Question

Whole genomic DNA was isolated from three individuals, digested separately with a restriction endonuclease to fragments and the fragments separated on agarose gel in an electric field. The gene of interest was isolated and analyzed using Southern blot technique. Each individual sample showed two bands. The bands were identical for two of the individuals. For the third, one band was identical to one band of the other two but the second band was of lower molecular weight than the others. This is an example of restriction fragment length polymorphism (RFLP) A reasonable explanation for this RFLP is that the gene in the third individual A. lacked the recognition sequence for the restriction endonuclease. B. had a mutation at the cleavage site. C. had an additional recognition site for the endonuclease. D. had a deletion of a segment of the gene. E. underwent a random cleavage.

Short answer

Answer: The most reasonable explanation for the observed RFLP is a deletion of a segment of the gene in the third individual, leading to a smaller fragment and a lower molecular weight band.

Step-by-step solution

Understand Southern blot technique and RFLP

Southern blot is a molecular biology technique used to identify specific DNA sequences in samples. The genomic DNA is isolated from the individuals, cut into fragments using restriction endonucleases, and separated on an agarose gel using electrophoresis. The fragments are then transferred to a membrane, and hybridization with a labeled probe specific for the desired gene is performed, enabling identification and analysis of that gene. RFLP, or Restriction Fragment Length Polymorphism, is a variation in the DNA sequence that results in different fragment sizes when DNA is digested with restriction enzymes. RFLP can result from mutations, insertions, or deletions, among other changes in the DNA sequence.

Analyze the given data

Review the experiment's results - Each individual's sample showed two bands, with one band being identical for all three individuals. For the third individual, a band of lower molecular weight than the other bands was observed. This indicates a variation in the DNA sequences between the individuals, which could result from a different number of recognition sites or changes in the gene region.

Review the answer choices and select the correct one

Now, let's evaluate each option and identify the most plausible explanation for the observed RFLP. A. The gene lacking the recognition sequence for restriction endonuclease is incorrect, as all samples produced two visible bands indicating successful cleavage of DNA fragments. B. A mutation at the cleavage site might not generate the second band in the third individual. Moreover, since we can see the second band in the third individual, we can rule out this option. C. If the third individual had an additional recognition site for the endonuclease, it would most likely produce more than two bands, which is not observed. Therefore, this option is incorrect. D. A deletion of a segment of the gene is the most plausible explanation for the observed RFLP. This would result in a smaller fragment (shorter DNA sequence) and lower molecular weight in the agarose gel. E. Random cleavage is an unlikely explanation since all individuals have two bands, indicating that the restriction enzyme cleavage is consistent in all individuals. Based on the given information, option D is the most reasonable explanation for the RFLP observed in this experiment. The gene in the third individual had a deletion of a segment, leading to a smaller fragment and a lower molecular weight band.

Key concepts

Southern Blot Technique

The Southern blot technique is a crucial method in molecular biology used to locate a particular sequence of DNA within a complex mixture. It involves several steps, starting with the extraction of genomic DNA from cells. The DNA is then digested with restriction endonuclease enzymes, which cut the DNA at specific sequences, resulting in various sized fragments.

After the DNA is fragmented, it undergoes agarose gel electrophoresis, where fragments are separated based on size. Smaller fragments move faster and thus travel further than larger ones. The DNA fragments are then transferred from the gel onto a membrane, maintaining their positions. A labeled DNA probe, which is a single-stranded sequence complementary to the DNA of interest, is then added. If the target sequence is present on the blot, the probe will bind to it, allowing for the visualization of the specific DNA sequence after appropriate detection methods are applied.

This technique can reveal important information, such as the number of copies of a gene within a genome or differences in a gene sequence, often related to genetic diseases or variations among individuals.

DNA Sequence Variation

DNA sequence variation is the foundation of genetic diversity among individuals. It refers to the differences in the nucleotide sequences that make up the DNA of different organisms. These variations can be as small as a single nucleotide polymorphism (SNP), or involve large stretches of DNA, and can result from mutations, insertions, deletions, or rearrangements of DNA segments.

Such variations may have no noticeable effect or they can significantly alter gene function and phenotype. In forensic science, paternity testing, and disease diagnostics, analyzing DNA sequence variations is essential for identifying genetic markers. This is where techniques like RFLP (Restriction Fragment Length Polymorphism) come into play, allowing researchers and clinicians to detect differences in DNA fragments after digestion with restriction enzymes, thereby identifying polymorphisms associated with genetic traits or conditions.

Restriction Endonuclease

Restriction endonucleases, or restriction enzymes, are proteins that recognize specific, short nucleotide sequences within DNA and cut the DNA at or near these sites. Over 3,000 restriction enzymes have been studied in detail, each targeting a specific sequence. These enzymes are naturally occurring in bacteria, serving as a defense mechanism against invading viral DNA.

The discovery of restriction enzymes was pivotal to the advancement of genetic engineering, as they allow scientists to cut and paste segments of DNA with precision. When used in RFLP analysis, the pattern of DNA fragments produced by restriction enzyme digestion can exhibit variations among individuals due to mutations in the recognition sites or changes in the DNA sequence between these sites, providing a powerful tool for genetic analysis and genotyping.

Agarose Gel Electrophoresis

Agarose gel electrophoresis is a common laboratory technique used to separate DNA or RNA fragments by size. The process involves placing a gel containing agarose, a polysaccharide matrix, in a buffer-filled chamber with electrodes at either end. Samples of DNA fragments are loaded into wells in the gel, and an electric current is applied. The negatively charged DNA fragments migrate towards the positive electrode.

DNA fragments separate based on size as they move through the agarose matrix; smaller fragments travel faster and thus go further than larger ones. After running the gel, the separated DNA fragments can be visualized, typically under UV light after staining with a dye that binds to nucleic acids. Agarose gel electrophoresis is essential for DNA analysis in cloning, sequencing, genotyping, and forensics, and it’s an integral step in the process of performing a Southern blot.