ch 10 (lectures 19-20) Flashcards
What are the basic steps to create a transgenic mouse using a jellyfish gene?
- Cut the genomic DNA of the jellyfish to isolate the gene of interest.
- Cut a DNA vector and insert the jellyfish gene into it.
- Transform E. coli with the plasmid and replicate it to make many copies (amplification).
- Screen to find the plasmid with the desired gene.
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Inject the plasmid into mouse cells to create a transgenic mouse (it has both mouse genes and foreign genes from another species) that expresses the jellyfish gene (e.g., glows in the dark).
(Note: The vector must contain a mouse-specific promoter and bacterial elements for replication and selection.)
What does in vitro mean?
In a test tube or outside a living organism.
What does in vivo mean?
Inside a living organism.
What is a vector in genetic engineering?
A plasmid or bacterial virus used to carry and deliver DNA sequences of interest.
What is recombinant DNA?
Artificially created DNA formed by combining DNA from different sources.
What are restriction enzymes?
Enzymes that recognize specific DNA sequences and cut the DNA.
What are the two types of ends produced by restriction enzymes?
Sticky (staggered) ends and blunt ends.
What is a DNA palindrome?
A sequence that reads the same in the 5′ to 3′ direction on both strands.
What are the basic steps for cloning a gene?
- Isolate the DNA fragment containing the gene of interest (in vitro)
- Insert it into a plasmid vector (in vitro)
- Transform bacteria and grow to amplify the plasmid with the gene (in vivo)
What are the steps in forming a recombinant DNA molecule?
- Cut both the plasmid and DNA fragment with the same restriction enzyme to create compatible ends (usually palindromic).
- Mix and incubate with DNA ligase to join the fragments.
- Transform the recombinant plasmid into bacteria to propagate and amplify.
What is a DNA library and how is it created?
A DNA library is a collection of DNA fragments that have been cloned into vectors (plasmids) using the same recombinant DNA method.
What is gel electrophoresis and what is it used for?
Gel electrophoresis is a technique used to separate DNA molecules by size, allowing for the selection of specific DNA fragments.
What is PCR (Polymerase Chain Reaction) and what does it require?
PCR is used to amplify DNA in a region of interest using two primers.
It requires:
* Template DNA
* Primers
* dNTPs
* Buffer + Mg²⁺
* Heat-resistant DNA polymerase (e.g., Taq polymerase)
Primers must be designed based on known DNA sequences.
What is cDNA and how is it synthesized from mRNA?
cDNA (complementary DNA) is synthesized from mRNA using reverse transcription.
The reaction requires:
* Template RNA
* Primers (usually an oligo-dT primer)
* dNTPs
* Buffer + Mg²⁺
* Reverse transcriptase
* Heat-resistant DNA polymerase (e.g., Taq polymerase)
How can PCR be used to produce DNA with sticky ends for cloning?
By designing PCR primers that include specific DNA sequences (such as restriction sites), sticky ends can be introduced into the PCR product. These added sequences enable easier cloning into vectors using restriction enzymes.
How can sticky ends be added to cDNA molecules for cloning?
Sticky ends can be added to cDNA by ligating synthetic DNA oligonucleotides containing restriction sites to the ends of the cDNA. This facilitates insertion into cloning vectors.
How is DNA amplification achieved using bacteria?
DNA is inserted into a plasmid and introduced into E. coli, which propagates and amplifies the DNA due to its short cell cycle and ease of cultivation.
What are the key features of a plasmid vector (like pUC18) used for cloning?
A plasmid vector needs:
* Origin of replication
* Selection marker (e.g., antibiotic resistance)
* Cloning site (a region for inserting DNA without disrupting plasmid function)
Often also includes:
* Reporter gene (to distinguish cloned vs. non-cloned plasmids)
* Polylinker (multiple unique restriction sites for flexibility in cloning)
How does blue/white selection work in cloning?
The polylinker is placed within the LacZ gene.
No insert: LacZ is functional → cleaves X-gal → blue colonies
Insert present: LacZ is disrupted → cannot cleave X-gal → white colonies
This allows easy visual separation of plasmids with insert (white) from those without (blue).
What are fosmids and BACs, and what are they used for?
Fosmids (F-plasmid + λ phage DNA) and BACs (F-plasmid) are cloning vectors that can carry large DNA inserts. They contain elements from the F’ factor and bacterial chromosome, allowing stable maintenance of big DNA fragments. They were mainly used in genome sequencing projects.
What is Gibson Assembly and what are its key steps in cloning?
Gibson Assembly is a cloning method that does not require restriction digestion.
It uses dsDNA fragments with 15–40 bp overlaps (homologous ends), which can be generated by PCR or synthesized.
Steps in Gibson Assembly:
* 5′ Exonuclease Processing: The overlapping ends are processed to create single-stranded regions.
* Annealing: The complementary single-stranded overhangs anneal (staggered ends come together).
* Gap Filling: DNA polymerase fills in the gaps.
* Ligation: DNA ligase seals any remaining nicks to complete the assembly.
What is plasmid synthesis and what are its typical size limits in modern cloning?
Plasmid Synthesis refers to the artificial construction of plasmid vectors by specialized companies.
It enables the design and creation of custom plasmids without traditional cloning methods.
The current size limit for synthesized plasmids is typically between 5–15 kb.
How can DNA probes be used to find a vector with the desired cloned DNA?
A short radioactive ssDNA probe is hybridized to denatured DNA from colonies or spots.
If the probe binds (due to sequence complementarity), the signal can be detected on film.
* This allows you to identify which colony contains your gene of interest.
* You can then align the film with the original plate and select the positive colony for further propagation.
Similar techniques can be used to detect RNA as well.
How can antibodies be used to find a vector with the desired cloned gene?
Antibodies are used to detect proteins expressed from the cloned gene.
Colonies or plaques are screened with an antibody that specifically binds the protein of interest.
A signal (e.g., color or radioactivity) identifies colonies expressing the target protein.
This method works with phage or plasmid vectors, making it useful for identifying clones when the gene is expressed as protein.
