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Question 1:

What is the purpose of Sanger sequencing?

A) Determining the size of DNA fragments
B) Amplifying specific DNA sequences
C) Identifying genetic mutations
D) Determining the nucleotide sequence of DNA

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Explanation: The correct answer is D) Determining the nucleotide sequence of DNA. The purpose of Sanger sequencing is to determine the nucleotide sequence of a DNA fragment. Sanger sequencing, also known as chain termination sequencing, uses DNA synthesis with chain-terminating dideoxynucleotides to generate a set of DNA fragments of varying lengths. These fragments are then separated by size through gel electrophoresis, and the sequence is determined by reading the bands of different nucleotides at each position.

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Question 2:

What is the role of dideoxynucleotides in Sanger sequencing?

A) To amplify the DNA fragments
B) To label the DNA fragments
C) To terminate DNA synthesis
D) To digest the DNA fragments

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Explanation: The correct answer is C) To terminate DNA synthesis. In Sanger sequencing, dideoxynucleotides (ddNTPs) are incorporated during DNA synthesis. These ddNTPs lack a 3'-OH group required for the formation of a phosphodiester bond with the next nucleotide, leading to termination of DNA synthesis. By including low concentrations of ddNTPs along with regular dNTPs, DNA synthesis stops randomly at different positions, resulting in a series of terminated fragments that represent the DNA sequence.

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Question 3:

What is the role of DNA polymerase in Sanger sequencing?

A) To amplify the DNA fragments
B) To label the DNA fragments
C) To terminate DNA synthesis
D) To generate new DNA strands

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Explanation: The correct answer is D) To generate new DNA strands. DNA polymerase is an essential enzyme in Sanger sequencing. It catalyzes the synthesis of new DNA strands complementary to the template DNA. DNA polymerase incorporates nucleotides during DNA synthesis, extending the growing DNA strand. In the presence of both regular dNTPs and low concentrations of ddNTPs, DNA polymerase randomly incorporates ddNTPs, leading to termination of DNA synthesis and the generation of terminated fragments of different lengths.

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Question 4:

How are the DNA fragments separated in Sanger sequencing?

A) By size exclusion chromatography
B) By gas chromatography
C) By capillary electrophoresis
D) By liquid chromatography

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Explanation: The correct answer is C) By capillary electrophoresis. In Sanger sequencing, the DNA fragments generated through DNA synthesis with ddNTPs are separated by capillary electrophoresis. Capillary electrophoresis uses an electric field to move charged molecules, such as DNA fragments, through a capillary filled with a gel matrix. The gel matrix helps separate the DNA fragments based on their size, with shorter fragments moving faster than longer fragments. The separated fragments are then detected and analyzed to determine the DNA sequence.

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Question 5:

What is the role of fluorescent dyes in Sanger sequencing?

A) To amplify the DNA fragments
B) To label the DNA fragments
C) To terminate DNA synthesis
D) To digest the DNA fragments

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Explanation: The correct answer is B) To label the DNA fragments. Fluorescent dyes play a crucial role in Sanger sequencing by labeling the DNA fragments. Each ddNTP used in the sequencing reaction is tagged with a different fluorescent dye. As the terminated DNA fragments are separated by capillary electrophoresis, the fluorescent dyes emit light of different wavelengths when excited by a laser. The emitted light is then detected, and the sequence is determined based on the order of the emitted fluorescent signals from the labeled DNA fragments.