In Vitro Cloning - The Polymerase Chain Reaction Flashcards
What is the polymerase chain reaction
A method of copying fragments of DNA.
It is automated, making it both rapid and efficient
What does the polymerase chain reaction require (PCR)
The DNA fragment - to be copied
DNA polymerase- an enzyme capable of joining nucleotides together (taq polymerase is obtained from bacteria and can be used at high temps )
Primers - short sequences of nucleotides that have set bases complementary to those at one end of each of the two DNA fragments
Nucleotides - which contain each of the four bases found in DNA
Thermocycler - a computer controlled machine that varies temperatures precisely over a period of time
Describe the three stages of the polymerase chain reaction
- Separation of the DNA strand - the DNA fragments, primers and DNA polymerase are placed in a vessel in the thermocycler. The temp is increased to 95 degrees, causing two strands of the DNA fragments to separate due to the breaking of the hydrogen bonds between the two DNA strands.
- Addition of the primers - the mixture is cooled to 55 degrees, causing the primers to join to their complementary bases at the end of the DNA fragment. The primers provide the starting sequence for DNA polymerase to begin DNA copying because DNA polymerase can only attach nucleotides to the end of an existing chain. Primers also prevent the two separate strands from simply rejoining
- Synthesis of DNA - the temperature is increased to 72 degrees. This is the optimum temperature for the DNA polymerase to add complementary nucleotides along each of the separated DNA stands. It begins at the primer on both strands and adds the nucleotides in sequence until it reaches the end of the chain
Because both strands are copied simultaneously there are now two copies of the original fragment. Once the two DNA strands are completed, the process is repeated by subjecting them to the temperature cycle again, resulting in four strands
How long does the temperature cycle take
Two mins
What are the advantages of in vitro cloning
Extremely rapid - valuable where only a minute amount of DNA is available (crime scenes for forensics )
It does not require living cells - all that is required is a base sequence of DNA that need amplification. No complex culturing techniques, requiring time and effort, are needed.
What are the advantages of in vivo gene cloning
It is particularly useful where we wish to introduce a gene into another organism - as it involves the use of vectors, once we have introduced the gene into a plasmid, this plasmid can be used to deliver the gene into another organism, such as human being. This is done in a technique called gene therapy
It involves almost no risk of contamination - a gene that has been cut by the same restriction endonuclease can match the sticky ends of the opened up plasmid. Contaminant DNA will therefore not be taken up by the plasmid. (In vitro cloning requires a very pure sample because any contaminant DNA will also be multiplied and could lead to a false result)
It is very accurate - DNA copied has few errors.
It cuts out specific genes- very precise procedure
It produces transformed bacteria that can be used to produce large quantities of gene products - transformed bacteria can produce proteins for commercial or medical use
Explain the role of primers
They attach to the end of a DNA strand that is to be copied and provide the starting sequences for DNA polymerase to begin DNA cloning. DNA polymerase can only attach nucleotides to the end of an existing chain. They also prevent the two separate strands from rejoining
State what type of bond is broken when DNA strands are separated in the pcr
Hydrogen bonds
It is important in the pcr that the fragments of DNA used are not contaminated with any other biological material. Suggest a reason why
Biological contaminants may contain DNA and this DNA would also be copied
What are the benefits of recombinant DNA technology
Microorganisms can be modified to produce a Range of substances
Microorganisms can be used to control pollution
Genetically modified plants can be transformed to produce specific substance in a particular organ in a plant
Genetically modified crops can be engineered to have financial and environmental advantages
Genetically modified crops can help prevent certain diseases
Genetically modified animals are able to produce expensive drugs relatively cheaply
Replacing defective genes to cure certain genetic disorders
Genetic fingerprinting in forensic science
What are the risks of recombinant DNA technology
Can’t predict what ecological consequences will be of releasing genetically engineered organism into the environment
A recombinant gene may pass from organism it was placed in, to a completely different one
Any manipulation of the dna of a cell will have consequences for the metabolic pathways within a cell
Genetically modified bacteria often have antibiotic resistance marker genes that have been added
What if a genetic engineered gene mutated
What’s the long term consequences of introducing new gene combinations
Financial consequences
How far can we take the technique of replacing defective genes
Could lead to a wrong conviction with DNA fingerprinting
Is it immoral
