Ch. 15 - Recombinant DNA Technology and Its Applications

Sanders - Genetic Analysis: An Integrated Approach 3rd Edition

Sanders3rd EditionGenetic Analysis: An Integrated ApproachISBN: 9780135564172당신이 사용하는 게 아니라요?교과서 변경

Sanders 3rd Edition

Ch. 15 - Recombinant DNA Technology and Its Applications

문제 13

15장, 문제 13

It is often desirable to insert cDNAs into a cloning vector in such a way that all the cDNA clones will have the same orientation with respect to the sequences of the plasmid. This is referred to as directional cloning. Outline how you would directionally clone a cDNA library in the plasmid vector pUC18.

검증된 단계별 안내

insert step 1: Choose appropriate restriction enzymes that cut the plasmid vector pUC18 at two different sites, ensuring that the cDNA can be inserted in a specific orientation.

insert step 2: Design the cDNA insert with compatible ends that match the overhangs created by the restriction enzymes used on the plasmid vector.

insert step 3: Digest the plasmid vector pUC18 with the selected restriction enzymes to create linearized vector with sticky ends.

insert step 4: Ligate the cDNA insert into the digested plasmid vector, ensuring that the complementary sticky ends facilitate the insertion in the desired orientation.

insert step 5: Transform the ligation mixture into competent bacterial cells and screen for colonies that contain the plasmid with the correctly oriented cDNA insert.

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주요 개념

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cDNA and cDNA Libraries

cDNA, or complementary DNA, is synthesized from mRNA through the process of reverse transcription. A cDNA library is a collection of cDNA clones that represent the expressed genes of a particular cell type or tissue at a specific time. This library allows researchers to study gene expression and function by providing a means to clone and analyze the coding sequences of genes.

Cloning Vectors

Cloning vectors are DNA molecules used to transport foreign genetic material into a host cell for replication and expression. The plasmid vector pUC18 is a commonly used cloning vector that contains features such as an origin of replication, a selectable marker (usually antibiotic resistance), and multiple cloning sites (MCS) for inserting DNA fragments. Understanding the structure and function of pUC18 is essential for successful cloning.

Directional Cloning

Directional cloning is a technique that ensures the inserted DNA fragment is oriented in a specific direction relative to the vector's sequences. This is typically achieved by using two different restriction enzymes that create compatible ends on the vector and the insert, allowing for the precise orientation of the cDNA insert. This method is crucial for maintaining the correct reading frame and ensuring proper expression of the cloned gene.

관련 실천

교과서 질문

Compare and contrast methods for making transgenic plants and transgenic Drosophila.

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교과서 질문

Three independently assorting STR markers (A, B, and C) are used to assess the paternity of a colt recently born to a quarter horse mare. Blood samples are drawn from the mare, her colt, and three possible male sires (S₁, S₂, and S₃). DNA at each marker locus is amplified by PCR, and a DNA electrophoresis gel is run for each marker. Amplified DNA bands are visualized in each gel by ethidium bromide staining. Gel results are shown below for each marker. Calculate the PI and CPI based on these STR markers, using the following population frequencies: A₁₂ = 0.12, A₁₀ = 0.18; B₁₈ = 0.08, B₁₂ = 0.17; C₁₆ = 0.11, C₁₄ = 0.20.

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교과서 질문

Three independently assorting STR markers (A, B, and C) are used to assess the paternity of a colt recently born to a quarter horse mare. Blood samples are drawn from the mare, her colt, and three possible male sires (S₁, S₂, and S₃). DNA at each marker locus is amplified by PCR, and a DNA electrophoresis gel is run for each marker. Amplified DNA bands are visualized in each gel by ethidium bromide staining. Gel results are shown below for each marker. Evaluate the data and determine if any of the potential sires can be excluded. Explain the basis of exclusion, if any, in each case.

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교과서 질문

The bacteriophage lambda genome can exist in either a linear form or a circular form.

Diagram the resulting fragments as they would appear on an agarose gel after electrophoresis.

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교과서 질문

The bacteriophage lambda genome can exist in either a linear form or a circular form.

How many fragments will be formed by restriction enzyme digestion with XhoI alone, with XbaI alone, and with both XhoI and XbaI in the linear and circular forms of the lambda genome?

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교과서 질문

A major advance in the 1980s was the development of technology to synthesize short oligonucleotides. This work both facilitated DNA sequencing and led to the advent of the development of PCR. Recently, rapid advances have occurred in the technology to chemically synthesize DNA, and sequences up to 10 kb are now readily produced. As this process becomes more economical, how will it affect the gene-cloning approaches outlined in this chapter? In other words, what types of techniques does this new technology have potential to supplant, and what techniques will not be affected by it?

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