Gene Technology Flashcards
What is recombinent DNA technology?
The combining of different organism’s DNA, whcih could enable scientists to manipulate and alter genes to improve industrial processes and medical treatments.
What three methods are there of creating DNA fragments?
- Reverse transcription
- Restriction endonucleases
- Gene machines
Describe the reverse transcription method of creating DNA fragments.
- A cell that produces the protein of interest is selected. These cells should have large amounts of mRNA for this protein
- Add reveerse transcriptase, which joins free-floating nucleotides with complementary bases to the mRNA sequence
- This creates single stranded DNA (cDNA)
- To make this DNA double stranded, DNA polymerase must be added
What is an advantage and disadvantage of reverse transcription as a method of creating DNA fragments?
+Because it is using mRNA, introns are already spliced meaning they are not present and wont interfere with translation.
+Lots of mRNA available to make cDNA
-More steps, so more time consuming and technically more difficult
What are restriction endonucleases, and where do they naturally occur?
Enzymes that cut up DNA, these can occur in bacteria
How do different types of restriction endonucleases differ from one another?
They have different active sites, meaning that they cut the DNA at certain specific locations, known as recognition sequences.
What are “blunt ends” of DNA
Some restriction endonucleases cut at the same location in a double strand creating a “blunt end”, which are unable to join DNA with complementary base pairs.
What are “sticky ends” of DNA?
Some restriction endonucleases cut to create staggered ends which are palindromic (and known as sticky ends) and are able to join to DNA with complementary base pairs
Describe the method of creating DNA fragments using the gene machine.
- Scientist identify the amino acid sequence of a protein of interest and work out the mRNA and DNA sequence
- The DNA sequence is entered into the computer, checking for biosafety and biosecurity that the DNA being produced is safe and ethical.
- The computer can create small sections of overlapping single strands of nucleotides that make up the gene, called oligonucleotides, which can then be joined to create the DNA for the entire gene
What is an advantage and disadvantage of restriction endonuclease as a method of creating DNA fragments?
+Sticky ends on DNA fragment make it easier to insert to make recombinent DNA
-Still contains introns
What is an advantage and disadvantage of gene machines as a method of creating DNA fragments?
+Can design exact DNA fragment you eanr, with sticky ends, labels and preferential codons
-Need to know the sequence of amino acids or bases
Describe anitbiotic resistant marker genes
- A desired gene is cur using restriction endonuclease
- A plasmid from a bacterium is cut by restriction endonuclease in the middle of a gene for antibiotic resistance
- These two are mixed using Lipase, and hopefully the gene is integrated.
- Place this culture in an antibiotic medium, and where the bacteria doesnt grow is where the gene has been taken up by plasmids.
Describe fluorescent marker genes.
- A gene from a jellyfish producing Green fluorescent protein (GFP) is cut out and placed into a plasmid using lipase
- The desired gene is then transplanted into the centre of the GFP gene
- Any bacterial cell that has taken up the plasmid with the gene that isnto be cloned will not be able to produce GFP and will not fluoresce.
Describe the method of the Polymerase Chain reaction
- Extract DNA (eg white blood cells from blood)
- Heat DNA to 90°C to separate strands
- Anneal DNA, work out the beginning of the sequence and create a primer
- Attach Taq DNA polymerase
- Extend by adding free floating nucleotides
- Repeat 22-30 times.
What Enzyme is used to extend DNA in PCR?
DNA polymerase from the heat-stable bacteria Thermus Aquatica, which is able to withstand rhe heat required to separate DNA strands
