Ch 9: Biotechnology and Recombinant DNA Flashcards
What is the function of restriction enzymes?
- Cut DNA at defined positions close to or within their recognition sequence
- Typically recognize 4, 6, or 8 base sequences
True or false. Blunt ends of DNA can only ligate with specific blunt ends.
False. Any blunt ends can ligate together
True or false. Stick-end DNA overhangs must be complementary in order to ligate.
True
Why do bacteria produce restriction enzymes?
They restrict the ability of foreign DNA (such as bacteriophage DNA) to infect/invade the host cell by cleaving it
How is host DNA modified to protect it from its own restriction enzymes?
Methylation of their sequences at C or A nucleotides
What E. coli plasmid vector is commonly used for cloning?
pUC19
What protein does the gene lacZ encode for?
β-galactosidease
What protein does the gene ampR encode for?
Ampicillin resistance
What stain is used to visualize DNA on an agarose gel?
Ethidium Bromide
What are some methods of transformating DNA into cells in the lab? (5)
- Electroporation
- Chemical transformation
- Protoplast fusion
- Gene gun
- Microinjection
What is the difference between transformation and transfection?
Transformation: uptake of DNA into a prokaryotic cell; term means something different in mammalian cells
Transfection: uptake of DNA into mammalian cells
True or false. Most cell types don’t naturally transform.
True
How does electroporation work?
- Controlled short, but powerful electrical pulse induces temporary hydrophilic pores in in the cell membrane
- DNA can enter the cell
- Applicable to all cells
- Organisms with cell walls may require conversion to protoplast
Explain how chemical transformation works in E. coli
- Cells incubated in ice-cold calcium chloride
- DNA is added
- Mild heat shock
Explain how chemical transformation works for yeast
- Incubated in ice-cold lithium chloride
- DNA added
- Mild heat shock
Rates of protoplast fusion increase in the presence of _____
Polyethyleneglycol (PEG)
How does a gene gun (or biolistic transformation) work?
- Microscopic gold or tungsten particles (1 μm) coated with DNA propelled into cells with burst of helium
- If a particle lands on the nucleus the DNA may be incorporated into the chromosome
In what organisms is biolistic transformation typically used? (3)
- Plant cells
- C. elegans
- Yeast mitochondria
How does microinjection for transformation work?
- Uses glass micropipette to inject DNA into the cell
True or false. Microinjection is most commonly used for bacterial and fungal transformations.
False. Bacteria and fungi are too small. This method is impractical.
What is a DNA library?
A collection of DNA fragments of one organism, each carried by a plasmid or virus and cloned in an appropriate host
What are the two types of DNA libraries?
- Genomic library: DNA fragments representing an entire organism’s genome
- cDNA library: only complementary DNA molecules synthesized from mRNA molecules in a cell
What nucleotide sequence does the restriction enzyme Sau3A recognize?
GATC
In genomic library construction, how many genome equivalents are necessary to ensure that all genomic sequences will be represented with 95% probability?
5 genome equivalents
In genomic library construction, how many genome equivalents are necessary to ensure that all genomic sequences will be represented with 99% probability?
10 genome equivalents
cDNA is made from mRNA by what enzyme?
Reverse transcriptase
What are oligonucleotides (ssDNA)?
Synthetic DNA that can be made chemically (not enzymatically) on a solid matrix using a DNA synthesis machine
What are some limitations of ssDNA?
- Limited to less than 200 bases in length
- Gene sequence will be ambiguous when only the protein sequence is available
What cell types are commonly used to make gene products?
- E. coli
- Saccharomyces cerevisiae
- Plant cells and whole plants
- Mammalian cells
What are some disadvantages of using E. coli to make gene products?
- Need to eliminate endotoxins from the products, since E. coli is gram-negative
- Does not normally secrete products; need to lyse the cells to get products, which is expensive on an industrial scale
What bacteria is alternatively used instead of E. coli to harvest gene products via secretion rather than lysing?
Bacillus subtilis
What are some advantages of using Saccharomyces cerevisiae to make gene products? (4)
- Easily grown with well-understood genomics
- Can carry plasmids
- Greater chance of expressing and correctly modifying eukaryotic genes and proteins
- Likely to continuously secrete products
What are some advantages of using plant cells to make gene products? (3)
- May express eukaryotic genes easily
- Low risk of product contamination by mammalian pathogens
- Large scale low-cost production
What are some advantages of using mammalian cells to make gene products? Disadvantage?
Adv:
- May express eukaryotic genes easily
- Well suited to make proteins for medical use; secreted and low risk of toxins
Disadv:
- Harder to grow
What are some therapeutic applications of rDNA?
- Human enzymes and other proteins (insulin, etc.)
- Subunit vaccines
- DNA vaccines
- Gene therapy
What is gene therapy?
The introduction, removal, or change in genetic material in the cells of a patient to treat an inherited or developed disease.
Insulin’s subunits are composed of _____ amino acids.
21 - 30
(per subunit, insulin has 2)
What vector is showing promise for use in gene therapy?
Adenovirus vectors
_______ is a eukaryotic biological process in which RNA molecules inhibit gene expression
RNA intererence (RNAi) or gene silencing
What is the difference between gene editing and gene therapy?
Editing: remove, disrupt, or correct faulty elements of DNA within the gene
Therapy: replaces the entire gene
What 3 components are required for gene editing?
- Cas9
- Guide RNA
- New designed DNA
CRISPR stands for:
clustered regularly interspaced short palindromic sequences
_____% of the human genome encodes for product, while the remainder is “junk DNA”
2%
What are some of the components of “junk DNA” in the human genome? (7)
- miRNA genes
- Short tendem repeats
- Introns
- Telomeres
- Transposons
- Viral remnants
- Pseudogenes
_____ is the science of understanding gene function through computer assisted analysis.
Bioinformatics
The bacterium _______ infects specific plants at wound sites with plasmid DNA (Ti plasmid) that contains genes for opine synthesis and tumor production (crown gall)
Agrobacterium tumefaciens
