DNA Technologies Flashcards
(16 cards)
RECOMBINANT DNA TECHNOLOGY
What is recombinant DNA technology?
Transferring DNA fragments from one organism to another.
Why can DNA fragments from one organism be transferred to another organsim and produce the same protein?
The gentic code, the transcription factors and the translation mechanisms are universal the gene can still be translated when moved to another organism.
What are the three steps in recombinant technology?
- Production of DNA fragments.
- Amplifying the DNA fragments.
PRODUCTION OF DNA FRAGMENTS
How can reverse trancriptase be used to create DNA fragments?
- Reverse trancriptase can be used to make complementary RNA, using mRNA and free nucleotides.
- DNA polymerase cam then be used to make double-stranded cDNA.
How can restriction endonuclease enzymes be used to create DNA fragments?
- They hydrolise phosphodiseter bonds to cut the DNA at specific base sequences called recognition sites.
- The sites are palindromic and the cutting of them produces sticky ends.
What does palindromic means?
The DNA base sequence is read the same in both directions.
How can a gene machine be used to create DNA fragments? What are the benefits of this method?
- It can be used to create any base sequence that codes for any polypeptide.
- It does this by creating short, single stranded oglinucleotides that are joined together to form a gene.
- Allows for the removal of introns and produces genes more quickly and accurately.
THE AMPLIFICATION OF DNA FRAGMENTS VIA IN VIVO AND IN VITRO METHODS.
What is the name of an in vivo method to amplify DNA fragments?
The polymerase chain reaction.
Describe what happens during the polymerase chain reaction?
- A reaction mixture is made and it contains DNA fragments, that acts as a template, DNA nucleotides, DNA polymerase and primers.
- The reaction mixture is heated to 95 degrees in order to break the hydrogen bonds between the DNA strands and separate them.
- Cool the mixture to 55 degrees so the complemtary primers can stick to the DNA strands.
- Heat the mixture to 70 degrees so DNA polymerase can bind to the DNA strands and add new nucleotides by forming phosphodiester bonds between them.
What is an in vitro method to amplify the DNA fragments?
Transforming cells
Describe what happens in the transformation of cells?
- A promoter regions ,which helps RNA polymerase bind to the gene and transcribe it, and a terminator region, which prevents RNA polymerase from binding to the gene and transcribing are added to the fragment.
- The DNA fragments is inserted into a vector, like a plasmid. This is done by the plasmids being cut by the sam restriction endonuclease as the one used to produce the DNA fragments, so the sticky ends of both are complementary. The sticky ends bind together due to complementary base pairing. Lipase then form phosphodieter bonds between the two sugar-phosphate backbones.
- The plasmid now contains the the DNA fragment, with the promoter and terminator regions attached to it, and a marker gene. The gene can code for something like fluorescence or antibiotic resistance.
- The vector helps carry the DNA fragment into a host cell.
- This is done by the bacteria and plasmid being dissolved in a calcium chloride solution and suddenly heated to increases the permeability of the membrane to the plasmids. The plasmids are taken into te host cells.
- The host cells then are culture to produce may of the DNA fragments. These can be removed if needed, or transcribed and the mRNA can be used to make a protein.
What methods are used to identify which cells have been transformed in which won’t?
- If the cells have taken in a plasmid that has a marker gene that codes for fluorescence, it will glow under UV light.
2 If the cells have taken in a plasmid that has a marker gene that codes for antibiotic reitsnace, they will not be killed if an antibiotic is added to the agar plate.
