Chapter 20: Problem 11
In the context of recombinant DNA technology, of what use is a probe?
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Chapter 20: Problem 11
In the context of recombinant DNA technology, of what use is a probe?
These are the key concepts you need to understand to accurately answer the question.
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In humans, congenital heart disease is a common birth defect that affects approximately 1 out of 125 live births. Using reverse transcription PCR (RT- PCR) Samir Zaidi and colleagues I(2013) Nature 498: 220.2231 determined that approximately 10 percent of the cases resulted from point mutations, often involving histone function. To capture products of gene expression in developing hearts, they used oligo(d'T) in their reverse transcription protocol. (a) How would such a high \(96 \mathrm{T}\) in a primer influence annealing temperature? (b) Compared with oligo(d'T) primers, a pool of random sequence primers requires a trickier assessment of annealing temperature. Why? (c) If one were interested in comparing the quantitative distribution of gene expression in say, the right and left side of a developing heart, how might one proceed using RT-PCR?
You have recovered a cloned DNA segment from a vector and determine that the insert is 1300 bp in length. To characterize this cloned segment, you isolate the insert and decide to construct a restriction map. Using enzyme I and enzyme II, followed by gel electrophoresis, you determine the number and size of the fragments produced by enzymes I and II alone and in combination, as recorded in the following table. Construct a restriction map from these data, showing the positions of the restriction-enzyme cutting sites relative to one another and the distance between them in units of base pairs. \(\begin{array}{cc}\text { Enzyme } & \text { Restriction Fragment Sizes (bp) } \\\ \text { I } & 350,950 \\ \text { II } & 200,1100 \\ \text { I and II } & 150,200,950\end{array}\)
A widely used method for calculating the annealing temperature for a primer used in PCR is 5 degrees below the melting temperature, \(T_{m}\left(^{\circ} \mathrm{C}\right),\) which is computed by the equation \(81.5+0.41 \times(\% \mathrm{GC})-(675 / N),\) where \(96 \mathrm{GC}\) is the percentage of GC nucleotides in the oligonucleotide and \(N\) is the length of the oligonucleotide. Notice from the formula that both the GC content and the length of the oligonucleotide are variables. Assuming you have the following oligonucleotide as a primer, $$5'-TTGAAAATATTTCCCATTGCC-3'$$ compute the annealing temperature for PCR. What is the relationship between \(T_{m}\left(^{\circ} \mathrm{C}\right)\) and \(\% \mathrm{GC} ?\) Why? (Note: In reality, this computation provides only a starting point for empirical determination of the most useful annealing temperature.)
If you performed a PCR experiment starting with only one copy of double- stranded DNA, approximately how many DNA molecules would be present in the reaction tube after 15 cycles of amplification?
In a typical PCR reaction, describe what is happening in stages occurring at temperature ranges (a) \(92-95^{\circ} \mathrm{C},\) (b) \(45-65^{\circ} \mathrm{C},\) and (c) \(65-75^{\circ} \mathrm{C}\)
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