21.2 - In Vivo Gene Cloning — The Use Of Vectors Flashcards
What are the two ways DNA fragments can be cloned?
In vivo: Transferring fragments to a host cell using a vector.
In vitro: Using the polymerase chain reaction (PCR).
What is necessary to find the DNA fragment with the required gene?
A DNA probe
What are recognition sites in DNA cloning?
Recognition sites are specific DNA sequences cut by restriction endonucleases, often in a staggered fashion, leaving sticky ends.
Why are sticky ends important?
- Sticky ends are complementary and can pair with any other sticky ends cut by the same restriction endonuclease.
- DNA ligase binds the phosphate-sugar backbone, uniting the fragments into one.
- Sticky ends allow the DNA of one organism to be combined with the DNA of another.
What additional DNA regions must be added to a DNA fragment for transcription to occur?
- Promoter region: Binds RNA polymerase and transcription factors to begin transcription.
- Terminator region: Releases RNA polymerase to stop transcription.
What is a vector, and what is its role?
A vector is a carrying unit used to transport the DNA fragment into the host cell.
What is the most common type of vector?
Plasmids—circular DNA found in bacteria, often carrying antibiotic-resistance genes.
How is DNA inserted into plasmids?
- Restriction endonucleases cut the plasmid and DNA fragment using the same enzyme to ensure complementary sticky ends.
- DNA ligase permanently joins the DNA fragment and plasmid, forming recombinant DNA.
What is transformation in DNA cloning?
The process of reintroducing plasmids into bacterial cells.
How is transformation achieved?
- Plasmids and bacterial cells are mixed in a medium containing calcium ions.
- Temperature changes make bacterial membranes permeable, allowing plasmids to enter.
Why don’t all bacterial cells take up the desired plasmids?
- Few bacterial cells (as low as 1%) take up plasmids.
- Some plasmids close without incorporating the DNA fragment.
- DNA fragments may join to form their own plasmid
What is the first step in identifying bacterial cells with plasmids?
Grow all bacterial cells on a medium containing ampicillin.
- Bacteria with plasmids survive due to ampicillin resistance.
- Bacteria without plasmids die.
Why doesn’t the first step confirm the presence of the desired gene?
Some plasmids survive by closing without incorporating the desired gene.
What are marker genes, and why are they used?
Marker genes are used to identify bacterial cells that have taken up the desired gene. They can:
1) Provide resistance to an antibiotic.
2) Produce a fluorescent protein.
3) Produce an enzyme with an identifiable action.
How are antibiotic-resistance genes used as markers?
- The required gene is inserted into an antibiotic-resistance gene (e.g., tetracycline resistance).
- Cells with the required gene lose resistance to tetracycline.
What is replica plating, and why is it used?
A technique to identify living bacterial colonies containing the required gene without killing them.
How are fluorescent markers used in DNA cloning?
- A green fluorescent protein (GFP) gene from jellyfish is inserted into the plasmid.
- The required gene is transplanted into the GFP gene.
- Bacterial cells with the desired gene do not fluoresce, while those without it fluoresce under a microscope.
Why is the method of fluorescent markers advantageous?
It is rapid, does not require replica plating, and does not kill the bacterial cells with the desired gene
How are enzyme markers used in DNA cloning?
- The required gene is inserted into a gene that produces lactase.
- Lactase turns a colorless substrate blue.
- Bacterial cells with the required gene do not produce lactase and cannot turn the substrate blue
How are bacterial cells without the required gene identified?
Bacteria without the required gene turn the substrate blue and are excluded.
