DNA Technology

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Producing DNA Fragments

Techniques have been developed to isolate genes, clone them and tranfer them into microorganisms. 

These microorganisms are then grow to provide a factory fo the continuous production of a desired protein

The DNA of two different organisms that has been combined- called Recombinant DNA 

The organism produced- called Genetically Modified Organism (GMO)

The process of making a protein using the DNA technology of gene transfer and clning invloves a number of stages:

  • Isolation of the DNA fragments that have the gene for the desired protein 
  • Insersion of the DNA fragment into a vector 
  • Identification of the host cells that have successfully taken up the gene by use of gene markers
  • Growth/Cloning of the population of host cells
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Using Reverse Transcriptase

The genetic information of retroviruses is in the form of RNA 

They are able of synthesising DNA from RNA using reverse transcriptase 

The process of using transcriptase to isolate a gene:

·A cell that readily produces the protein is selected 

·These cells have large quantities of the relevant mRNA which is extracted 

·Reverse transcriptase is used to make DNA from RNA. This DNA is known as complementary DNA (cDNA) because it is made up of nucleotides that are complementary to the mRNA 

·To make the other strand of DNA the enzyme DNA polymerase is used to build up the complementary nucleotides on the cDNA template. This double strand of DNA is the required gene

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Using Restriction Endonucleases

Restriction Endonucleases cut a DNA double strand at a specific sequences of bases- called recognition sequence

Sometimes this leaves two straight edges-known as blunt ends

Other restriction endonucleases cut in a staggered fashion leaving DNA exposed with unpared bases- known as sticky ends

Two sequences are opposites to one another- called palindrome

 

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Importance Of Sticky Ends

If the same restriction endonuclease is used to cut DNA then all the fragments will be complementary to one another 

This means that they can be joined using these sticky ends

Once they have paired at an enzyme- DNA Ligase -is used to join up the phosphate-sugar backbone of the DNA 

This also means that DNA of different species can be combined using this technique 

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Insertion Of DNA Fragment Into A Vector

The vector is used to transport DNA into the host cell

The most commonly used is a plasmid

Plasmids almost always contain genes for antibiotic resistance

Restriction endonucleases are used to cut this out of the plasmid

This leaves sticky ends that are complementary to the sticky ends of the DNA fragment

They are then incorporated with the plasmid 

The join is made permenant with DNA Ligase 

These plasmids now have Recombinant DNA 

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Introduction Of DNA Into Host Cells

Once the DNA has been introduced it is then reintroduced to the bacterial cells

TransformationThe plasmids and bacterial cells are mixed in a medium with calcium ions and heated- bacteria becomes permeable- plasmid passes through 

Not all the bacterial cells with posses the DNA because:

  • Only a few bacterial cells take up the plasmids (sometimes only 1%)
  • Some plasmids will have closed up again without incorporating the DNA fragment

How to find out if the plasmid has been taken up:

  • All bacterial cells are grown in a medium with the antibiotic
  • Those that have taken up the plasmid will be resistant
  • These will secrete an enzyme to break down the antibiotic
  • Those that have not taken up the plasmid will not be resistant and die

Some bacteria take up the plasmids without incorporating the new gene and survived 

These cells are identified by gene markers

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Polymerase Chain REaction

The Polymerase Chain Reaction to copy fragments of DNA occurs in 3 Stages:

Stage 1- Separation Of The DNA Strand. The DNA fragments, primers and DNA polymerase are placed in a vessel in the thermocycler and the temperature is increased to 95C to separate the strands

Stage 2- Addition (annealing) Of The Primers. The mixture is cooled to 55C causing the primers to anneal to their complementary bases at the end of the DNA fragment. The primers provide the starting sequences for DNA polymerase to begin DNA copying as DNA polymerase can only attach nucleotides to the end of an existing chain. Primers also prevent the two separate strands from rejoining

 Stage 3- Synthesis Of DNA. The temperature is increased to 72C. Optimum temperature for the DNA polymerase to add complementary nucleotides along each of the separated DNA strands. It begns at the primer on both stands and adds the nucletides in sequence until it reaches the end of the chain

The process takes 2 minutes and creates two strands of DNA 

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Genetically Modified Microorganisms

Antibiotics

  • Produced naturally by bacteria.
  • Genetic engineering increased the quantity and the rate at which they are produced

Hormones

  • Insulin is needed daily by 2 million diabetics for them to lead normal lives.
  • Insulin was taken from cows and pigs but could can an immune response. Now bacterial cells with the human insulin producing gene.
  • This method has little chance of rejection and does not kill animals. 

