20 Biotechnology Flashcards
What is recombinant DNA?
DNA molecules from from segments of two different sources
What is recombinant DNA?
DNA molecules from from segments of two different sources
What is a common gene cloning occurs is formed? (not virus)
With bacterial ‘plasmids’
What does ’gene cloning’ refer to?
The production of multiple copies of the same gene
What is it called when genes are duplicated for research?
‘Gene cloning;
How is gene cloning performed with plasmids?
The plasmid from a bacterium i.e. E coli is extracted.
The gene of interest is taken from another cell and added to the plasmid, forming recombinant DNA.
The plasmid is then returned to the bacterium which divides to form new copies of the plasmid and thus gene of interest.
What is a typical bacterium used for gene cloning using plasmids?
E. coli
What is the most common method to produce recombinant DNA?
The use of ‘restriction enzymes’
How is bacterial DNA protected from degradation by its own restriction enzymes?
By the addition of methyl groups (-CH3) to adenines or cytosines within the sequences recognized by the enzymes.
How are restriction enzymes used to produce recombinant DNA?
‘Restriction enzymes’ bind to specific base pair sequences called ‘restriction site.’
Within this site they break the DNA at particular points. This forms two ‘restriction fragments.’
Due to the way the DNA was broken the ends probably overlap i.e. ==⎻ and _==. This leaves an unpaired region called the ’sticky end.’
Two restriction fragments from different DNA molecules can combine if their sticky ends are complementary. DNA ligase seals the strands.
What does ’restriction site’ refer to?
The specific base sequence that a particular restriction enzymes binds to.
What is the place that the restriction enzymes bind to called?
The restriction site
What are the resultant strands of DNA after restriction called?
Restriction fragments
What is a DNA molecule that can carry foreign DNA into a host cell called?
A ‘cloning vector’
What is a cloning vector?
A DNA molecule that can carry foreign DNA into a host cell where it can replicate.
What is special about bacterial plasmids derived from E.coli?
They often have genetically engineered amp R and LacZ genes
What does ’ampR’ refer to?
A gene often added to bacterial plaids to make them resistant to the antibiotic ampicillin.
How can it be determined whether bacteria are recombinant? (non-genetic method)
The plasmids are engineered so that they have the ampR gene (ampicilin resistance) and the LacZ gene to break down lactose. Conversely the original host bacteria are engineered to not have the LacZ gene and thus can’t break down lactose.
The first step in determining whether the bacteria are recombinant is to dose them with ampicillin. Only those who are resistant could have taken up the plasmid.
Second, the presence of X-gal in the medium allows us to distinguish colonies with recombinant plasmids from those with nonrecombinant plasmids. Colonies containing non- recombinant plasmids have the lacZ gene intact and will produce functional β-galactosidase. These colonies will be blue because the enzyme hydrolyzes the X-gal in the medium, forming a blue product. The others will be white
What does X-gal’’ refer to?
A substance which when broken down forms a blue product. Otherwise it is white.
Often used to determine if bacteria are recombinant.
What is a collection of an organism’s gene in the form of plasmids called?
A plasmid library/genomic library
What does ’genomic library’ refer to?
A collection of all an organism’s DNA in an accessible form i.e. in many plasmids
What does ‘plasmid library’ refer to?
A collection of all an organism’s DNA in the form of many plasmids
What vector is typically used for library construction?
Bacterial artificial chromosome (BAC) although plasmids can still be used.
What does ’BAC’ refer to?
Bacterial artificial chromosome
What are Bacterial artificial chromosomes?
A specialised vector in which all but the genes necessary for replication are removed.
What is the fundamental different between normal plasmids and BAC?
In plasmids 90% of the DNA is normal non-recombinat DNA.
However in BACs the genes carried are 1000 times larger than the vector itself.
Besides Plasmid libraries, what is a common way of collecting all of an organism’s DNA?
With cDNA (complementary DNA) libraries
What does ‘cDNA’ stand for?
Complementary DNA
What is cDNA?
DNA which is formed from mRNA. It is therefore complementary to the RNA and thus identical to the original DNA
What is the fundamental way cDNA libraries differ from plasmid libraries?
Plasmid libraries contain the entirety of an organism’s DNA.
Conversely cDNA is contracted from mRNA and thus contains only the expressed genes. This means that is does not include promoter regions etc., silenced genes or introns.
How is cDNA formed?
Reverse transcrip- tase is added to a test tube containing mRNA isolated from a certain type of cell.
Reverse transcriptase makes the first DNA strand using the mRNA as a template and a stretch of dT’s (lots of Thymine) as a DNA primer.
mRNA is degraded by another enzyme.
DNA polymerase synthesizes the second strand, using a primer in the reaction mixture.
Thus cDNA is formed without introns.
What is a common method for detecting a specific sequence of DNA?
Nucleic acid hybridisation
What is nucleic acid hybridisation used for?
Detecting a specific sequence of DNA
How does nucleic acid hybridisation work?
The DNA wanting to be screened is stored in ‘multiwell plates’ i.e. those things we used when doing food tests or mixing paints. cells form each are transferred onto specific places on a nylon membrane.
’Nucleic acid probes’ are synthesised that are complementary for the sequence to be found. They are then tagged with radiation etc.
The nucleic acid probes are then added to the multi well plates so that they bind to complementary sequences. Any excess are washed away.
Photographic plate is then placed over the multi wells. Any regions where the probes are present will cause dark spots on the film, indicating that the desired sequence is at that location.
What is a common issue when adding eukaryotic genes to bacteria? How can this be solved
The eukaryotic genes often have promoter regions that are are not recognised but the bacteria and thus not expressed.
This is solved with ‘expression vectors’
What are expression vectors?
A cloning vector that has a highly active bacterial promoter upstream of the restriction site so that the downstream eukaryotic gene is still expressed.
How are recombinant plasmids formed?
With restriction enzymes.
Why is the use of cDNA beneficial?
- It shortens the gene segment and thus allows more to be placed in one vector
- Prokaryotes do not posses the capability to remove introns. Therefore the cDNA form, which lacks introns, is used when RNA splicing must occur for the gene to function.
What are the basic ways DNA is added to a cells besides with plasmids?
Electroporation and with Agrobacterium or with tiny needles
What is a electroporation?
A electric pulse is applied to cells to create temporary holes in the plasma membrane through which DNA can enter and hopefully enter the genome
In what organisms can electroporation be performed?
Animals and bacteria
In what species is Agrobacterium used to add DNA?
Plants only
What is Agrobacterium?
Agrobacterius tumiferens is a soil bacteria (rhizobacterium)
What is a common gene cloning occurs is formed? (not virus)
With bacterial ‘plasmids’
