L5-6 Recombinant DNA Technology Flashcards
Understand the principles and potential applications of recombinant DNA technologies.
Recombinant DNA is when two pieces of DNA are…
ligated artificially to perform a modified function
The resources required to create recombinant DNA are (4)…
- Enzymes
- Vector
- Cells
- DNA or RNA raw material
Four groups of enzymes are used in the production of recombinant DNA…
- Restriction endonuclease (site-specific cleavage)
- DNA ligase (the ‘glue’)
- TAQ polymerase (used in PCR)
- Reverse transcriptase (converts RNA back to DNA)
Restriction endonucleases originated as bacterial self-defence mechanisms. They tend to recognise what sort of sequences?
4-8 base pair palindromic sequences
Clevage can be symmetrical (resulting in blunt ends), or…
Asymmetrical (resulting in ‘sticky ends’)
Vectors are a means of introducing exogenous DNA into…
Host cells
Plasmids are commonly used as…
Vectors
Vectors other than plasmids include (2)…
Bactereophages and Cosmid/phagemids
Specially made plasmids often have a variety of ? to ensure compatibility with a variety of restriction enzymes
Restriction sites (known as a Multiple Cloning Site, MCS, or a polylinker)
A specific method for isolating the gene we want to insert involves…
Separating strands, attaching primers and amplifying with PCR
There are two ‘random’ ways to isolate the required gene, these are…
- Isolate the DNA, cleave, leaving random fragments
2. Isolate the mRNA, use reverse transcriptase, producing a cDNA library
cDNA stands for…
Complementary DNA
In order to introduce a vector into the cell, it must be made…
Competent
A cell may be made competent via…
🔥 Heat shock + CaCl
Adding exogenous DNA to the cell is known as…
Transformation
A further way to check if the cells cultured contain the recombinant DNA is to…
Use a hybridisation probe (ssDNA) for the sequence of interest (Southern blot)
CFU stands for…
Colony-Forming Unit
For a recombinant gene to successfully expressed as a recombinant protein, it must contain a…
Promoter recognised by the cell’s RNA polymerase
How do we ensure the insert is joined to the plasmid in the correct orientation?🔁
Use a different restriction endonuclease for each end
plasmid—-⧑ new gene ◑—plasmid
Define: intron
A non-coding region of DNA removed during maturation of mRNA by spliceosomes
Reverse transcriptase is used to obtain cDNA from mRNA, rather than using spliceosomes, because…
Splicesomes are complex and difficult to assemble/utilise
We can choose from four main Expression Systems to express our recombinant DNA into a protein (4)…
- Bacterial
- Yeast
- Insect
- Mammalian
Insect cells are a good alternative to mammalian cells as (5)…
They are capable of high-level protein expression
Correct folding of mammalian proteins
Post-translational modification
Cheaper than mammalian cells
Fewer ethical issues
