Recombinant DNA technology Flashcards
How were human diseases treated before DNA technology?
A number of diseases result from being unable to produce various metabolic chemicals.
Many of these are proteins, so are the product of a gene.
Treatment previously involved extracting the chemical from a donor and introducing it to the patient, which risked rejection and risk of infection, and is expensive.
What is recombinant DNA technology?
There are advantages to produce large quantities of proteins, so techniques have been developed to isolate genes, clone and transfer them into microorganisms.
These are then grown to provide continuous production of the protein.
The DNA from 2 different organisms that has been combined is recombinant DNA.
The resulting organism is transgenic or genetically modified organism.
Why is DNA of an organism accepted by a different species and functions normally?
The genetic code is the same in all organisms (universal).
Making proteins is also universal in that the mechanisms of transcription and translation are the same in all organisms.
So transferred DNA can be transcribed and transferred in the cells of the transgenic organism and proteins coded for in the same way.
What are the stages of using DNA technology of gene transfer and cloning?
Isolation of the DNA fragments that have the gene for the desired protein.
Insertion of the fragment into a vector.
Transformation - transfer of DNA into suitable host cells.
Identification of the host cells that have successfully taken up the gene by gene markers.
Cloning/growth of the population of host cells.
What are the methods of producing DNA fragments?
Conversion of mRNA to cDNA using reverse transcriptase.
Using restriction endonucleases to cut fragments containing the desired gene from DNA.
Creating the gene in a gene machine, based on known protein structure.
What are retroviruses?
A group of viruses e.g. HIV, where the coded information is RNA.
In a host cell they are able to synthesise DNA from their RNA using the enzyme reverse transcriptase, which catalyses the production of DNA from RNA.
How is reverse transcriptase used to isolate a gene?
A cell that readily produces the protein is selected. (E.g. B cells of islets of langerhans from the pancreas to produce insulin).
These cells have large quantities of the relevant mRNA, which is more easily extracted.
Reverse transcriptase makes complementary DNA from RNA. cDNA is called because it is made up of the nucleotides that are complementary to the mRNA.
To make the other DNA strand, DNA polymerase builds up the complementary nucleotides on the cDNA template.
How do bacteria use restriction endonucleases?
Bacteria are frequently infected by viruses that inject their DNA into them in order to take over the cell.
Some bacteria defend themselves by producing enzymes that cut up the viral DNA - restriction endonucleases.
What are restriction endonucleases?
Each type cuts a DNA double strand at a specific sequence of bases called the recognition sequence.
Sometimes this cut occurs between two opposite base pairs.
This leaves two straight edges - blunt ends.
What do other types of restriction endonucleases do?
They cut DNA staggered, leaving an uneven cut in which each strand of the DNA has exposed, unpaired bases - sticky ends.
The sequences of unpaired bases that remain are opposites of on another - a palindrome.
How does the gene machine work (1)?
The desired sequence of nucleotide bases is determined from the desired protein. The amino acid sequence of the protein is determined, and the mRNA codons looked up and cDNA triplets worked out.
The desired sequence of nucleotide bases is fed into a computer and checked for biosafety and biosecurity to ensure it meets international and ethical standards.
How does the gene machine work - oligonucleotides (2)?
The computer designs a series of small, overlapping single strands of nucleotides - oligonucleotides, which can be assembled into the desired gene.
In an automated process, each oligonucleotide is assembled by adding one nucleotide at a time in the required sequence.
The oligonucleotides are then joined to make a gene, then replicated using polymerase reaction.
This also constructs the complementary strand of nucleotides to make the double stranded gene, then multiplied to give many copies.
How does the gene machine work - insertion (3)?
Using sticky ends, the gene can be inserted into a bacterial plasmid, which acts as a vector for the gene to be stored, cloned or transferred.
The genes are checked using sequencing techniques and those with errors are rejected.
What are the advantages of the gene machine?
Any sequence of nucleotides can be produced, in a short time and with great accuracy.
The artificial genes are also free of introns, and other non-coding DNA, so can be transcribed and translated by prokaryotic cells.
How are the fragment genes cloned?
The fragments are cloned so there is sufficient quantity for medical or commerical use, by:
In vivo - by transferring the fragments to a host cell using a vector.
In vitro - using the polymerase chain reaction.