Ch. 17 - Recombinant DNA Technology

Klug - Essentials of Genetics 10th Edition

Klug10th EditionEssentials of GeneticsISBN: 9780135588789Not the one you use?Change textbook

Klug 10th Edition

Ch. 17 - Recombinant DNA Technology

Problem 14

Chapter 17, Problem 14

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?

Verified step by step guidance

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.

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.

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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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

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

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In the context of recombinant DNA technology, of what use is a probe?

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

Textbook Question

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?

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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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