Gene technologies


What are gene technologies

Techniques used to study genes and their functions

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Describe the polymerase chain reaction

Step one
A reaction is set up that contains the DNA sample , free nucleotides , primers and DNA polymerase

Step two

The DNA mixture is heated to .95 degrees Celsius to break the hydrogen bonds between he two strands of DNA. The Mixture is then cooled to50-65 degrees Celsius so that the primers can bind to the strands

Step three
The reaction mixture Is heated to 72 degrees Celsius so DNA polymerase can work . The DNA polymerase lines up free DNA nucleotides alongside each template strand. Complementary base pairing means new complementary base pairing means new complementary strands are formed

Step four
Two new copies of the fragment of DNA are formed and one cycle of PCR is complete. When the cycle starts again the mixture is heated to 95 degrees Celsius and this time all four strands are used as templates

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Define the following

Primer - short pieces of DNA that are complementary to the bases at the start of the fragment you want

DNA polymerase- an enzyme that creates new DNA strands

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Describe restriction enzymes

Some sections of DNA have palindromic sequences of nucleotides. These sequences consist of anti parallel base pairs

Restriction enzymes recognise specific palindromic sequences and cut the DNA at these places. Different restriction enzymes cut at different specific recognition sequences , because the shape of the recognition is complementary to an enzymes active site

If recognition sequences are present at either side of the DNA you want , you can use restriction enzymes to separate it from the rest of the DNA. The DNA sample is incubated with specific restriction enzymes, which cuts the DNA fragment via a hydrolysis reaction. Sometimes the cut leaves sticky ends. Small tails of unpaired bases at each end of the fragment. Sticky ends can be used to bind the DNA fragment together to another piece of DNA that has DNA with complementary sequences.

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Describe gel electrophoresis

This is a technique used to separate out DNA fragments by size. First a fluorescent tag is added to all the DNA fragments so they can be viewed under a uv light. The DNA mixture is placed into a well in a slab of gel covered in buffer solution that conducts electricity . An electrical current is then passed through the gel- DNA fragments are negatively charged so they move towards the positive electrode at the far end of the gel so the DNA fragments separate according to size. The DNA are viewed as bands under a uv light.

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DNA probes

They are used to identify DNA fragments that contain specific sequences of bases

They are short single strands of DNA. They have a specific base that's complementary to the target sequence - this means a DNA probe will hybridise which means bind to the target sequence if it'd present in a sample of DNA

A DNA probe also has a label attached, so that it can be detected. The two most common labels are a radioactive label or a fluorescent label

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How are fluorescent label probes used

Step one

A sample of DNA is digested into fragments using restriction enzymes and seperated using electrophoresis

Step two

The separated DNA fragments are then transferred to a nylon membrane and unbolted with a fluorescently labelled DNA probe. If the target sequence is present the DNA probe will hybridise

Step three

The membrane is then exposed to uv light and if the target sequence is present there will be a fluorescent band

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What is genetic engineering

It is the manipulation of an organisms DNA. Organisms that have their DNA altered are called transformed organisms and they have recombined DNA

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Describe the process of gene cloning

Part one
Obtain DNA containing desired gene
The fragment can be isolated using a restriction enzyme

Part two

Insert DNA into vector DNA e,g plasmid
The vector is isolated and cut open using the same restriction enzyme that was used to isolate the DNA fragment containing the desired gene. This means that the sticky ends of the vector DNA are complemmentary to the sticky ends of the DNA fragment containing the gene

The vector DNA and DNA fragment are mixed together with DNA ligase. DNA ligase joins the sugar phosphate backbones of the two bits of DNA. This is called ligation

The new combination of bases in the DNA is called recombinant DNA

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Describe the process of gene cloning

Part three - transforming cells

The vector with the recombinant DNA is used to transfer the gene into the bacterial cell . If a plasmid vector is used the host cells have to be persuaded to take in the plasmid vector and it's DNA

With a bacteriophage vector, the bacteriophage will infect the host bacterium by injecting its DNA into it. The phage DNA with desired gene in it then inter generates into the bacterial DNA.

Part four
Not all host cells will have taken up the vector and it's DNA. Marker genes can be used to identify the transformed cells.
Marker genes can be inserted into vectors at the same time as the desired gene. This means any transformed host cells will contain the desired gene and marker gene

The host cells are grown on agar plates and each cell divides and replicates it's DNA, creating a colony of cloned cells. Transformed cells will produce colonies where all the cells contain the desired gene and marker gene. The markers gene can code for antibiotic resistance - if the host cells are grown on agar plates containing the specific antibiotic , only transformed cells that have ,asker gene will survive and grow

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Describe the process of gene cloning

Marker genes can also code for fluorescent when the agar plate is placed under a uv light only transformed cells will fluoresce

The transformed cells will be able to produce ten desired gene product

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The benefits of taking up plasmids

Come microrganisms can naturally take up plasmids from their surroundings.this is beneficial for the microrganisms because the plasmids often contain useful genes. This means the microorganisms gain useful characteristics, so they're more likely to have an advantage over other microorganisms which increases their chance of survival. Plasmids can contain many useful things

Genes that cods for resistance to antibiotics
Genes that help microorganisms invade hosts
Gene that mean microorganisms can use different nutrients

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Positives and negatives of invivo

Larger fragments of DNA can be cloned
Less technically difficult
Fewer mutations

DNA fragment must be isolated
Slow process
Uses a lot of lavatory space

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Positives and negatives of invitro

Only replictes DNA fragment of interest isolation isn't needed
Faster process
Uses less lab space
You can use older DNA

Can only rlicate a small DNA fragment and is expensive it can introduce more mutations

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