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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
All textbooksSanders 3rd EditionCh. 15 - Recombinant DNA Technology and Its ApplicationsProblem 19a
Chapter 15, Problem 19a

You have isolated a genomic clone with an EcoRI fragment of 11 kb that encompasses the CRABS CLAW gene. You digest the genomic clone with HindIII and note that the 11-kb EcoRI fragment is split into three fragments of 9 kb, 1.5 kb, and 0.5 kb.
Does this tell you anything about where the CRABS CLAW gene is located within the 11-kb genomic clone?

Verified step by step guidance
1
Step 1: Understand the problem setup. You have a genomic clone containing an 11-kb EcoRI fragment that includes the CRABS CLAW gene. When digested with HindIII, this fragment is split into three smaller fragments of 9 kb, 1.5 kb, and 0.5 kb. The goal is to determine if this digestion provides information about the location of the CRABS CLAW gene within the 11-kb fragment.
Step 2: Recognize that restriction enzymes like EcoRI and HindIII cut DNA at specific recognition sites. The fact that HindIII splits the 11-kb fragment into three pieces indicates that there are two HindIII recognition sites within the EcoRI fragment.
Step 3: Consider the sizes of the HindIII fragments (9 kb, 1.5 kb, and 0.5 kb). These sizes represent the distances between the HindIII recognition sites within the 11-kb EcoRI fragment. The CRABS CLAW gene must be located within one of these three fragments.
Step 4: To determine the location of the CRABS CLAW gene, additional experiments would be needed, such as hybridization with a probe specific to the CRABS CLAW gene or sequencing of the fragments. These techniques can identify which HindIII fragment contains the gene.
Step 5: Conclude that while the HindIII digestion provides a map of the EcoRI fragment, it does not directly reveal the exact location of the CRABS CLAW gene. However, it narrows down the possibilities to one of the three HindIII fragments (9 kb, 1.5 kb, or 0.5 kb).

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Key Concepts

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

Restriction Enzymes

Restriction enzymes, like EcoRI and HindIII, are proteins that cut DNA at specific sequences. Understanding how these enzymes work is crucial for analyzing DNA fragments. In this scenario, EcoRI cuts the genomic clone into an 11-kb fragment, while HindIII further digests it into smaller pieces, providing insights into the structure and location of genes within the DNA.
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Mapping with Markers

Genomic Cloning

Genomic cloning involves isolating and amplifying specific DNA fragments from an organism's genome. The 11-kb EcoRI fragment containing the CRABS CLAW gene is a result of this process. Analyzing the sizes of the fragments produced by subsequent digestion helps determine the arrangement of genes and regulatory elements within the cloned DNA.
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Positional Cloning

Gene Mapping

Gene mapping is the process of determining the location of genes on a chromosome. The fragmentation pattern observed after HindIII digestion suggests that the CRABS CLAW gene is likely located within the 11-kb EcoRI fragment, as the sizes of the resulting fragments can indicate the proximity of the gene to the restriction sites. This information is essential for understanding gene organization and function.
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Related Practice
Textbook Question

The restriction enzymes XhoI and SalI cut their specific sequences as shown below:

Can the sticky ends created by XhoI and SalI sites be ligated? If yes, can the resulting sequences be cleaved by either XhoI or SalI?

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Textbook Question

The bacteriophage ϕX174 has a single-stranded DNA genome of 5386 bases. During DNA replication, double-stranded forms of the genome are generated. In an effort to create a restriction map of ϕX174, you digest the z-stranded form of the genome with several restriction enzymes and obtain the following results. Draw a map of the ϕX174 genome.

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Textbook Question

To further analyze the CRABS CLAW gene, you create a map of the genomic clone. The 11-kb EcoRI fragment is ligated into the EcoRI site of the MCS of the vector shown in Problem 18. You digest the double-stranded form of the genome with several restriction enzymes and obtain the following results. Draw, as far as possible, a map of the genomic clone of CRABS CLAW.

What restriction digest would help resolve any ambiguity in the map?

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Textbook Question

You have isolated a genomic clone with an EcoRI fragment of 11 kb that encompasses the CRABS CLAW gene (see Problem 18). You digest the genomic clone with HindIII and note that the 11-kb EcoRI fragment is split into three fragments of 9 kb, 1.5 kb, and 0.5 kb.

Restriction enzyme sites within a cDNA clone are often also found in the genomic sequence. Can you think of a reason why occasionally this is not the case? What about the converse: Are restriction enzyme sites in a genomic clone always in a cDNA clone of the same gene?

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Textbook Question

You have identified a 0.80-kb cDNA clone that contains the entire coding sequence of the Arabidopsis gene CRABS CLAW. In the construction of the cDNA library, linkers with EcoRI sites were added to each end of the cDNA, and the cDNA was inserted into the EcoRI site of the MCS of the vector shown in the accompanying figure. You perform digests on the CRABS CLAW cDNA clone with restriction enzymes and obtain the following results. Can you determine the orientation of the cDNA clone with respect to the restriction enzyme sites in the vector? The restriction enzyme sites listed in the dark blue region are found only in the MCS of the vector.

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Textbook Question

You have isolated another cDNA clone of the CRABS CLAW gene from a cDNA library. The cDNA was directionally cloned using the EcoRI and XhoI sites. You sequence the recombinant plasmid using primers complementary to the T7 and T3 promoter sites flanking the MCS. The first 30 to 60 bases of sequence are usually discarded since they tend to contain errors.

Will the long stretch of T residues in the T3 sequence exist in the genomic sequence of the gene?

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