Recombinant DNA technology Flashcards
(12 cards)
What is a GMO?
Genetically modified organism
What is recombinant DNA
The method of joining 2 or more DNA molecule to create a hybrid
What has recombinant DNA allowed for?
It has allowed for genes to be altered and transferred between organisms
What is a transgenic organism?
A GMO
Briefly outline the stages of recombinant DNA technology
- Isolation of DNA fragments (genes for the desired protein)
- Insertion of DNA fragments into vector
- Transformation of DNA into suitable host cell
- Identification of host cells that have successfully taken up the gene
- Growth/cloning of the population of host cells.
Briefly describe isolation and the 3 ways it is possible
- DNA is a long continuous molecule, the desired gene needs to be located, isolated and cut from the DNA to be inserted into another organism.
- Reverse transcriptase
- Restriction endonucleases
- Gene machine
Describe reverse transcriptase
- Take a cell that produces the desired protein and extract the mRNA
- Reverse transcriptase combines free DNA nucleotides to the mRNA which forms cDNA (complementary)
- The cDNA is a single stranded molecule, the use of DNA polymerase makes it double stranded, this double stranded DNA is the required gene
Describe restriction endonucleases
- Enzymes that cut DNA naturally occur in bacteria, cut up and destroy viral DNA
- There are different types of RE: each one has different active site shape that is complementary to a specific DNA sequence. This is known as recognition site and is where the enzyme will cut the DNA.
- Blunt ends: cuts in between 2 opposite pairs
- Palindrome (sticky ends): cuts in staggered fashion leaving unpaired bases known as sticky ends. This is beneficial as DNA joins easier with complementary base pairs
Describe gene machine
- This is done by scientists, the rest is done by a computer which makes it very quick
1. Examine proteins to identify amino acid sequence and from this work out the mRNA and DNA sequence
- The sequence is put into the computer and checked for biosafety and biosecurity (ethical/harmful)
- The computer creates small sections of overlapping single strands of nucleotides.
- They then get joined together to make 1 copy of the gene.
- PCR replicates gene and constructs complimentary strand
- Genes are checked, errors are rejected
What are the 2 regions that must be added onto the DNA fragments
Promotor region - added at start of fragment, sequence of DNA that acts as the binding site for RNA polymerase to allow transcription
Terminator region - added at the end of the gene, causes RNA polymerase to detach and stop transcription so only one gene is copied into mRNA at a time
What is a vector and what are the most common ones used?
Vector - something that can carry the isolated DNA fragments into the host cell
• most common vectors are plasmids (circular loops of DNA which are very small and only contain a few genes)
How is DNA inserted into a vector?
- The plasmid is cut open using restriction endonucleases
- This creates sticky ends
- The sticky ends on the plasmids are complementary to the sticky ends on the DNA fragments
- The ligase enzyme sticks together the DNA fragments and the cut plasmids by catalysing the condensation reaction to form phosphodiester bonds between nucleotides
