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What is the structure of a typical bacteriophage?
What is the structure of a typical bacteriophage?
What is the name of the bacterial species that bacteriophage lambda infects?
What is the name of the bacterial species that bacteriophage lambda infects?
Escherichia coli
Bacteriophage lambda DNA is a circular double-stranded molecule.
Bacteriophage lambda DNA is a circular double-stranded molecule.
False (B)
Which of the following is NOT a strategy for using lambda phage as a cloning vector?
Which of the following is NOT a strategy for using lambda phage as a cloning vector?
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Which of the following is a feature of a cosmid vector?
Which of the following is a feature of a cosmid vector?
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What is the maximum insert size that can be cloned into a cosmid vector?
What is the maximum insert size that can be cloned into a cosmid vector?
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What is the name of the genus of bacteria that is a vector for transferring genes into plants?
What is the name of the genus of bacteria that is a vector for transferring genes into plants?
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Agrobacterium tumefaciens is a gram-positive soil bacterium that naturally transforms plant cells, resulting in crown gall tumors.
Agrobacterium tumefaciens is a gram-positive soil bacterium that naturally transforms plant cells, resulting in crown gall tumors.
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What is the name of the DNA segment that is transferred from the Ti plasmid into the plant cell's genome?
What is the name of the DNA segment that is transferred from the Ti plasmid into the plant cell's genome?
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Which of the following is NOT a component of the Ti plasmid?
Which of the following is NOT a component of the Ti plasmid?
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The vir genes on the Ti plasmid are responsible for conjugative transfer of the T-DNA to the plant cell.
The vir genes on the Ti plasmid are responsible for conjugative transfer of the T-DNA to the plant cell.
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What is the name of the type of vector system that uses two plasmids, a disarmed Ti plasmid and a helper vector, to transfer genes into plants?
What is the name of the type of vector system that uses two plasmids, a disarmed Ti plasmid and a helper vector, to transfer genes into plants?
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What is the name of the vector system that uses a single plasmid that is integrated into the Ti plasmid to transfer genes into plants?
What is the name of the vector system that uses a single plasmid that is integrated into the Ti plasmid to transfer genes into plants?
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Which of the following is a feature of shuttle vectors?
Which of the following is a feature of shuttle vectors?
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What is the name of the type of artificial chromosome that is derived from the DNA of yeast?
What is the name of the type of artificial chromosome that is derived from the DNA of yeast?
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Which of the following is NOT a feature of a YAC vector?
Which of the following is NOT a feature of a YAC vector?
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The maximum insert size that can be cloned into a YAC is about 100 kbp.
The maximum insert size that can be cloned into a YAC is about 100 kbp.
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What is the name of the type of artificial chromosome that is derived from the DNA of bacteria?
What is the name of the type of artificial chromosome that is derived from the DNA of bacteria?
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BAC vectors contain a single copy of the F-plasmid origin of replication.
BAC vectors contain a single copy of the F-plasmid origin of replication.
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What is the name of the type of artificial chromosome that is derived from the DNA of the P1 bacteriophage?
What is the name of the type of artificial chromosome that is derived from the DNA of the P1 bacteriophage?
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What is the name of the process that involves the direct transfer of DNA into a cell using a small glass needle or micropipette?
What is the name of the process that involves the direct transfer of DNA into a cell using a small glass needle or micropipette?
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Lipofection is a method of gene transfer that involves the use of liposomes to deliver DNA into cells.
Lipofection is a method of gene transfer that involves the use of liposomes to deliver DNA into cells.
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What is the name of the technique that involves the use of high voltage electrical pulses to create pores in the cell membrane, allowing DNA to enter the cell?
What is the name of the technique that involves the use of high voltage electrical pulses to create pores in the cell membrane, allowing DNA to enter the cell?
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Which of the following is NOT a method of direct gene transfer?
Which of the following is NOT a method of direct gene transfer?
