Recombiant DNA Flashcards
What are the 5 stages of in vivo cloning?
Isolation
Insertion
Transformation
Identification with gene markers
Growth/cloning of population of host cells
How does reverse transcriptase used to isolate genes
mRNA is produced which codes for a gene
Reverse transcriptase then converts mRNA into complementary DNA (cDNA) as it has nucleotides that are complementary to the mRNA
DNA polymerase then makes the complementary DNA strand to the cDNA(makes it double stranded)
What are restriction endonucleases and how do they isolate genes?
enzymes that cut DNA at specific bases sequences called recognition sites
What type of cuts do restriction enzymes make?
Staggered cut-generates sticky ends made of single strand DNA overhangs
Blunt cut- both ends are double stranded
How is a gene machine used to isolate genes?
Use genome sequence to search desired sequence
Produe small section of DNA sequence (oligonucleotides) that span the gene sequence
Oligonucleotides overlap and join together to restore the original sequence
Gene is then inserted into a plasmid
Whats he process of insertion in gene technology?
Plasmid and gene are cut with the same restriction enzyme to create complemetary sticky ends
Fragment is then incubated with the plasmids
This then leads to base pairing between complementary sticky ends
DNA ligase creates single phosphodiester bonds
What non coding sequences are needed to ensure that the gene of interest is transcribed in insertion?
Promoter and terminator sequences
How does transformation in DNA technology work?
Mix together plasmids and bacterial cells in medium with calcium ions and change the temperature
This makes the bacterial cells more permeable
This can lead to the bacteria containing:
nothing
Vector+insert
No vector
How does identification work in DNA technology?
Gene markers are used to see if the DNA has been taken in to the plasmid
Gene markers include-flourescent dye, antibiotic resistance and enzyme
How is antibiotic resistance used as a gene marker? Use ampicillin as example
All the bacterial cells grown in medium that ocntais the antibiotic ampicillin
The bacterial cells that have taken up plasmids will have acquired the gene for antibiotic resistance
These bacterial cells will be able to break down the ampicillin and survive
The cells that haven’t taken up the plasmid will die
How do fluorescent markers work?
Transfer GFP gene from jellyfish to the plasmid,
desired gene is placed in the middle of GFP.
If bacteria takes up the plasmid, it won’t go fluorescent/glow
How do enzyme markers work?
Lactase will turn a colourless substance blue,
desired gene placed in the middle of gene that makes lactase. If bacteria takes up the plasmid, no lactase will be produced and substance won’t go blue
How does PCR work?(in vitro cloning)
PCR contains DNA polymerase, DNA nucleotides and primers and DNA fragments
DNA is heated to 95 degrees to break hydrogen bonds between strands
Annealing then takes place where two primers bind to the template DNA strands at a cooler temp(55 degrees) due to complementary base pairing
Temperature increased to 72 degrees to allow thermostable DNA polymerase to join free nucleotides to DNA strands creating phopshodiester bonds
Cycle repeats to generate billions of copies
What are primers?
Single stranded pieces of DNA which allow DNA polymerase to attach at start and end of target sequence
What are the advantages and disadvantages of PCR(in vitro cloning)
Advantages:
Quick
Doesn’t use living cells
Can generate billions of copies in a few hours
Disadvantages:
Because it’s so quick mistakes are amplified
DNA can also be contaminated by handler