Enzymes

  • Many enzymes used in the food industry are manufactured by genetically modified bacteria.
  • These include amylases that break down starch during beer production
  • lipases used to impove the flavour of cheeses
  • proteases used to tenderise meat.
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Genetically Modified Plants

Tomatoes

  • Enzyme that softens the tomato is removed so they can be easily stored and transported 

Herbicide Resistant Crops 

  • The fields are sprayed to kill the competing weeds but it does not affect the crop 

Disease-Resistant Crops

  • Introduced with genes to withstand infection

Pest-Resistant Crops 

  • Gene introduced that produces toxin to kill insects that feed on it 

Plants That Produce Plastics

  • Gene for producing raw materials for plastic production
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Genetically Modified Animals

Food animals like fish and sheep have a growth hormone added so that they can grow larger than normal at a faster rate. 

Domestic milk-producing animals are inserted with a gene for producing protiens in their milk. The gene is inserted into a fertilised egg. 

Some people have a disorder in one of the alleles that code for an anticlotting protein called anti-thrombin. They are at more risk of blood clots they are treated with anti-thrombin from donated blood. Only small amounts of anti-thrombin can be extracted from blood so using genetically modified goats is much more viable as more can be produced. (http://www.fda.gov/ucm/groups/fdagov-public/documents/image/ucm172410.gif)

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Cystic Firbrosis

Is the most common genetic disorder among white people affecting 1 in 20,000

It is a muated recessive allele caused by deletion of 3 Adenine bases 

The normal gene called cystic fibrosis trans-membrane-conductance regulator (CFTR) gene produces a protein of 1480 amino acids. The mutation takes one amino acid out 

CFTR is a chloride-ion channel protein that transports chloride ions out of epithelial cells and water naturally flows by osmosis- keeping membranes moist

The defective gene means that the protein is not produced or does not function normally causing the epithelial membranes to be dry and the mucus produced is viscous and sticky 

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Treatment Of Cystic Fibrosis Using Gene Therapy

Gene Replacement- The defective gene is replaced with a healthy gene 

Gene Supplementation- One or more copies of the healthy gene are added alongside the defective gene. The healthy allele is dominant the defective gene are masked

Germ-line therapy- Replace or supplementing the defective gene in the fertilised egg ensuring that all cells of the organism will develop normally as will their offspring. A permanent solution but has ethical and moral issues of long-term genetic change so is not legal.

Somatic-cell gene thrapy- Targets just the affected tissues like lungs. It is not in the egg or sperm cells so will not be passed on to future generations. As cells are constantly dying and being replaced the treatement must be repeated- even as often as every few days. Treatment has limited success. Long-term aim is to target stem cells so it can be effective in the lifespan of the individual. 

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Delivering Cloned CFTR Genes

Viruses called adenoviruses cause colds and other respiratory diseases by injecting their DNA into epithelial cell of the lungs so make useful vectors 

The process occurs as follows:

The adenoviruses are made harmless by interfering with a gene involved in their replication

These viruses are grown in epithelial cells in the lab along with plasmids that have been inserted with the healthy gene 

The healthy gene becomes incorporated into the DNA of the viruses 

These viruses are isolated from epithelial cells and purified 

The viruses with the healthy gene are introduced into the nosrils of patients 

The viruses inject their DNA containing the healthy gene into epithelial cells of the lungs 

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Inserting CFTR

Once the plasmid that took up the gene have been identified theey are wrapped in liped molecules before being inserted in the nostril

Genes are wrapped in lipid molecules becuase they easily pass though the phospholipids portion of epithelial cells 

These froms of delivery are not always effective because:

