Ch. 15 - Recombinant DNA Technology and Its Applications

Sanders - Genetic Analysis: An Integrated Approach 3rd Edition

Sanders3rd EditionGenetic Analysis: An Integrated ApproachISBN: 9780135564172Not the one you use?Change textbook

Sanders 3rd Edition

Ch. 15 - Recombinant DNA Technology and Its Applications

Problem 3

Chapter 15, Problem 3

Ligase catalyzes a reaction between the 5′ phosphate and the 3′ hydroxyl groups at the ends of DNA molecules. The enzyme calf intestinal phosphatase catalyzes the removal of the 5′5′ phosphate from DNA molecules. What would be the consequence of treating a cloning vector, before ligation, with calf intestinal phosphatase?

Verified step by step guidance

Understand the role of DNA ligase: DNA ligase catalyzes the formation of a phosphodiester bond between the 5′ phosphate group of one DNA strand and the 3′ hydroxyl group of another, effectively joining DNA fragments together.

Recognize the function of calf intestinal phosphatase (CIP): CIP removes the 5′ phosphate groups from DNA ends, leaving 5′ hydroxyl groups instead.

Consider the consequence of removing the 5′ phosphate groups on the cloning vector: Since ligase requires a 5′ phosphate to form the phosphodiester bond, the absence of this group on the vector DNA ends will prevent ligation from occurring.

Analyze the practical implication: Treating the cloning vector with CIP before ligation will prevent the vector from self-ligating (re-circularizing without an insert), which is often used to reduce background colonies in cloning experiments.

Summarize the overall effect: The vector DNA will be unable to ligate to itself or to any insert DNA lacking a 5′ phosphate, thus promoting the insertion of DNA fragments that have intact 5′ phosphates during the cloning process.

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Key Concepts

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

DNA Ligase Function

DNA ligase is an enzyme that joins DNA strands by catalyzing the formation of a phosphodiester bond between the 3′ hydroxyl group of one nucleotide and the 5′ phosphate group of another. This activity is essential for sealing nicks in the DNA backbone during replication and cloning.

Role of 5′ Phosphate in Ligation

The 5′ phosphate group on DNA ends is critical for ligation because DNA ligase requires it to form the phosphodiester bond. Without the 5′ phosphate, ligase cannot join DNA fragments, preventing the formation of continuous DNA strands.

Effect of Calf Intestinal Phosphatase (CIP) Treatment

Calf intestinal phosphatase removes 5′ phosphate groups from DNA ends, thereby preventing self-ligation of cloning vectors. This treatment reduces background colonies by ensuring that only DNA fragments with 5′ phosphates (e.g., inserts) can be ligated into the vector.

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Textbook Question

The human genome is 3×10⁹ bp in length.

How many fragments would be predicted to result from the complete digestion of the human genome with the following enzymes: Sau3A (˘GATC), BamHI (G˘GATCC), EcoRI (G˘AATTC), and NotI (GC˘GGCCGC)?

Textbook Question

You have constructed four different libraries: a genomic library made from DNA isolated from human brain tissue, a genomic library made from DNA isolated from human muscle tissue, a human brain cDNA library, and a human muscle cDNA library.

Which of these would have the greatest diversity of sequences?

Textbook Question

The human genome is 3×10⁹ bp in length.

How would your initial answer change if you knew that the average GC content of the human genome was 40%?

Textbook Question

Compare and contrast the terms Paternity Index (PI) and Combined Paternity Index (CPI). How does each contribute to paternity determination?

Textbook Question

What is the statistical principle underlying genetic health risk assessment? Why are these assessments not predictive of disease occurrence?

Textbook Question

What is the exclusion principle? How is it used in forensic genetic analysis and in paternity determination?