Biotechnology- Principles and Processes
Components of Vectors
- Vectors are the DNA molecules used as vehicles to transfer foreign DNA into the host organism.
- The components of vectors include:
- Origin of replication (ORI): Allows replication of the vector within the host cell.
- Selectable marker: Allows selection of host cells containing the vector.
- Cloning site: Allows insertion of foreign DNA into the vector.
- Plasmid backbone: Provides stability and replication ability to the vector.
Origin of Replication (ORI)
- ORI is a specific DNA sequence recognized by host cell enzymes for replication initiation.
- It allows the vector to autonomously replicate within the host cell.
- Vectors often contain a high-copy ORI, ensuring high efficiency of replication.
Selectable Marker
- Selectable markers are genes that confer a survival advantage to host cells containing the vector.
- Common selectable markers include antibiotic resistance genes (e.g., ampicillin resistance).
- Host cells containing the vector can be selected by growing them in a medium containing the corresponding antibiotic.
Cloning Site
- Cloning sites, also known as multiple cloning sites (MCS), are specific DNA sequences where foreign DNA can be inserted.
- MCS often contain recognition sites for various restriction enzymes.
- After insertion of the foreign DNA, host cell enzymes can join the vector and foreign DNA together, creating a recombinant DNA molecule.
Plasmid Backbone
- Plasmid backbone is the remaining DNA sequence of the vector after insertion of foreign DNA.
- It provides stability and replication ability to the vector.
- The plasmid backbone also contains regulatory sequences for gene expression.
Biotechnology- Principles and Processes
- DNA extraction is a crucial step in biotechnology for obtaining DNA from various sources.
- The process typically involves:
- Cell lysis: Breaking down the cells to release DNA.
- Removal of proteins, lipids, and other cellular debris.
- Precipitation of DNA using alcohol or other solvents.
Polymerase Chain Reaction (PCR)
- PCR is a widely used technique in biotechnology to amplify a specific DNA sequence.
- The process involves the repeated cycles of:
- Denaturation: Heating DNA to separate the two strands.
- Primer annealing: Cooling DNA to allow primers to bind to the target sequence.
- Primer extension: DNA polymerase adds nucleotides to extend the DNA strand.
- Each cycle doubles the amount of the target DNA sequence.
Gel Electrophoresis
- Gel electrophoresis is a technique used to separate DNA fragments based on their size.
- The process involves:
- Loading DNA samples onto a gel made of agarose or polyacrylamide.
- Applying an electric current that causes DNA to migrate through the gel.
- Smaller DNA fragments migrate faster, resulting in distinct bands on the gel.
- DNA bands can be visualized using fluorescent dyes or stains.
Restriction Digestion
- Restriction digestion is a technique used to cut DNA at specific sequences using restriction enzymes.
- Restriction enzymes recognize specific DNA sequences and cut the DNA at or near those sequences.
- The resulting DNA fragments can be used for DNA sequencing, cloning, or other downstream applications.
DNA Ligation
- DNA ligation is the process of joining DNA fragments together using DNA ligase enzyme.
- The DNA fragments to be joined should have compatible ends (sticky ends or blunt ends) after restriction digestion.
- The ligase enzyme catalyzes the formation of phosphodiester bonds, permanently joining the DNA fragments.
Biotechnology- Principles and Processes
- Transformation is the process of introducing foreign DNA into bacterial cells.
- The foreign DNA often contains a selectable marker, allowing selection of transformed cells.
- Transformation can be achieved by:
- Heat shock: Briefly exposing the host cells and DNA to high temperature.
- Electroporation: Applying an electric field to create temporary pores in the cell membrane.
- Chemical methods: Using chemicals to increase cell membrane permeability.
- After transformation, not all cells will take up the foreign DNA.
- The selectable marker can be used to select cells that have successfully taken up the DNA.
- Cells that have taken up the vector with the selectable marker will grow on a medium containing the corresponding antibiotic.
- Screening is done to identify transformed cells containing the desired cloned DNA.
- Common methods for screening include:
- Blue-white screening: Utilizes a reporter gene (e.g., lacZ in E. coli) to distinguish transformed cells.
- PCR-based screening: Amplifies specific DNA sequences to confirm the presence of desired DNA.
- DNA sequencing: Determines the nucleotide sequence of the cloned DNA.
Expression of Cloned DNA
- After the identification of transformed cells containing the desired cloned DNA, it can be expressed.
- Expression of the cloned DNA can be achieved by:
- Expression vectors: Vectors designed to promote transcription and translation of the cloned DNA.
- Gene promoters: Regulatory sequences that initiate transcription of the cloned DNA.
- Inducible systems: Systems that allow control over when and how much of the cloned DNA is expressed.