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What is the name of the technique that involves the use of a gene gun to deliver DNA into cells?
What is the name of the technique that involves the use of a gene gun to deliver DNA into cells?
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DNA extraction is a procedure used to isolate RNA from the nucleus of cells.
DNA extraction is a procedure used to isolate RNA from the nucleus of cells.
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Which of the following is NOT a step involved in DNA extraction?
Which of the following is NOT a step involved in DNA extraction?
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What is the name of the method of DNA extraction that uses phenol and chloroform to separate DNA from other cellular components?
What is the name of the method of DNA extraction that uses phenol and chloroform to separate DNA from other cellular components?
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Which of the following is NOT a method of DNA extraction?
Which of the following is NOT a method of DNA extraction?
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Solid-phase extraction is a method of DNA extraction that uses a solid substrate, such as silica resins or beads, to isolate DNA.
Solid-phase extraction is a method of DNA extraction that uses a solid substrate, such as silica resins or beads, to isolate DNA.
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RNA extraction is a more difficult process than DNA extraction because RNA is less stable and more susceptible to degradation than DNA.
RNA extraction is a more difficult process than DNA extraction because RNA is less stable and more susceptible to degradation than DNA.
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What is the name of the technique used to separate DNA or RNA molecules based on their size?
What is the name of the technique used to separate DNA or RNA molecules based on their size?
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The pore size in an agarose gel is controlled by the concentration of agarose used to make the gel.
The pore size in an agarose gel is controlled by the concentration of agarose used to make the gel.
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Which of the following is NOT a factor that affects the electrophoretic migration rate of nucleic acids?
Which of the following is NOT a factor that affects the electrophoretic migration rate of nucleic acids?
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What is the name of the process by which two single-stranded nucleic acids with complementary sequences associate to form a double-stranded molecule?
What is the name of the process by which two single-stranded nucleic acids with complementary sequences associate to form a double-stranded molecule?
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Which of the following is NOT a method for marking probes?
Which of the following is NOT a method for marking probes?
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Study Notes
Bacteriophage Lambda
- Bacteriophage lambda is a bacterial virus that infects Escherichia coli.
- Its structure includes a head, tail, and tail fibers.
- Viral DNA is linear, double-stranded, and 48.5 kbp with 12 base pair sticky ends.
- The sticky ends are complementary, allowing them to hybridize.
Bacteriophage Lambda as a Cloning Vector
- Lambda phage is used as a vector using two strategies.
- Insertional vectors: The insert DNA is cloned into the multiple cloning site (MCS) within the lacZ coding sequence. Cloning capacity is 13 kbp.
- Replacement vectors (or substitution vectors): A central stuffer is swapped with the insert DNA. Cloning capacity is 23 kbp.
- Substitution vectors are generally preferred but more complex to implement.
Cosmid as a Cloning Vector
- Cosmids are artificial vectors, essentially plasmids with one or two cos sites from lambda phage.
- The cos sites allow in vitro packaging of cosmid DNA into lambda phage particles.
- Insert size can be up to 45 kbp.
- Cosmids contain plasmid features (origin of replication, multiple cloning site, selectable marker) and lambda phage features (cos sites).
Advantages and Limitations of Cosmids
- Advantages: Cloning of large inserts (up to 45 kbp), infection process (rather than transformation), maintenance of recombinant phage particles, and cosmid reproduction as a large plasmid without destroying the infected bacteria.
- Limitations: Complex realization and DNA rearrangements can occur.
Agrobacterium as a Vector
- Agrobacterium tumefaciens is a gram-negative soil bacterium that naturally transforms plant cells, resulting in crown gall tumors.
- Infection occurs through breaks or wounds.
- Tumor formation results from T-DNA integration into the plant genome.
Ti Plasmid of Agrobacterium
- Virulent strains of A. tumefaciens harbor large plasmids (140-235 kbp) often called Ti plasmids, with elements like:
- T-DNA (with left and right borders).
