Steps in Cloning a Gene

Приволжский исследовательский медуниверситет

16 Sept 201601:42

Summary

TLDRThe video script outlines the cloning process of a gene, starting with DNA isolation from an organism. It details the purification and fragmentation using restriction enzymes, which create cohesive ends. These DNA fragments are then inserted into plasmids with compatible cohesive ends, forming a DNA library. The library is introduced into bacterial host cells through transformation, allowing for the identification and isolation of the desired gene within colonies grown on agar medium.

Takeaways

🔬 The first step in gene cloning is isolating DNA from the organism containing the desired gene.
🧬 Isolated DNA is purified and fragmented using a restriction enzyme.
✂️ Restriction enzymes produce staggered cuts in specific DNA sequences, creating fragments with cohesive ends.
🔗 Each DNA fragment has single-stranded sequences at its ends that can hybridize with other DNA fragmented by the same enzyme.
🧪 The fragmented DNA is incorporated into plasmids, which have a single restriction site.
⚙️ When cleaved by the restriction enzyme, plasmids generate cohesive ends that align with the DNA fragments.
🔗 DNA ligase is used to form phosphodiester bonds between plasmid and DNA fragments, completing the insertion.
🦠 Plasmids are incorporated into bacterial host cells through transformation, each carrying different DNA segments.
📚 These cells collectively represent a DNA library containing segments from the original organism.
🔍 Desired cloned gene colonies can be identified and isolated after plating the transformed cells on agar medium.

Q & A

What is the first step in cloning a gene?
-The first step in cloning a gene is to isolate the DNA from the organism that contains the desired gene.
Why is the DNA purified after isolation?
-The DNA is purified to remove contaminants and ensure only the desired DNA is used for further steps in the cloning process.
What role do restriction enzymes play in gene cloning?
-Restriction enzymes cut the DNA at specific sequences, producing fragments with cohesive ends that can hybridize with other DNA fragments.
What are cohesive ends and why are they important?
-Cohesive ends are single-stranded sequences of nucleotides on the ends of DNA fragments. They are important because they allow the DNA fragments to hybridize with DNA fragmented by the same restriction enzyme.
How are the DNA fragments incorporated into plasmids?
-The DNA fragments are incorporated into plasmids by aligning the cohesive ends of the plasmid and DNA fragments, then using the enzyme DNA ligase to form phosphodiester bonds between them.
What type of plasmids are used in cloning?
-The type of plasmid used in cloning has a single restriction site that, when cleaved by the restriction enzyme, generates the same cohesive ends as the DNA fragments to be cloned.
What is the purpose of DNA ligase in the cloning process?
-DNA ligase is used to form phosphodiester bonds between the cohesive ends of the plasmid and the DNA fragments, sealing the fragments into the plasmid.
How are the plasmids introduced into bacterial host cells?
-The plasmids are introduced into bacterial host cells through a process called transformation, where the bacterial cells take up the plasmids.
What is a DNA library in the context of gene cloning?
-A DNA library is a collection of bacterial cells, each containing a different segment of DNA from the original organism. Taken together, these cells represent the entire DNA of the organism.
How can the desired cloned gene be identified and isolated?
-The bacterial cells are plated on an agar medium, and the colony containing the desired cloned gene can be identified and isolated for further study.

Outlines

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🧬 DNA Cloning Process Overview

The paragraph outlines the fundamental steps involved in cloning a gene. It begins with the isolation of DNA from an organism containing the gene of interest. The DNA is then purified and fragmented using restriction enzymes, which create specific sequences with cohesive ends. These DNA fragments are incorporated into plasmids, circular DNA molecules used as vectors in cloning, that have a single restriction site. The cohesive ends of both the plasmids and the DNA fragments align, and DNA ligase is used to form phosphodiester bonds, effectively joining them. The plasmids are then introduced into bacterial host cells through transformation, creating a DNA library where each cell contains a different segment of the original organism's DNA. This library is grown on agar medium, and the colony containing the desired gene can be identified and isolated for further use.

Mindmap

Keywords

💡Gene

A gene is a sequence of DNA that contains the instructions to make a specific protein or set of proteins. In the video, the gene of interest is the one scientists want to clone, meaning they want to replicate its exact sequence to study or use it for further applications.

💡DNA

DNA (Deoxyribonucleic acid) is the molecule that carries genetic information in living organisms. In this process, DNA is isolated from the organism that contains the desired gene. This is the starting material for gene cloning and manipulation.

💡Restriction enzyme

A restriction enzyme is a protein used in molecular biology to cut DNA at specific sequences. In the cloning process, these enzymes create staggered cuts, leaving 'cohesive ends' that are important for combining DNA fragments with plasmids.

💡Cohesive ends

Cohesive ends are the single-stranded ends of DNA fragments that are produced when DNA is cut by restriction enzymes. These ends can easily pair with complementary sequences, allowing DNA fragments to join together. In the cloning process, cohesive ends enable the DNA fragment and plasmid to be combined.

💡Plasmid

A plasmid is a small, circular piece of DNA typically found in bacteria. Plasmids are used as vectors in gene cloning to carry the DNA fragment of interest into a host cell. The plasmid contains a restriction site that allows it to be opened and incorporate the DNA fragment.

💡DNA ligase

DNA ligase is an enzyme that joins DNA fragments by forming phosphodiester bonds between them. In gene cloning, it is used to link the cohesive ends of DNA fragments with the cohesive ends of a plasmid, completing the cloning process.

💡Transformation

Transformation is the process of introducing plasmids into bacterial cells. In this context, after the DNA fragments are inserted into the plasmids, the plasmids are introduced into bacterial cells so that they can replicate and form a 'DNA library' for future analysis.

💡DNA Library

A DNA library is a collection of bacterial cells, each containing a different fragment of the original organism's DNA. This library allows researchers to screen for and isolate specific genes, such as the one they are attempting to clone, from the entire genome.

💡Host cells

Host cells, often bacterial cells in the context of gene cloning, are the cells that take up the plasmid DNA. These cells replicate, producing many copies of the plasmid and, by extension, the gene of interest, allowing scientists to analyze or further manipulate the gene.

💡Phosphodiester bonds

Phosphodiester bonds are the chemical bonds that connect nucleotides within a strand of DNA. DNA ligase forms these bonds between the cohesive ends of DNA fragments and plasmids during the cloning process, allowing the DNA fragments to be stably incorporated into the plasmid.

Highlights

The first step in cloning a gene is to isolate the DNA from the organism containing the desired gene.

The isolated DNA is purified and fragmented using a restriction enzyme.

Restriction enzymes used in cloning produce staggered cuts in specific sequences of DNA, generating fragments with cohesive ends.

Each DNA fragment has a single-stranded sequence of nucleotides on its ends that can hybridize with DNA fragmented by the same restriction enzyme.

The fragmented DNA is incorporated into plasmids, which have a single restriction site.

Plasmids cleaved by the restriction enzyme generate cohesive ends, matching the ends of the DNA fragments.

Cohesive ends from the plasmid and DNA fragments align, and DNA ligase forms phosphodiester bonds.

The plasmids are introduced into bacterial host cells through a process called transformation.

Each bacterial cell contains a different segment of DNA from the original organism.

These transformed cells collectively represent a DNA library.

The cells are plated on an agar medium to allow for growth and identification.

Colonies of cells containing the desired cloned gene can be identified and isolated from the agar medium.

The restriction enzymes enable precise manipulation of DNA fragments for gene cloning.

The process forms the basis for genetic research, biotechnology applications, and gene therapy.

DNA libraries enable the study of gene sequences and expression in various organisms.