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Ch. 20 - Recombinant DNA Technology
Klug - Concepts of Genetics 12th Edition
Klug12th EditionConcepts of Genetics ISBN: 9780135564776Not the one you use?Change textbook
All textbooksKlug 12th EditionCh. 20 - Recombinant DNA TechnologyProblem 20
Chapter 20, Problem 20

We usually think of enzymes as being most active at around 37°C, yet in PCR the DNA polymerase is subjected to multiple exposures of relatively high temperatures and seems to function appropriately at 65–75°C. What is special about the DNA polymerase typically used in PCR?

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1
Understand that the DNA polymerase used in PCR is not the typical enzyme found in most organisms, which usually function best around 37°C (human body temperature).
Recognize that PCR requires repeated heating steps (denaturation at around 95°C), so the DNA polymerase must be stable and active at higher temperatures (65–75°C for annealing and extension).
Identify that the DNA polymerase used in PCR is derived from thermophilic bacteria, such as Thermus aquaticus, which naturally live in hot environments like hot springs.
Know that this thermophilic DNA polymerase is heat-stable, meaning it does not denature at high temperatures and can maintain its enzymatic activity during the PCR cycles.
Conclude that the special property of the DNA polymerase used in PCR is its thermostability, allowing it to function effectively at elevated temperatures required for the PCR process.

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Key Concepts

Here are the essential concepts you must grasp in order to answer the question correctly.

Thermostability of DNA Polymerase

Thermostability refers to an enzyme's ability to remain stable and functional at high temperatures. The DNA polymerase used in PCR, such as Taq polymerase, is derived from thermophilic bacteria and can withstand repeated heating cycles without denaturing, allowing it to synthesize DNA efficiently at elevated temperatures.
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PCR Thermal Cycling

PCR involves repeated cycles of heating and cooling to denature DNA, anneal primers, and extend new DNA strands. The high temperatures (usually 65–75°C for extension) require an enzyme that can function optimally under these conditions, which is why a thermostable DNA polymerase is essential for the process.
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Source of DNA Polymerase in PCR

The DNA polymerase used in PCR is typically isolated from thermophilic organisms like Thermus aquaticus. These organisms live in hot environments, so their enzymes have evolved to function at high temperatures, making their polymerases ideal for PCR applications.
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Related Practice
Textbook Question

Although the capture and trading of great apes has been banned in 112 countries since 1973, it is estimated that about 1000 chimpanzees are removed annually from Africa and smuggled into Europe, the United States, and Japan. This illegal trade is often disguised by simulating births in captivity. Until recently, genetic identity tests to uncover these illegal activities were not used because of the lack of highly polymorphic markers (markers that vary from one individual to the next) and the difficulties of obtaining chimpanzee blood samples. A study was reported in which DNA samples were extracted from freshly plucked chimpanzee hair roots and used as templates for PCR. The primers used in these studies flank highly polymorphic sites in human DNA that result from variable numbers of tandem nucleotide repeats. Several offspring and their putative parents were tested to determine whether the offspring were 'legitimate' or the product of illegal trading. The data are shown in the following Southern blot.

Examine the data carefully and choose the best conclusion.

a. None of the offspring is legitimate.

b. Offspring B and C are not the products of these parents and were probably purchased on the illegal market. The data are consistent with offspring A being legitimate.

c. Offspring A and B are products of the parents shown, but C is not and was therefore probably purchased on the illegal market.

d. There are not enough data to draw any conclusions. Additional polymorphic sites should be examined.

e. No conclusion can be drawn because 'human' primers were used.

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Textbook Question

To estimate the number of cleavage sites in a particular piece of DNA with a known size, you can apply the formula N/4ⁿ where N is the number of base pairs in the target DNA and n is the number of bases in the recognition sequence of the restriction enzyme. If the recognition sequence for BamHI is GGATCC and the λ\lambda phage DNA contains approximately 48,500 bp, how many cleavage sites would you expect?

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Textbook Question

In a typical PCR reaction, describe what is happening in stages occurring at temperature ranges

(a) 92-26 °C

(b) 45-65 °C and

(c) 65-75 °C

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Textbook Question

Traditional Sanger sequencing has largely been replaced in recent years by next-generation and third-generation sequencing approaches. Describe advantages of these sequencing methods over first-generation Sanger sequencing.

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Textbook Question

How is fluorescent in situ hybridization (FISH) used to produce a spectral karyotype?

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Textbook Question

What is the difference between a knockout animal and a transgenic animal?

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