9. If our primers were about 20nt in length. How often would you expect a 20-mer
ID: 275405 • Letter: 9
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9. If our primers were about 20nt in length. How often would you expect a 20-mer oligonucleotide to match a target sequence by chance? A primer designed to amplify one gene could amplify other genes in a more systematic way than by chance. How? 10. When designing primers, you can specify the melt temperature of the primers. The melt temperature is the temperature at which half of the primer-template duplex DNA dissociates to single strands. a. What feature of the primer sequence is the main determinant of melt temperature? b. You are working with a gene that has primers that anneal at 54C. You decide that you want to test an additional gene at the same time in one PCR reaction. Therefore, you need to design primers that will work with those same PCR conditions. When designing primers for the new gene, would you designate the primer melt temperature to be higher or lower than 54C? Explain. In your answer, first explain what is happening in the PCR reaction at 54C.Explanation / Answer
9. the probability of a random primer to match a target sequence by chance would be approximately (1/4)20
However, all the nucleotides in the primer need not match the target sequence. Therefore, the probability would be somewhat lower than the expected.
if the complementary sequence (of the primer) is present in more than one gene, all the genes containing complementary sequences may be amplified. Since 100% complementarity is not required for the binding of the primer to its target sequence, primer can also bind to a region that contains 15 out of 20 complementary nucleotides.
10. GC content of the primer dictates the melting temperature. Therefore, this acts as the main determinant.
Annealing temperature is the phase of PCR where the primers bind to the target sequences.
Annealing temperature is usually set 4-6oC lower than the melting temperature of the primer.
Therefore, 54oC is lower than the melting temperature of the primer.
Hence, the melting temperature should be approximately 5oC higher than 54oC.
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