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level: 21.1 Producing DNA fragments

Questions and Answers List

level questions: 21.1 Producing DNA fragments

QuestionAnswer
What is a gene?- Section of DNA that code for specific polypeptides and RNA
Genetic modification- Process of inserting a gene into the genetic makeup of another organism
Transgenic organism- Organism that has had genes inserted in order to make specific polypeptides
Reverse transcriptase- An enzyme that carries out transcription in reverse - Transcription = DNA to mRNA - Reverse transcription = mRNA to DNA
Restriction enzyme- An enzyme that digests DNA - They obtained from bacteria that use them to defend against viruses - They recognise palindromic(can read it either way is same) sequences of 4-6 bases - Make blunt or sticky ends
Blunt ends- Only cut phosphodiester bonds
Sticky ends- Cut phosphodiester and hydrogen bonds
How do sticky ends stick?- Sticky end pairs with comp. base, DNA ligase cements the bonds between nucleotides by condensation - DNA ligase used in repairing broken DNA - The DNA is now called recombinant DNA - If the plasmid is cut using the same restriction enzyme it will have complementary sticky ends to the gene
Recombinant DNA- Where the DNA of the 2 different organisms is combined, the product is known as recombinant DNA
GM process (1) producing DNA fragments- One method -> use of reverse transcriptase - mRNA -> DNA - Forms comp. strand of DNA called complementary DNA - Forms another strand using DNA polymerase
GM process (2) producing DNA fragments- Another method of producing DNA fragments is to use restriction enzymes - Named source of DNA (hair follicle...) - Digested/hydrolysed using restriction enzymes - Use a thermocycler - Heat to (95) to break h bonds - Cool to (55) allow primers to attach - Function of primers (allow polymerase to attach, locate specific seq) - Heat to (72) optimum temp for DNA polymerase - Free nucleotides bind with comp. nucleotides - Repeated many times
GM process (3) producing DNA fragments- Some of these leave fragments with 2 blunt or sticky ends - The sequences of bases on uneven ends are palindromic?
A vector- A gene carrier that the gene is spliced into - Plasmids are vectors, they are small pieces of circular DNA found in bacteria
Transformation- Ice cold - Incubate at 42oC for 2 minutes - Ice cold
Finding the bacteria- Splice in 2 genes - the genes we want and a gene marker - Gene markers can be : - Fluorescent 'genes' - Antibiotic resistant genes - A particular enzyme (may cause a colour change)
Explain how modified plasmids are made by genetic engineering and how the use of markers enable bacteria containing these plasmids to be detected.- Isolate wanted gene using restriction enzymes to get DNA and produce sticky ends - Use ligase to join wanted gene to plasmid - Also include marker gene e.g. antibiotic resistance - Add plasmid to bacteria to grow (colonies) then (replica) plate onto medium where the marker gene is expressed - Bacteria not killed have antibiotic resistance gene and (probably) the wanted gene
Why are gene markers necessary in gene cloning- To know what plasmids with the genes were taken up by the bacteria
One advantage of using fluorescent markers rather than antibiotic gene markers- Results can be obtained more easily and more quickly - Because with antibiotic resistance markers, the bac cells with the gene are killed, so replica plating is necessary to obtain cells with the gene - With fluorescent gene markers, the bac cells are not killed and so there is no need to carry out replica plating
Strengths of GM- GM bacteria can be used to produce insulin - GM used to make vaccines - Genes could be introduced for gene therapy
Weaknesses of GM- GM crops may contain allergens - Introducing genes could lead to eugenics - Could decrease biodiversity