  • Adenoviruses may cause infections 
  • Patients may develop immunity 
  • The liposome aerosol may not be fine enough to pass through the tiny bronchioles in the lungs 
  • Even when the CFTR gene is successfully devlivered very few are actually expressed
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Gene Therapy For Immunodeficiency

Severe combined immunodeficiently (SCID) is a rare inherited disorder where they do not have a cell- mediated immune system or produce antibiotics 

Was found in only young children because they usually died of infection before they reached maturity 

The disorder is caused by a defect in the gene that codes for the enzyme ADA that destroys toxins that would otherwise kill white blood cells 

Patients have to be raised in a strictly sterile environment of an isolation tent and recieve bone marrow transplants and injections of ADA 

The gene is isolated and inserted into a retrovirus are grown in host cells and mixed with the patients Tcells which inject a copy of the normal ADA gene into the Tcells that are then reintroduced into the patients blood 

Tcells only live for 6-12 months 

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DNA Probes

Is a short single stranded section of DNA that have a label attched that makes it easily identifiable 

Two most commonly used-

Radioactively Labelled Probes- Made up of nucleotides with the isotope 32P. The probe is identifiable using a photographic plate that is exposed by radioactivity

Fluorescently Labelled Probes- emit light under certain conditions

DNA probes are used to identify genes in the following way-

  • A DNA probe  is made with complementary bases to the DNA sequence
  • The DNA is treated to separate its two strands
  • The separated DNA strands are mixed with the probe which binds to the complementary bases on one of the strands- DNA Hybridisation
  • The site at which the probe binds can be identified by the radioactivity or fluorescence  
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DNA Sequencing

The Stanger Method- Used modified nucleotides that cannot attach to the next base in the sequence when they are being joined together- act as terminators ending the synthesis of a DNA strand 

Four different terminator nucleotides are used each with one of the four bases 

The first stage is to set up for four test tubes-

  • Many single-stranded fragments of DNA to be sequenced act as a template for the synthesis of its complementary stand
  • A mixture of nucleotides with the bases 
  • A small quantity of one of the four terminator nucleotides 
  • A primer to start the process of DNA synthesis. This primer is radioactively labelled or fluorescently labelled 
  • DNA polymerase tocatalyse DNA synethesis
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Gel Electrophoresis

The DNA fragments are placed on to an agar gel and a voltage is applied accross it

The resistance of the gel means that the larger the fragments the more slowly they move

A sheet of photographic film is placed over the agar gel for several hours 

The radioactivity from each DNA fragment exposes the film and shows where it is on the gel

(http://www.michaelsharris.com/12ubio/pix/elctrophoresis.jpg)

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Restriction Mapping

Invoves cutting DNA with a series of different restriction endonucleases that are separated by gel electrophoresis. The recognition sites can be determined by the patterns of fragments that are produced.

For example: Plasmid with 100,000 bases and the endonucleases cuts the plasmid but because it was circular if only one of the restriction endonucleases at a time then we will always get a single piece of DNA that is 100kb long regardless of which endonuclease used. 

Two cuts mean that we will always be left with two fragments of DNA from a circular plasmid and the length of each of the two fragments depends on which two restriction endonucleases are used to make the cut 

What will be the length if two pairs are used together? 

1 cut is 10kb in length and he 2nd cut to the 1st cut is (30+60)=90kb long 

We know that the fragemnt is 100kb in length so the 2nd fragment is 90kb in length

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Summary Of Genetic Screening

1 Genetic libraries store the DNA sequences the genes responsible for diseases 

2 Fragment of DNA with complementary bases to the mutated gene is produced

3 DNA probe is formed by radioactively labelling the DNA fragment

4 PCR techniques are used to produce multiple copies of DNA probe

5 Probe is aded to single-stranded DNA fragments from the person being screened

6 Donor has the mutated gene- some have nucleotide sequence that is complementary to the probe that will bind to its complementary bases on the donor DNA 

7 These DNA fragments will be labelled with the probes- can be seen from the DNA fragements by the used of X-ray film 

8 Complementary fragments- In the DNA probe- taken up and the X-ray film will be exposed 

9 Complementary fragments- not present the DNA probe-  not be taken up and the X-ray film will be unexposed

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