In Vitro Cloning - The Polymerase Chain Reaction Flashcards Preview

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Flashcards in In Vitro Cloning - The Polymerase Chain Reaction Deck (11)
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1

What is the polymerase chain reaction

A method of copying fragments of DNA.

It is automated, making it both rapid and efficient

2

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

3

Describe the three stages of the polymerase chain reaction

1. 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.

2. 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

3. 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

4

How long does the temperature cycle take

Two mins

5

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.

6

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

7

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

8

State what type of bond is broken when DNA strands are separated in the pcr

Hydrogen bonds

9

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

10

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

11

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