- Virulence (vir) region genes involved in T-DNA transfer.
- Origin of replication.
- Region enabling conjugative transfer.
- O-cat region (for opine catabolism).
Ti Plasmid Based Vectors
- Binary systems: Uses two vectors: a disarmed Ti plasmid (with gene of interest but no vir genes), and a helper vector (with vir genes).
- Co-integrated vectors: Involves three vectors; a disarmed Ti plasmid, an intermediate vector, and the helper vector.
Retroviral Cloning Vectors
- Retroviral vectors are capable of reverse-transcribing RNA into DNA, enabling stable integration into the host genome.
- Useful for introducing foreign genes into target cells.
- Widely used vectors (based on RNA viruses) include murine leukemia viruses and lentiviruses.
Retroviral Vector Genome
- Contains:
- LTR (long terminal repeat)
- PBS (primer binding site)
- PPT (polypurine track)
- "pol" gene
- "gag" gene
- "env" gene
- Packaging signal site
- 5' and 3' untranslated regions
Shuttle Cloning Vectors
- Shuttle vectors can replicate in two different organisms (e.g., bacteria and yeast).
- They include two origins of replication and two selection markers, one for each organism.
Artificial Chromosome Cloning Vectors
- Include Yeast artificial chromosomes (YACs), P1 artificial chromosomes (PACs), and bacterial artificial chromosomes (BACs).
- Useful for genome sequencing, functional characterization of genomic regions, and transduction of large DNA segments.
YACs
- Genetically engineered yeast chromosomes.
- Consist of two telomeric sequences, a yeast centromere, a yeast ARS, and selectable markers (e.g., URA3).
- Average cloning capacity of 100 kbp to 2 Mbp.
BACs
- Designed for cloning large DNA fragments (100-300 kbp) in E. coli.
- Contain a single copy F-plasmid origin of replication.
- Useful for carrying various genetic material.
P1-derived Artificial Chromosomes (PAC)
- Derived from the DNA of P1 bacteriophages, and bacterial artificial chromosome.
- Can carry larger amounts (100-300 kb) of DNA.
Human Artificial Chromosomes (HAC)
- Mini-chromosomes constructed artificially in human cells.
- Have their own self-replicating and segregating systems, behaving independently from host chromosomes.
Expression Vectors
- Plasmids or viruses designed for expressing genes (to produce protein).
- Have elements like origin of replication, multiple cloning site (MCS), regulatory elements (promoters, enhancers, terminators, initiation sites, stop codons), and selectable markers.
Methods for Direct DNA Transfer into Eukaryotic Cells (non-viral)
- Calcium Phosphate: DNA forms a precipitate taken up by the cells via endocytosis.
- Electroporation: High voltage electrical pulses create transient pores in the plasma membrane, allowing DNA entry.
- Microinjection: DNA is directly injected into cells using a fine glass needle/micropipette.
- Liposomes: Hollow spheres of phospholipid that carry DNA into cells.
- Gene Gun: Propel DNA-coated particles into cells; useful for plant tissues.
Nucleic Acid Extraction
- Isolates DNA from cellular components for further investigations (PCR, sequencing, etc.).
- Two main methods:
- Phenol-chloroform extraction
- Solid-phase extraction
Gel Electrophoresis
- Separates DNA fragments based on size.
- Agarose is a linear polymer gel used.
Molecular Hybridization
- Association of complementary single-stranded nucleic acids.
- Hydrogen bonds (A-T, C-G) form a double-strand or duplex.
- Probes are used for identifying specific nucleic acid sequences. They can either use radioactive or non-radioactive tags.
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Description
Explore the fascinating world of bacteriophage lambda as both a viral entity and a crucial cloning vector in genetic engineering. This quiz covers its structure, usage strategies as insertional and replacement vectors, and the role of cosmids in molecular biology. Test your knowledge on these essential tools in biotechnology!
