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Summary A* Recombinant DNA technology notes
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A* Recombinant DNA technology notes
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Recombinant DNA technologyWhy should we mess with DNA?
-to understand how organisms work
-to design new industrial processes
-for medical applications e.g., producing insulin
What is the normal role of reverse transcriptase?
an enzyme that retroviruses use to synthesise DNA from RNA because their genetic information is
RNA
How is reverse transcriptase used to isolate a gene?
Step one- what type of cell needs to be found?
A cell that readily produces the desired protein
How is this cell identified?
It will have large amounts of relevant mRNA which is extracted
How is RNA then converted into DNA?
Use mRNA as a template for reverse transcriptase to convert RNA to DNA (single stranded copy-
cDNA)
How is double stranded DNA then formed?
Using DNA polymerase with cDNA as a template
What is the product?
A double stranded copy of the gene required
What is the normal role of restriction endonucleases?
Bacteria use enzyme as a defence mechanism to break down the DNA inserted into them by invading
viruses
How do blunt end restriction endonucleases work?
The cut occurs between 2 opposite base pairs leaving 2 straight edges called blunt ends e.g.,
recognition sequence CCC GGG
How do sticky end restriction endonucleases work?
The cut base pairs leave each strand of DNA having exposed single stranded (unpaired) bases known
as sticky ends, the bases sequence overhang is a palindrome
Compare the 2 ways of producing DNA fragments
-restriction endonucleases cut DNA from a genome whereas reverse transcriptase uses mRNA as a
template
, -restriction endonucleases produce fragments with sticky ends whereas using reverse transcriptase
results in cDNA
-using restriction endonucleases contains introns
-using reverse transcriptase uses DNA polymerase
Why do DNA fragments need to be cloned?
To create sufficient quantity for medical or commercial use
What 2 ways are used to do this?
In vivo and in vitro cloning
What is in vivo cloning?
Techniques used to identify genes and clone them into microorganisms
What does it involve?
Transferring the fragments of DNA into a host cell which will then secrete the desired polypeptide
What are the stages designed to produce genetically modified organisms?
1. isolation of DNA fragment with desired gene
2. insertion of DNA fragment into vector
3. transformation- transfer DNA into host
4. identification of success using gene markers
5. growth/cloning a population of host cells
How is the fragment prepared?
Extra lengths of DNA need to be added
What is a promoter region?
A binding site for RNA polymerase to initiate transcription and site of transcriptional factor binding
What is a terminator region?
A sequence which RNA polymerase and end transcription
How is DNA inserted into the plasmid?
DNA and plasmid are both cut using the same restriction endonuclease leaving complementary
sticky ends, these can be joined permanently using DNA ligase enzyme so now recombinant DNA
Describe stage two- transformation
Plasmid reintroduced to bacterial cell by mixing together in a medium of calcium ions and changing
temperature, causes bacterial cell membrane to become permeable, allowing the plasmid to pass
through into cytoplasm
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