and why they are required
- PCR is quick, efficient and easy to perform. However, there are some situations when cell-based cloning is preferred over PCR to amplify a DNA sequence. Mention two of them.Explain why a positive control and negative control are included in PCR experiments. Explain the three steps involved in each cycle of polymerase chain reaction.Why is loading dye added to the DNA sample for gel electrophoresis? Explain the function of the following components in a PCR reaction:− Primer, dNTP, MgCl, Taq polymerase, buffer.Briefly present experimental and practical benefits of using PCR in DNA cloning process. Give some examples of clinical applications.