Flashcards in DNA Technology Deck (13):
The diagram shows matching parts of the base sequences for the mRNA produced by the gene for the softening enzyme and that produced by the inserted gene.
Softening gene mRNA …AAUCGGAAU…
Inserted gene mRNA …UUAGCCUUA…
Suggest how the inserted gene reduces the production of the softening enzyme.
Inserted gene/mRNA complementary to normal gene
Binds to it to prevent protein synthesis/prevents mRNA binding to ribosomes.
Will not stop all translation, some mRNA reaches ribosomes/ because not all mRNA is bound by inserted gene mRNA.
The process of making a protein using the DNA technology of gene transfer and cloning involves a number of stages...
1. Isolation of the DNA fragments that have the gene for the required protein.
2. Insertion of the DNA fragment into a vector.
3. Transformation, that is, the transfer of DNA into host cell.
4. Identification of the host cells that have successfully taken up the gene by use of gene markers.
5. Growth/ cloning of population of host cells.
What are the good properties of DNA?
- double helix held by H bonds so very stable
- sugar-phosphate backbone protects bases from external chemical damage
- DNA molecules long but coiled tightly hence a lot of genetic information can fit into a small space in the nucleus
- passed on from generation to generation without change due to double helix stability
Gene technology is basically all the techniques that can be used to study and alter genes and their function. Examples include:
- The polymerase chain reaction (PCR)- produces lots of identical copies of a specific gene
- In vivo gene cloning
- DNA probes- used to identify specific genes
What is the specific site restriction enzymes cut at called and what does palindrome mean
palindrome - two sequences are opposite to eachother
1) The plasmid is cut with the same restriction enzyme as the gene and hence it'll be cut at the same specific sequence and their sticky ends will be complementary and will anneal.
2) When the gene is inserted into the plasmid, DNA ligase is used to join the sugar-phosphate backbone= RECOMBINANT DNA
3) The bacteria/host cell and the plasmid are now mixed together - TRANSFORMATION
What are the three types of GENE MARKERS
1) Antibiotic resistance gene
Insert gene into middle of antibiotic resistance gene, use two resistance genes.
Grow bacteria on agar plates - the ones that survive on one antibiotic but not on the other have taken up the plasmid and the gene has succesfully inserted into the plasmid.
Transfer gene from jellyfish that produces green fluorescent protein --> bacteria that have taken up plasmid containing the gene will not produce protein/not fluorescent
3) Enzyme markers
eg. lactase which turns a specific colourless substrate blue, successful bacteria will not turn substrate blue
Why is using fluorescent markers good?
Bacteria you want arent killed like in antibiotic resistance, hence no need for replicate plating = rapid
INVITRO CLONING ( PCR- Polymerase chain reaction)
1) separation of DNA strand - add dna fragment, primers and dna polymerase to a thermocycler.
Temp: 95 --> breaks H bonds hence separates strands
2) addition/annealing of primers
Temp: 55 --> causes primers to anneal to their complementary bases at start of fragment = provides starting point for DNA polymerase
3) synthesis of DNA
Temp: 72 ---> optimum temperature for dna polymerase, it begins at primers on both strands causing formation of phosphoester bonds (joining backbone)
What do primers do
1) specify start and end points of a gene for dna polymerase to being replication
2) prevent target gene from reannealing
Why is pcr good?
copies 2 strands of DNA simultaneously . therefore number of copies of dna where n=number of repeats is 2^n
Advantages of invivo cloning
-very accurate, fewer errors that invitro cloning which will copy errors through subsequent cycles
-precise cuts out a specific gene
-bacteria produce can make large quantities of gene products
-No risk of contamination - cuts with same restriction enzyme so contaminant DNA isnt taken up whereas in invitro cloning contaminant DNA is also replicated
-useful to introduce gene via vectors into another organise - eg. humans in gene therapy