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Biotechnology- Principles and Processes
Components of Vectors
- Vectors are the DNA molecules used as vehicles to transfer foreign DNA into the host organism.
- The components of vectors include:
- Origin of replication (ORI): Allows replication of the vector within the host cell.
- Selectable marker: Allows selection of host cells containing the vector.
- Cloning site: Allows insertion of foreign DNA into the vector.
- Plasmid backbone: Provides stability and replication ability to the vector.
Origin of Replication (ORI)
- ORI is a specific DNA sequence recognized by host cell enzymes for replication initiation.
- It allows the vector to autonomously replicate within the host cell.
- Vectors often contain a high-copy ORI, ensuring high efficiency of replication.
Selectable Marker
- Selectable markers are genes that confer a survival advantage to host cells containing the vector.
- Common selectable markers include antibiotic resistance genes (e.g., ampicillin resistance).
- Host cells containing the vector can be selected by growing them in a medium containing the corresponding antibiotic.
Cloning Site
- Cloning sites, also known as multiple cloning sites (MCS), are specific DNA sequences where foreign DNA can be inserted.
- MCS often contain recognition sites for various restriction enzymes.
- After insertion of the foreign DNA, host cell enzymes can join the vector and foreign DNA together, creating a recombinant DNA molecule.
Plasmid Backbone
- Plasmid backbone is the remaining DNA sequence of the vector after insertion of foreign DNA.
- It provides stability and replication ability to the vector.
- The plasmid backbone also contains regulatory sequences for gene expression.
- DNA extraction is a crucial step in biotechnology for obtaining DNA from various sources.
- The process typically involves:
- Cell lysis: Breaking down the cells to release DNA.
- Removal of proteins, lipids, and other cellular debris.
- Precipitation of DNA using alcohol or other solvents.
Polymerase Chain Reaction (PCR)
- PCR is a widely used technique in biotechnology to amplify a specific DNA sequence.
- The process involves the repeated cycles of:
- Denaturation: Heating DNA to separate the two strands.
- Primer annealing: Cooling DNA to allow primers to bind to the target sequence.
- Primer extension: DNA polymerase adds nucleotides to extend the DNA strand.
- Each cycle doubles the amount of the target DNA sequence.
Gel Electrophoresis
- Gel electrophoresis is a technique used to separate DNA fragments based on their size.
- The process involves:
- Loading DNA samples onto a gel made of agarose or polyacrylamide.
- Applying an electric current that causes DNA to migrate through the gel.
- Smaller DNA fragments migrate faster, resulting in distinct bands on the gel.
- DNA bands can be visualized using fluorescent dyes or stains.
Restriction Digestion
- Restriction digestion is a technique used to cut DNA at specific sequences using restriction enzymes.
- Restriction enzymes recognize specific DNA sequences and cut the DNA at or near those sequences.
- The resulting DNA fragments can be used for DNA sequencing, cloning, or other downstream applications.
DNA Ligation
- DNA ligation is the process of joining DNA fragments together using DNA ligase enzyme.
- The DNA fragments to be joined should have compatible ends (sticky ends or blunt ends) after restriction digestion.
- The ligase enzyme catalyzes the formation of phosphodiester bonds, permanently joining the DNA fragments.
Biotechnology- Principles and Processes
- Transformation is the process of introducing foreign DNA into bacterial cells.
- The foreign DNA often contains a selectable marker, allowing selection of transformed cells.
- Transformation can be achieved by:
- Heat shock: Briefly exposing the host cells and DNA to high temperature.
- Electroporation: Applying an electric field to create temporary pores in the cell membrane.
- Chemical methods: Using chemicals to increase cell membrane permeability.
- After transformation, not all cells will take up the foreign DNA.
- The selectable marker can be used to select cells that have successfully taken up the DNA.
- Cells that have taken up the vector with the selectable marker will grow on a medium containing the corresponding antibiotic.
- Screening is done to identify transformed cells containing the desired cloned DNA.
- Common methods for screening include:
- Blue-white screening: Utilizes a reporter gene (e.g., lacZ in E. coli) to distinguish transformed cells.
- PCR-based screening: Amplifies specific DNA sequences to confirm the presence of desired DNA.
- DNA sequencing: Determines the nucleotide sequence of the cloned DNA.
Expression of Cloned DNA
- After the identification of transformed cells containing the desired cloned DNA, it can be expressed.
- Expression of the cloned DNA can be achieved by:
- Expression vectors: Vectors designed to promote transcription and translation of the cloned DNA.
- Gene promoters: Regulatory sequences that initiate transcription of the cloned DNA.
- Inducible systems: Systems that allow control over when and how much of the cloned DNA is expressed.