Multiple Choice
Which of the following lists the steps of genetic cloning in the proper order?
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18. Molecular Genetic Tools
Genetic Cloning
2:04 minutesProblem 1b
Textbook Question
What steps make PCR a chain reaction that can produce millions of copies of a specific DNA molecule in a matter of hours without using host cells?
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Understand the principle of PCR (Polymerase Chain Reaction): PCR is a technique used to amplify a specific DNA sequence exponentially. It relies on the use of a DNA polymerase enzyme, primers, and a thermal cycler to create millions of copies of the target DNA sequence.
Step 1: Denaturation - Heat the DNA sample to a high temperature (usually around 94-98°C) to break the hydrogen bonds between the two strands of the DNA, resulting in the separation of the double-stranded DNA into single strands.
Step 2: Annealing - Cool the reaction mixture to a lower temperature (typically 50-65°C) to allow short, synthetic DNA primers to bind (anneal) to their complementary sequences on the single-stranded DNA. These primers define the specific region of DNA to be amplified.
Step 3: Extension - Raise the temperature to an optimal level for the DNA polymerase enzyme (usually around 72°C). The DNA polymerase extends the primers by adding nucleotides to synthesize the complementary DNA strand, starting from the primer.
Repeat the cycle - The denaturation, annealing, and extension steps are repeated multiple times (usually 20-40 cycles). Each cycle doubles the amount of DNA, leading to an exponential increase in the number of copies of the target DNA sequence.
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Key Concepts
Here are the essential concepts you must grasp in order to answer the question correctly.
Polymerase Chain Reaction (PCR)
PCR is a molecular biology technique used to amplify specific DNA sequences, generating millions of copies from a small initial sample. It involves repeated cycles of denaturation, annealing, and extension, allowing for exponential amplification of the target DNA. This method is crucial for various applications, including cloning, genetic analysis, and forensic science.
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Denaturation, Annealing, and Extension
These three steps are the core of the PCR process. Denaturation involves heating the DNA to separate its strands, annealing allows primers to bind to the target sequence at lower temperatures, and extension is where DNA polymerase synthesizes new DNA strands by adding nucleotides. This cycle is repeated multiple times to exponentially increase the amount of DNA.
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DNA Polymerase
DNA polymerase is an enzyme essential for DNA replication and amplification. In PCR, a heat-stable variant, such as Taq polymerase, is used to withstand the high temperatures of denaturation. This enzyme synthesizes new DNA strands by adding nucleotides complementary to the template strand, enabling the rapid production of DNA copies during the extension phase of PCR.
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