Passage
Mitochondria are thought to have been independent bacterial organisms that were engulfed and integrated into eukaryotic cells approximately two billion years ago. Most of the mitochondrial genome was lost or transferred into the large central genome of the eukaryotic nucleus, leaving only a residual genome within each mitochondrion.Because multiple mitochondria are found in the cell, mitochondrial DNA (mtDNA) mutations result in a condition known as heteroplasmy, or the intracellular mixture of wild-type mtDNA and mutant mtDNA. Just as the cellular ratio of wild-type to mutant mtDNA varies, the overall mtDNA content within a tissue or organ is also highly variable. For this reason, disease-causing mutant mtDNA manifests phenotypically only when the majority of mitochondria within a given tissue express the deleterious allele. In addition, the variability in mtDNA content makes it possible for individuals with the same mitochondrial mutation to display drastically different clinical symptoms.To investigate how heteroplasmy influences disease manifestation, researchers analyzed an A to G substitution at nucleotide 3243 in the tRNA^leu gene of mtDNA. This mutation has been associated with hereditary hearing loss, diabetes mellitus, and a syndrome characterized by mitochondrial encephalomyopathy, lactic acidosis, and strokelike episodes.Family members who were confirmed to have the A3243G mutation were tested using an audiometry examination to assess hearing impairment, as shown in Table 1. The subjects carried no other genetic mutations known to cause hearing loss.Table 1 Audiometry Results From Family Members with the A3243G Mutation
The blood samples of these subjects were collected, and the 3243 region of the tRNA^leu gene was amplified via standard PCR. The PCR products were subsequently treated with the restriction enzyme ApaI and visualized on a PAGE gel. The normal undigested PCR product is 161 bp in length, but the PCR product amplified from the mutant A3243G is digested into two fragments by ApaI.
Figure 1 PAGE visualization of PCR products following ApaI digestion
Adapted from Hadjivasiliou Z, Pomiankowski A, Seymour RM, Lane N. Selection for mitonuclear co-adaptation could favour the evolution of two sexes. Proc Biol Sci. 2012;279(1734) :1865-72.
-Based on the passage, which of the following reagents was most likely used during PCR?

Question

Passage
Mitochondria are thought to have been independent bacterial organisms that were engulfed and integrated into eukaryotic cells approximately two billion years ago. Most of the mitochondrial genome was lost or transferred into the large central genome of the eukaryotic nucleus, leaving only a residual genome within each mitochondrion.Because multiple mitochondria are found in the cell, mitochondrial DNA (mtDNA) mutations result in a condition known as heteroplasmy, or the intracellular mixture of wild-type mtDNA and mutant mtDNA. Just as the cellular ratio of wild-type to mutant mtDNA varies, the overall mtDNA content within a tissue or organ is also highly variable. For this reason, disease-causing mutant mtDNA manifests phenotypically only when the majority of mitochondria within a given tissue express the deleterious allele. In addition, the variability in mtDNA content makes it possible for individuals with the same mitochondrial mutation to display drastically different clinical symptoms.To investigate how heteroplasmy influences disease manifestation, researchers analyzed an A to G substitution at nucleotide 3243 in the tRNA^leu gene of mtDNA. This mutation has been associated with hereditary hearing loss, diabetes mellitus, and a syndrome characterized by mitochondrial encephalomyopathy, lactic acidosis, and strokelike episodes.Family members who were confirmed to have the A3243G mutation were tested using an audiometry examination to assess hearing impairment, as shown in Table 1. The subjects carried no other genetic mutations known to cause hearing loss.Table 1 Audiometry Results From Family Members with the A3243G Mutation

The blood samples of these subjects were collected, and the 3243 region of the tRNA^leu gene was amplified via standard PCR. The PCR products were subsequently treated with the restriction enzyme ApaI and visualized on a PAGE gel. The normal undigested PCR product is 161 bp in length, but the PCR product amplified from the mutant A3243G is digested into two fragments by ApaI.

Figure 1 PAGE visualization of PCR products following ApaI digestion
Adapted from Hadjivasiliou Z, Pomiankowski A, Seymour RM, Lane N. Selection for mitonuclear co-adaptation could favour the evolution of two sexes. Proc Biol Sci. 2012;279(1734) :1865-72.
-Based on the passage, which of the following reagents was most likely used during PCR?

Quizplus · Accepted Answer

11ecba2d_0f82_125e_a3bf_a5d8f8db3021_MD0008_00 Polymerase chain reaction (PCR) uses thermal cycling to amplify small DNA fragments, which can then be screened for mutations or used in further genomic analysis. PCR reagents include the following: A source DNA template (containing deoxyribonucleotides) that includes the target region to be amplified and its adjacent flanking sequences (Choice C) Primer pairs designed from the oligonucleotide sequence of the regions flanking the target sequence A thermostable DNA polymerase (ie, not denatured at high temperatures) to replicate the DNA template using a pool of supplied deoxyribonucleotide triphosphates (dNTPs) A buffer solution with positively charged ions (cations) to provide an optimal environment for DNA polymerase to function (ie, cations bind the negatively charged phosphates on the DNA backbone and those on dNTPs, neutralizing the negative charge of DNA and stabilizing primer-template binding) In PCR, thermal separation (denaturation) of the DNA template is accomplished by exposing the sample to high temperatures. Subsequent cooling allows primers to anneal to the single-stranded flanking ends of the target region. DNA polymerase uses the primers to elongate the new daughter strands in the 5′ to 3′ direction. These steps are repeated to obtain millions of copies of the target DNA segment in a short time. (Choice A) Deoxyribonucleotide triphosphates, not ribonucleoside triphosphates, are used during PCR to synthesize new DNA strands. dNTPs arefree nucleic acids that contain deoxyribose (sugar) whereas ribonucleoside triphosphates are used during the transcription of RNA as they contain ribose (sugar). (Choice B) RNA polymerase is not used during PCR as this enzyme synthesizes new RNA molecules from DNA during transcription (ie, does not participate in DNA replication). (Choice D) GC-rich DNA fragments tend to be more stable at high temperatures; therefore, primers must have a substantial amount of GC content (40%-60%) to avoid premature denaturation during PCR. Educational objective: Polymerase chain reaction (PCR) is a thermal cycling technique used to amplify DNA fragments. PCR reagents include a source DNA template, GC-rich primer pairs, a thermostable DNA polymerase, and a buffer solution with positively charged ions.