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Questions and Answers
What is the first step in generating a transgenic organism?
What is the first step in generating a transgenic organism?
- Isolation/amplification of selected genes. (correct)
- Breeding for offspring with desired genetic modification.
- Delivery of recombinant DNA into a host cell.
- Fusion of selected gene with selected vector.
Which of the following methods is primarily used to introduce new restriction enzyme (RE) sites for cloning?
Which of the following methods is primarily used to introduce new restriction enzyme (RE) sites for cloning?
- Gel electrophoresis.
- Transcription.
- Polymerase Chain Reaction (PCR). (correct)
- DNA ligation.
What process is used to select genetically modified host cells?
What process is used to select genetically modified host cells?
- PCR amplification.
- Cloning.
- Screening. (correct)
- Fusion.
What is the approximate size of the average human protein-coding gene?
What is the approximate size of the average human protein-coding gene?
Which of these is NOT a reason for cloning DNA?
Which of these is NOT a reason for cloning DNA?
What is involved in the fusion of a selected gene with a vector?
What is involved in the fusion of a selected gene with a vector?
Which step follows after the delivery of recombinant DNA into a host cell in the process of generating a transgenic organism?
Which step follows after the delivery of recombinant DNA into a host cell in the process of generating a transgenic organism?
How many base pairs approximately make up the human genome?
How many base pairs approximately make up the human genome?
What is the purpose of using PCR in gene modification?
What is the purpose of using PCR in gene modification?
Which step directly follows the isolation/amplification of selected genes in the process of generating a genetically modified organism?
Which step directly follows the isolation/amplification of selected genes in the process of generating a genetically modified organism?
What is the primary role of vectors in genetic engineering?
What is the primary role of vectors in genetic engineering?
What characteristic is common to plasmids used as cloning vectors?
What characteristic is common to plasmids used as cloning vectors?
Which statement best describes the copy number of plasmid vectors?
Which statement best describes the copy number of plasmid vectors?
Which of the following is NOT a common type of vector used in genetic engineering?
Which of the following is NOT a common type of vector used in genetic engineering?
What is a key feature of a vector that assists in the selection of host cells with successfully delivered DNA?
What is a key feature of a vector that assists in the selection of host cells with successfully delivered DNA?
How are plasmids generally structured?
How are plasmids generally structured?
What is a key feature of the plasmid vector pBR322?
What is a key feature of the plasmid vector pBR322?
What is the purpose of the antibiotic resistance genes in pBR322?
What is the purpose of the antibiotic resistance genes in pBR322?
How does the removal of the rop gene affect the plasmid copy number?
How does the removal of the rop gene affect the plasmid copy number?
What is the function of the BamHI site in the cloning process?
What is the function of the BamHI site in the cloning process?
What characteristic differentiates colonies based on plasmid insertion?
What characteristic differentiates colonies based on plasmid insertion?
What is the main advantage of relaxed replication in plasmids?
What is the main advantage of relaxed replication in plasmids?
What modification to pBR322 led to an improvement in cloning efficiency?
What modification to pBR322 led to an improvement in cloning efficiency?
Which statement about pBR322 is true?
Which statement about pBR322 is true?
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Study Notes
Generating Transgenic Organisms
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Process Steps:
- Isolate and amplify selected genes.
- Fuse the selected gene with a suitable vector to recombine DNA.
- Deliver the recombinant DNA into a host cell.
- Select the genetically modified host.
- Breed the modified host for offspring with desired genetic traits.
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Applications:
- DNA cloning.
- Recombinant protein production.
- Development of desirable phenotypes.
Generating Recombinant DNA
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Isolation of Genes:
- Any DNA from any organism can be cloned, including genomic DNA, mRNA, or synthesised DNA.
- Purposes for cloning include protein expression, sequencing, and functional studies.
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Human Genomic Context:
- The human genome consists of approximately 3.2 billion base pairs (bp).
- Average size of a human protein-coding gene, including introns, is about 62,000 bp, which is roughly 0.002% of the genome.
- The human insulin gene has a length of 1,425 bp.
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Polymerase Chain Reaction (PCR):
- PCR involves three steps: denaturation, annealing, and extension.
- It can be used to introduce new restriction enzyme (RE) sites for cloning and to create point mutations in target sequences.
Vectors in Genetic Engineering
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Purpose of Vectors:
- Vectors serve as molecular carriers for foreign DNA into host cells.
- They can replicate within host cells, facilitating gene expression.
- Often designed to allow transcription and translation of the foreign DNA.
- Can carry antibiotic resistance genes to aid selection of successfully modified cells.
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Common Types of Vectors:
- Plasmids.
- Viral vectors (e.g., phages).
- Cosmids.
- Artificial chromosomes.
Plasmids as Cloning Vectors
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Characteristics of Plasmids:
- Naturally occurring, extrachromosomal DNA found in bacteria, 1-250 kb in size.
- Typically exist as closed circular double-stranded DNA.
- Can carry genes that confer useful traits, such as antibiotic resistance.
- Copy number depends on replication type:
- Stringent: 1-2 copies per cell.
- Relaxed: More than 50 copies per cell.
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Example - Plasmid Vector pBR322:
- One of the earliest plasmids for DNA cloning, established in 1977.
- Contains an origin of replication for plasmid replication.
- Features a gene (rop) to regulate copy number (~20 copies/cell).
- Includes two antibiotic resistance genes (tetracycline and ampicillin) for easy selection.
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Improved Plasmid Vector:
- Derived from pBR322, incorporates a Lac promoter/operator.
- Can generate 500-700 copies per cell after removal of the rop gene, enhancing cloning efficiency and selection of colonies.
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