In Vivo DNA amplification Flashcards
What is the name of this method to amplify a DNA fragment?
Culture of transformed host cells
Main idea behind this method
Put DNA fragment into vector which enters host cell
The gene will be expressed to create the protein required by translation
And DNA replication and cell division will naturally lead to many copies of the gene be made = amplified
3 steps of this method
Insert DNA fragments into vectors using restriction endonuclease + vectors
Transform host cell by vectors
Use marker genes to detect which host cells were successfully genetically modified
Vector
Something that carries a DNA fragment into a host cell
Examples of vectors
A plasmid
= small circular loop of DNA containing few genes found in prokaryotes
Where are plasmids obtained from?
Cut out of bacteria
How to prepare vector to insert a DNA fragment in?
Cut open vector using the same restriction endonuclease used to obtain DNA fragment
SO cuts vector to produce same sticky ends as on DNA fragment
What are sticky ends?
Single stranded DNA stretches at the end of a plasmid/target DNA molecule that can complementary base pair with each other due to being cut by same restriction endonuclease
SO BOTH CUT AT SAME BASE SEQUENCE = complementary
How does is cut fragment of DNA incorporated TEMPORARILY into the vector?
By complementary base pairs at the sticky ends
They are complementary so can form temp H bonds because they were cut at same base sequence by restriction endonuclease
How is cut DNA fragment actually incorporated into the vector after forming temporary Hydrogen bonds?
By enzyme DNA ligase
Joins the fragment and plasmid together via phosphodiester bonds between nucleotides = called annealing
Annealing
WHEN DNA ligase joins together DNA fragment and plasmid together by forming phosphodiester bonds between nucleotides
Recombinant
A DNA molecule made of 2 or more molecules of DNA joined together
So describes the vector
Why must restriction endonucleases be used to obtain DNA fragments?
Produces sticky ends at a specific base sequence (according to active site of the restriction endonuclease)
So by complementary base pairs can be put into the plasmid
What else needs to be inserted into the plasmid vector?
Promoter and terminator base sequence for specific inserted DNA fragment
Why is a promoter base sequence needed to be inserted into vector?
So transcription factors can bind and stimulate transcription by RNA polymerase
So when in host cell, the DNA fragment can actually be translated
Where is the promoter base sequence needed in the vector?
At the start of the DNA fragment upstream in the direction RNA polymerase acts
Why is a terminator base sequence needed to be inserted into vector?
Triggers RNA polymerase to detach from DNA fragment so ONLY 1 GENE AT A TIME IS TRANSCRIBED INTO MRNA
When in the host cell
Where is terminator needed in vector?
At the end of the DNA fragment
How to add the correct promoter and terminator base sequence to vector?
Use a plasmid that already has the correct promoter and terminator base sequences
The gene can then be inserted between them using restriction endonuclease
Promoter + terminator must be….
Appropriate for host cell
Is the terminator and stop codon the same?
NO
Stop codon = triggers mRNA to detach from ribosome during translation
Terminator = Triggers RNA polymerase to detach from gene during transcription
What do we do to the host cell before the vector can be inserted
Make host cell surface membrane MORE PERMEABLE
By mixing with Ca2+ ions and heat shocked
So vector with DNA fragment incorporated inside reaches host cells cytoplasm
Genetically modified
Describes cells or organisms that are genetically transformed
Aka had foreign DNA from fragment inserted into plasmid transforms the host cell
What do we USE to ensure the host cell actually took up the vector containing DNA fragment and the DNA fragment was actually incorporated into the plasmid?
By using marker genes
Tend to be antibiotic resistance genes
