Making DNA fragments
Method 1- Reverse transcriptase
mRNA is isolated from cells, mixed with free DNA nucleotides and reverse transcriptase.
The reverse transcriptase uses the mRNA as a template, to make a new strand of complmentry DNA (cDNA), this will only contain exons as the original mRNA has been spliced.
The cDNA will code for a specific gene.
Making DNA fragments
Method 2- Polymerase Chain Reaction (PCR)
Reaction mixture containing DNA sample, free nucleotides, primers and DNA polymerase set up.
Heated to 95c to break hydrogen bonds between strands of DNA. Cooled so primers can bind to strands.
Primers bind to the start of the gene as they are complimentry, allow DNA polymerase a starting point. Mixture heated so DNA polymerase can work. Free nucleotides line up and DNA polymerase joins them together.
Two new copies of the fragment of DNA are formed on one cycle. Cycle repeated.
Making DNA fragments
Method 3- using restriction endonuclease enzymes
Restriction endonucleases are enzymes that recognise specific palindromic sequences (antiparallel base pairs) and cut at these points.
Different restriction endonucleases cut at different points.
If recognition sequences present at each side of the DNA fragment you want, you can use restriction endonucleases to seperate it from the rest of the DNA.
Somtimes when they cut they leave sticky ends- small tails of unpaired bases at each end of the fragment. Sticky ends can be used to bind DNA fragments to another piece of DNA that has sticky ends with complmentry sequences.
Part 1- making recombinant DNA
Vector DNA is isolated.
Vector cut open using the same restriction endonuclease that was used to isolate the DNA fragment containing the target gene meaning sticky ends are complimentry.
Vector DNA and DNA fragment mixed together with DNA ligase, which joins the sticky ends together. Process called ligation.
The new combination of DNA vector and fragment is called recombinant DNA.
Part 2- Transforming cells
The vector with the recombinant DNA is used to transfer the gene into host cells, these host cells that have taken up the gene of interest are said to be transformed.
If a plasmid vector is used host cells may have to be persuauded to take up the plasmid vector and its DNA, for example by heat shock.
Part 3- Identifying transformed cells
Not all host cells take up the vector and its DNA, marker genes can be used to identify transformed cells.
Marker genes should be inserted at the same time as the cell is being cloned.
Host cells grown on agar plates, cells will replicate and form colonies of cloned cells. Transformed cells will produce colonies with marker gene and cloned gene.
Marker gene may be for antibiotic resistance- if host cells are grown on agar playes containing the specific anitbiotic, only transformed cells that have marker gene will survive and grow.
Identified transformed cells are allowed to grow more, producing lots and lots of copies of the cloned gene.
In vivo advantages:
Cloning can prodice mRNA and protein
Can produce modified DNA
Large fragments can be cloned
Relatively cheap method
In vivo disadvantages:
DNA fragment has to be isolated
May not want modified DNA
In vitro (PCR) advantages:
Can produce lots of DNA
DNA produced isnt modified(advantage if you dont want it)
Only replicates DNA fragment of interest, no isolation
In vitro disadvantages:
Can only replicate small fragments
Also known as recombinant DNA technology, manipulating an organisms DNA.
Transformed microorganisms can be made using the same technology as in vivo cloning.
Transformed plants can also be produced- a gene that codes for a desirable characteritic is inserted into a plasmid. Plasmid added to bacterium which is used as a vector.Transformed plant will have the desirable charactertic coded for by that gene.
The benefits of transformed organisms:
Higher yields in agriculture, used to reduce risk of famine and malnutrition.
Resistant to pests or drought. Pest resistant need fewer pesticides, cost less, less eutrophication. Drought resistant survive in drought prone areas.
In industry used a s biological catalysts. Can be produced from transformed organisms so they can be produed in large quantities for less money.
Medicine, Drugs using recombinant DNA can be produced quickly, cheaply and in large quantities. Cheaper so available to more people.
The disadvantages of transformed organisms:
Agriculture, get monoculture so more vulnerable to disease, can get a superweed!
Industries, lead to toxins in food.
Medicine, People could limit technology that could be saving lives.
Unethical can make designers babies.which is playing God.
Globablly, large companines can control some forms of genetic engineering and use this to grow larger, putting smaller businesses out of work.