Researchers are screening bacterial chromosomes for mutations affecting protein function. After using DNA sequencing analysis and analyzing the gel electrophoresis results, they identify a sequence with a single point mutation in which the fourth nucleotide has been changed from adenine to cytosine. The sequence is shown below.5′-TTACG-3′Which of the following gel electrophoresis diagrams obtained from DNA sequencing analysis represents this sequence?

Question

Researchers are screening bacterial chromosomes for mutations affecting protein function.  After using DNA sequencing analysis and analyzing the gel electrophoresis results, they identify a sequence with a single point mutation in which the fourth nucleotide has been changed from adenine to cytosine.  The sequence is shown below.5′-TTACG-3′Which of the following gel electrophoresis diagrams obtained from DNA sequencing analysis represents this sequence?

Quizplus · Accepted Answer

11ecba2d_1013_2eeb_a3bf_7317f9ca167c_MD0008_00 Researchers use DNA sequence analysis to determine the specific nucleotide order of a DNA molecule. One method of DNA sequence analysis is the dideoxy (Sanger) sequencing method, which is carried out as follows: The target DNA is isolated from cells or tissue and amplified using PCR or gene cloning to ensure that an adequate amount of DNA is available for the sequencing process. The amplified DNA sample is split into four separate PCR tubes, each containing a primer complementary to the DNA sequence of interest, DNA polymerase, and the four deoxyribonucleotides (dNTPs). In addition, each tube contains one fluorescently labeled (or radiolabeled) dideoxynucleotide (ddNTP): ddATP, ddTTP, ddGTP, or ddCTP. Unlike dNTPs, ddNTPs lack a free hydroxyl group and are unable to form phosphodiester bonds with additional nucleotides, terminating DNA replication. Therefore, differential incorporation of ddNTPs (in the 5′ to 3′ direction) during PCR generates DNA fragments of varying lengths. Gel electrophoresis is used to separate the fragments by size, and bands are visualized via fluorescence or radiolabeling. Because longer fragments migrate slower than shorter fragments in a gel, early incorporation of ddNTPs will result in shorter fragments that migrate farther in the gel, and later incorporation of ddNTPs will result in longer fragments that travel a shorter distance in the gel. The bottom band in the gel represents the first nucleotide at the 5′ end of the sequence, and the top band represents the last nucleotide at the 3′ end of the sequence. In the given scenario, researchers have used DNA sequencing analysis to identify a mutated sequence of bacterial DNA. The mutated sequence (5′-TTACG-3′) is represented by the gel given in Choice B. (Choice A) This gel shows the sequence of interest with an additional point mutation, a single base pair change in a DNA molecule. The second T has been changed to a G (pyrimidine to purine). (Choice C) The sequence identified in this gel is complementary to the mutated sequence. (Choice D) This gel depicts the sequence in the wrong orientation (3′ to 5′). Educational objective: The Sanger (dideoxy) method of DNA sequencing analysis uses PCR along with the incorporation of ddNTPs and gel electrophoresis to determine the nucleotide order of target DNA molecules.

Researchers Are Screening Bacterial Chromosomes for Mutations Affecting Protein Function