Ch. 20 - Recombinant DNA Technology

Klug - Concepts of Genetics 12th Edition

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Klug 12th Edition

Ch. 20 - Recombinant DNA Technology

Problem 32a

Chapter 20, Problem 32a

In humans, congenital heart disease is a common birth defect that affects approximately 1 out of 125 live births. Using reverse transcription PCR (RT-PCR), Samir Zaidi and colleagues [(2013) Nature 498:220.223] determined that approximately 10 percent of the cases resulted from point mutations, often involving histone function. To capture products of gene expression in developing hearts, they used oligo(dT) in their reverse transcription protocol.
How would such a high %T in a primer influence annealing temperature?

Verified step by step guidance

Understand the role of oligo(dT) primers in reverse transcription: Oligo(dT) primers are designed to bind to the poly-A tail of mRNA molecules, which is rich in adenine (A) bases. These primers are used to initiate the synthesis of complementary DNA (cDNA) during reverse transcription.

Recognize the relationship between nucleotide composition and annealing temperature: The annealing temperature of a primer is influenced by its nucleotide composition. Primers with a higher percentage of guanine (G) and cytosine (C) bases (GC content) tend to have higher annealing temperatures due to the stronger hydrogen bonding between G-C pairs compared to A-T pairs.

Analyze the impact of high %T in the primer: A primer with a high percentage of thymine (T) bases, such as oligo(dT), will have a lower GC content. This results in weaker overall hydrogen bonding and a lower melting temperature (Tm), which directly affects the annealing temperature during PCR.

Consider the implications for experimental design: The lower annealing temperature required for oligo(dT) primers means that the reverse transcription step must be optimized to ensure efficient binding and synthesis of cDNA. Researchers must carefully adjust the temperature settings to accommodate the primer's properties.

Relate this to the study's context: In the study by Samir Zaidi and colleagues, the use of oligo(dT) primers was essential for capturing gene expression products in developing hearts. Understanding the primer's influence on annealing temperature helps ensure the accuracy and reliability of the reverse transcription process in their experiments.

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Key Concepts

Here are the essential concepts you must grasp in order to answer the question correctly.

Reverse Transcription PCR (RT-PCR)

RT-PCR is a laboratory technique used to convert RNA into complementary DNA (cDNA) using the enzyme reverse transcriptase. This process allows researchers to amplify specific RNA sequences, making it easier to study gene expression. In the context of congenital heart disease, RT-PCR can help identify mutations affecting gene function by analyzing the expression levels of relevant genes.

Annealing Temperature

The annealing temperature is the temperature at which primers bind to their complementary DNA sequences during PCR. It is crucial for the specificity and efficiency of the amplification process. A higher percentage of thymine (T) in a primer can lower the melting temperature (Tm), which may require adjustments to the annealing temperature to ensure optimal binding and reduce non-specific amplification.

Point Mutations and Histone Function

Point mutations are changes in a single nucleotide in the DNA sequence, which can significantly impact gene function and regulation. In the context of histone function, these mutations can alter how DNA is packaged and expressed, potentially leading to congenital heart disease. Understanding the relationship between point mutations and histone modifications is essential for elucidating the genetic basis of such conditions.

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Point Mutations

Related Practice

Textbook Question

A widely used method for calculating the annealing temperature for a primer used in PCR is 5 degrees below the melting temperature, Tₘ(°C), which is computed by the equation 81.5+0.41×(%GC)−(675/N), where %GC is the percentage of GC nucleotides in the oligonucleotide and N is the length of the oligonucleotide. Notice from the formula that both the GC content and the length of the oligonucleotide are variables. Assuming you have the following oligonucleotide as a primer,

5′-TTGAAAATATTTCCCATTGCC-3′

Compute the annealing temperature for PCR. What is the relationship between %GC and? Why? (Note: In reality, this computation provides only a starting point for empirical determination of the most useful annealing temperature.)

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Textbook Question

Most of the techniques (blotting, cloning, PCR, etc.) are dependent on hybridization (annealing) between different populations of nucleic acids. The length of the strands, temperature, and percentage of GC nucleotides weigh considerably on hybridization. Two other components commonly used in hybridization protocols are monovalent ions and formamide. A formula that takes monovalent Na⁺ ions (M[Na⁺]) and formamide concentrations into consideration to compute a Tₘ (temperature of melting) is as follows:

Tₘ=81.5+16.6(log M[Na+])+0.41(%GC)−0.72(%formamide)

For the following concentrations of Na⁺ and formamide, calculate the Tₘ. Assume 45% GC content.

[Na⁺] % Formamide

0.825 20

0.825 40

0.165 20

0.165 40

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Textbook Question

Most of the techniques described in this chapter (blotting, cloning, PCR, etc.) are dependent on hybridization (annealing) between different populations of nucleic acids. The length of the strands, temperature, and percentage of GC nucleotides weigh considerably on hybridization. Two other components commonly used in hybridization protocols are monovalent ions and formamide. A formula that takes monovalent Na⁺ ions (M[Na⁺]) and formamide concentrations into consideration to compute a Tₘ (temperature of melting) is as follows:

Tₘ=81.5+16.6(log M[Na+])+0.41(%GC)−0.72(%formamide)

Given that formamide competes for hydrogen bond locations on nucleic acid bases and monovalent cations are attracted to the negative charges on nucleic acids, explain why the Tₘ varies as described in part (a).

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Textbook Question

In humans, congenital heart disease is a common birth defect that affects approximately 1 out of 125 live births. Using reverse transcription PCR (RT-PCR), Samir Zaidi and colleagues [(2013) Nature 498:220.223] determined that approximately 10 percent of the cases resulted from point mutations, often involving histone function. To capture products of gene expression in developing hearts, they used oligo(dT) in their reverse transcription protocol.

Compared with oligo(dT) primers, a pool of random sequence primers requires a trickier assessment of annealing temperature. Why?

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Textbook Question

If one were interested in comparing the quantitative distribution of gene expression in, say, the right and left sides of a developing heart, how might one proceed using RT-PCR?

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Textbook Question

The U.S. Department of Justice has established a database that catalogs PCR amplification products from short tandem repeats of the Y chromosome (Y-STRs) in humans. The database contains polymorphisms of five U.S. ethnic groups (African-Americans, European Americans, Hispanics, Native Americans, and Asian-Americans) as well as the worldwide population.

Given that STRs are repeats of varying lengths, for example (TCTG)₉₋₁₇ or (TAT)₆₋₁₄, explain how PCR could reveal differences (polymorphisms) among individuals. How could the Department of Justice make use of those differences?

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