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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 19b
Chapter 15, Problem 19b

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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1
Understand the difference between genomic DNA and cDNA: Genomic DNA contains both coding (exons) and non-coding (introns, regulatory regions) sequences, while cDNA is synthesized from mRNA and represents only the coding regions (exons). This distinction is key to answering the question.
Consider why restriction enzyme sites might differ between genomic DNA and cDNA: Restriction enzyme sites are specific sequences of nucleotides. In genomic DNA, these sites can be present in introns or regulatory regions, which are absent in cDNA. Therefore, some restriction sites in the genomic DNA may not appear in the cDNA clone.
Analyze the converse scenario: Restriction enzyme sites in a cDNA clone are derived from the coding regions of the gene. These sites should generally be present in the genomic DNA, as the exons in cDNA are part of the genomic sequence. However, mutations or polymorphisms in the genomic DNA could alter or eliminate these sites.
Reflect on the role of alternative splicing: Alternative splicing can lead to different mRNA isoforms, which may result in cDNA clones with variations in sequence. These variations could affect the presence of restriction enzyme sites in the cDNA compared to the genomic DNA.
Summarize the implications: Restriction enzyme sites in genomic DNA are not always found in cDNA due to the absence of introns and regulatory regions in cDNA. Conversely, restriction sites in cDNA are typically present in genomic DNA, but exceptions can occur due to mutations or alternative splicing.

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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 are proteins that cut DNA at specific sequences, known as restriction sites. They are essential tools in molecular biology for cloning and analyzing DNA fragments. Different enzymes recognize different sequences, which can lead to varying fragment sizes when DNA is digested. Understanding how these enzymes work is crucial for interpreting results from genomic and cDNA clones.
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Genomic vs. cDNA Clones

Genomic clones contain the entire genomic DNA, including introns, exons, and regulatory regions, while cDNA clones are synthesized from mRNA and represent only the expressed genes (exons). This difference means that cDNA clones lack intronic sequences, which can affect the presence of restriction sites. Consequently, restriction sites found in genomic DNA may not always be present in cDNA clones due to splicing.
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Gene Structure and Splicing

Gene structure refers to the arrangement of exons and introns within a gene. During the process of splicing, introns are removed from the pre-mRNA transcript, resulting in a mature mRNA that only contains exons. This process can lead to the absence of certain restriction sites in cDNA clones that are present in the genomic DNA, as the sites may be located within introns that are not included in the final mRNA product.
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Related Practice
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. 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?

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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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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.

Can you identify which sequence portions are derived from the vector (specifically the MCS) and which are derived from the cDNA clone?

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