Polymerase Chain Reaction (PCR) was invented by Kary Mullis in 1983. This technique had indeed facilitated research in various areas of molecular biology and genetics. You would like to amplify a particular gene fragment from the yeast genome using Polymerase Chain Reaction (PCR). What are the THREE (3) main cycles in PCR? Discuss the processes at each PCR cycle mentioned.
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- The target sequence for a PCR amplification is shown below. When the final cycle of PCR has ended, the resulting amplicon will be how many base pair (bp) long? 5' ACGTGCGAGCACGTATATATGTCGCGTGCTGAGTGTAGCGTATGCGAATCACCGC 3' 3' TGCACGCTCGTGCATATATACAGCGCACGACTCACATCGCATACGCTTAGTGGCG 5' 31bp 39bp 42bp 47bp None of the above. In the PCR process, if we assume that each cycle takes5 minutes, how manyfold amplification would be accomplished in 1 hour?The temperature at which the primers and target DNA hybridize may be changed to influence the stringency of PCR amplification. What effect will changing the hybridization temperature have on the amplification? Let's say you have a certain yeast gene A and want to check whether it has a human equivalent. How might managing the hybridization's rigor benefit you?
- The exponential nature of PCR allows spectacular increases in the abundanceof a DNA sequence being amplified. Consider a 10-kbp DNA sequence in agenome of 1010 base pairs. What fraction of the genome does this sequence represent? That is, 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.You want to amplify the following sequence of DNA from a gene of interest that you are studying. To do this, you will use PCR. Design a forward and reverse primer. Show where the primers anneal to the template sequence. Template DNA: 5’ ATGACGGAATATAAGCTGGTGGTGGTGG---GGCTGCATGAGCTGCAAGTGTGTGCTCTCCTAA 3’ 3’ TACTGCCTTATATTCGACCACCACCACC---CCGACGTACTCGACGTTCACACACGAGAGGATT 5’ (PLEASE EXPLAIN STEP - BY STEP) Answer : Foward primer 5": Reverse primer 3"In the very first round of PCR using genomic DNA, the DNA primers prime synthesis that terminates only when the cycle ends (or when a random end of DNA is encountered). Yet, by the end of 20 to 30 cycles - a typical amplification - the only visible product is defined precisely by the ends of the DNA primers. Explain how.
- PCR errors during library amplification are one possible source of false positive results. If an error occurs in the first round of amplification, all the subsequent copies of that library fragment will also carry the variant, even though it is not present in the genome. However, reads from other library fragments spanning this same region will not have the variant, which means that identifying PCR copies, or clones, of library fragments during analysis can help to identify these types of errors. Based on what you know about PCR, which of the following statements would be true about the PCR clones in a sequencing library? A. PCR is subject to amplification bias, so reads derived from PCR clones will only map to regions that are not GC-rich. B. PCR is subject to amplification bias, so reads derived from PCR clones will only map to non-repetitive regions. C. PCR produces identical copies, so reads derived from PCR clones would map to the exact same location. D. PCR introduces many…Running a PCR Why is a Hotstart necessary and how is it achieved? Why are the three temperatures necessary for PCR? Explain the role of each. Why does the lid of the thermocycler need to be heated? Why did we conduct an amplification reaction with no DNA? When looking at your PCR results why do you only see one band? What size were each of your PCR products?What is the difference between reverse transcriptase PCR(RT-PCR) and standard PCR? For what purpose would youuse RT-PCR?
- Mention all the enzymes and reagents used in the everse Transcriptase Polymerase Chain Reaction (RT-PCR) technique, with their functions.Analyze the PCR products in Fig. 1A by describing your observations and the PCR result. Your analysis should be in maximum of 5 sentences in bullet form. Include the following in your analysis: Observations: Are bands present? If yes, what the approximate size in bp of each band? Results: Was amplifying the target gene successful?After four cycles of PCR, which type of PCR product predominates? Explain why.