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Chapter 20: Problem 28

What techniques can scientists use to determine if a particular transgene has been integrated into the genome of an organism?

Short Answer

Expert verified
Answer: The main methods for determining transgene integration are Polymerase Chain Reaction (PCR), Southern blot analysis, and Fluorescent in situ Hybridization (FISH). PCR provides information about the presence of the transgene, Southern blot analysis reveals the size and copy number, and FISH offers insight into the exact chromosomal location of the transgene.

Step by step solution

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1. Understanding Transgenes

A transgene is a gene or genetic material that has been transferred naturally or by any of a number of genetic engineering techniques from one organism to another. Determining whether a transgene has been successfully integrated into an organism's genome is an essential step in genetic engineering research.
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2. Polymerase Chain Reaction (PCR)

PCR is a widely used technique for amplifying a specific region of DNA. To determine the integration of a transgene, scientists design specific primers that bind to the transgene's sequence and the target organism's genome. The presence of the transgene in the PCR product confirms that the transgene has been integrated into the genome. PCR can quickly provide results, but it does not provide information about the copy number or the exact location of the integration.
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3. Southern Blot Analysis

Southern blot is a technique used to detect the presence of a specific DNA sequence in a DNA sample. In this method, DNA from the organism is extracted, digested with restriction enzymes, and separated by gel electrophoresis. The DNA is then transferred to a membrane and hybridized with a labeled probe that is complementary to the transgene sequence. A successful hybridization, detected through autoradiography or chemiluminescence, indicates the presence and integration of the transgene. Southern blot provides information about the size and copy number of the inserted transgene but may be time-consuming and labor-intensive.
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4. Fluorescent in situ Hybridization (FISH)

FISH is a technique used to visualize specific DNA sequences on chromosomes. A fluorescent-labeled probe specific to the transgene sequence is hybridized to the DNA within the cell or tissue sample. The probe will bind to the target sequence, and the integration of the transgene can be visualized under a fluorescence microscope. FISH provides information on the exact chromosomal location of the transgene, but the technique requires specialized equipment and expertise. Through these techniques, scientists can confirm the integration of a transgene into an organism's genome and gather valuable information about its location, copy number, and size.

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Most popular questions from this chapter

Review the Chapter Concepts list on page \(523 .\) All of these refer to recombinant DNA methods and applications. Write a short essay or sketch a diagram that provides an overview of how recombinant DNA techniques help geneticists study genes.

What are the advantages of using a restriction enzyme whose recognition site is relatively rare? When would you use such enzymes?

In this chapter we focused on how specific DNA sequences can be copied, identified, characterized, and sequenced. At the same time, we found many opportunities to consider the methods and reasoning underlying these techniques. From the explanations given in the chapter, what answers would you propose to the following fundamental questions? (a) In a recombinant DNA cloning experiment, how can we determine whether DNA fragments of interest have been incorporated into plasmids and, once host cells are transformed, which cells contain recombinant DNA? (b) When using DNA libraries to clone genes, what combination of techniques are used to identify a particular gene of interest? (c) What steps make \(P C R\) a chain reaction that can produce millions of copies of a specific DNA molecule in a matter of hours without using host cells? (d) How has DNA sequencing technology evolved in response to the emerging needs of genome scientists?

How do next-generation sequencing (NGS) and third. generation sequencing (TGS) differ from Sanger sequencing?

You have recovered a cloned DNA segment from a vector and determine that the insert is 1300 bp in length. To characterize this cloned segment, you isolate the insert and decide to construct a restriction map, Using enzyme I and enzyme II, followed by gel clectrophoresis, you determine the number and size of the fragments produced by enzymes I and II alone and in combination, as recorded in the following table. Construct a restriction map from these data, showing the positions of the restriction-enzyme cutting sites relative to one another and the distance between them in units of base pairs.
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