Genetic Engineeing Flashcards
Process of a gene being altered
- isolation of the gene interest
- isolation of genes into a carrier
- transfer the vector to the organism that need to modified
- Transformation of cells in the organism
How can you obtain a cells DNA
The cells have to be lysed with chemicals, then the nucleus will be lysed and the DNA will be released
Where are restriction enzymes naturally found
In bacterial cells
Define Restriction enzyme
Found naturally in bacteria, they are used to cut DNA at a specific known recognition site.
What types of ends can restriciton enzymes produce?
Sticky
Blunt
Define Recognition Site:
A sequence of nucleotides that form a specific site for a restriction enzyme to cut DNA.
Define Sticky Ends
A piece of DNA with that has been cut by restriction enzymes. The fragment will consist of a section of single stranded nucleotides, these fragments can form hydrogen bonds with a complementary strand.
Define Blunt Ends
A double stranded piece of DNA that has been cut by a restriction enzyme.
What is the calculation for linear DNA?
n cuts = n+1
What is the calculation for circular DNA?
n cuts
What is the role of DNA ligase
Catlayses the formation of covalent bonfd between sugar phosphate backbones of DNA fragments.
Define Recombinant DNA
A DNA molecule made outside of the cell with segments from different organisms
What is the key to recombinant DNA
The same restriction enzymes are used to cut open plasmids as to cut the desired DNA fragment this ensures there are complementary sticky ends.
Explain the process of making plasmid recmobinant
- Isolate the gene and cut with a restriction enzyme
- Then cut the plasmid with the same restriction enzyme as the gene was cut, to ensure they are complementary
- Mix together with ligase
What do bactiera need to be in order to take up the plasmid?
Competent
Explain why plasmids cant just go straight through the cell wall?
Plasmids have a neg charge
Cell wall/membrane has a negative charge
Therefore they repel eachother
Why is CaCl2 added?
To make the cell membrane soften in order for holes to easily be formed in the heat shock process
Why is there heat added in the process of bacterial transformation?
Heat shock makes holes
Define the process of gel electrophroesis
The process of seperating DNA fragments accordance to their size.
Which end of the agarose gel is the DNA attracted to?
Positive
Explain how the agarous gel works
It is highly pourous and acts like a sieve where the shortest DNA fragments are able to move faster through the pores, therefore it is harder for the larger fragments to fit through the pores and they lag behind.
What is the purpose of the buffer
- Allows for a stable environment including pH
- Allows for a temperature in that the agarose will not melt
- Contains ions which all for the electrical current to occur
Define Plasmids
A vector that consists of a circular loop of DNA. A plasmid will carry DNA from one organism to another/
Why are plasmids so useful
- Due to their circular shape and being small in size this causes plasmids to generally be harder to break
- Easy to manipulate
- Used for cloning they are replicated and divide when a bacteria does also
What is the purpose of the marker?
Acts as a standard to indicate how far the DNA have travelled
What must DNA be prepped with before undertaking electrophoresis
Glycerol must be added to ensure the DNA sinks into the well
A methlyline blue stain is added to track the overall migration of the DNA
What is the role of the arabanose sugar?
Required for flurosence to occur
Define competent
Treated in such a way that they are able to accept a plasmid vector
What is reverse transcriptase used for?
Synthesises Dna from an RNA template
-Performs the reverse of transcription
What is the process of microinjections
Involving the use of a microneedle to insert DNA into a living cell
Penetreates cell membrane and sometimes even nuclear membrane
used in cloning
What is a probe
Short single stranded piece of DNA
makes DNA visible
Explain briefly the process of southern blotting
- DNA placed in seperate wells and a restriction enzyme was added to each to produce DNA fragments
- Then underwent electrophoresis
- DNA is transferred to paper and it is denatured in the process (single strands)
- Placed then in a solution of radioactive probes
What is PCr
technique used to amplify particular DNA fragments
Why is PCR needed?
Many engineering genetic process require large amount of DNA
Explain the 4 steps in PCR
- Heat DNA to 95 degrees, DNA will denature and will form two
strands - DNA is then made the temperature of 55 degrees and primers will joins to the two strands via comp base pairing
- DNA heated to 72 degrrees and DNA polymerase intiates DNA synthesis
- Process is repeated.
What is DNA profiling
Using DNA to identify an individual
What are some benefits of DNA profiiling
- Sensitive
- Requires small qunaities of DNA
- Fragments difffers in size
- Hours to perform
What is DNA sequencing
Determining the sequence of nucleotides making up the lenght of DNA
- Restriction enzymes digest the strand
- Fragments isolated via gel electrophoresis
- sSequnece is determines by PCR or chemical analysis
