Sunday 24 February 2019

Gel Electrophoresis- Genetic Technology

Gel Electrophoresis

Gel electrophoresis is a technique used to separate DNA fragments (or other macromolecules, such as RNA and proteins) based on their size and charge.
The suffix phoresis means "migration" or "movement."  The prefix electro tells us that we are using electricity to make molecules migrate.
Electrophoresis involves running a current through a gel containing the molecules of interest. Based on their size and charge, the molecules will travel through the gel in different directions or at different speeds, allowing them to separate from one another.

Gel Electrophoresis of DNA
      DNA is extracted from a sample, such as sperm, hair, etc.
      DNA is chopped using enzymes called Restriction Endonucleases.
NB: Restriction endonucleases are enzymes that cut specific regions in the DNA sequence. They cut the sugar phosphate bone to give either sticky ends or blunt ends.
      Restriction Endonucleases cleave the DNA close to VNTR region.
NB: Variable tandem number repeat is a repeat sequence in the DNA that is different in each person.
      The DNA is ready for electrophoresis
NB: If you’re comparing DNA, cut both DNA with the same restriction enzyme.
      Place the DNA fragments in a well with an electric current applied using a teat pipette. The DNA should be on the negative side or the cathode since it is negatively charged. Phosphates in nucleotides of DNA have negative charge. This gives the DNA a negative charge. 
      All DNA molecules have the same amount of charge per mass. Because of this, gel electrophoresis of DNA fragments separates them based on size only.Since DNA is negatively charged, it will travel to the positive side. Smaller fragments travel furthest as they have smaller mass. Larger fragments are slower and would travel the least.
      When a gel is stained with a DNA-binding dye and placed under UV light, the DNA fragments will glow, allowing us to see the DNA present at different locations along the length of the gel.
      Probes can also be added to make the film go dark when placed on an X-Ray film
      A well-defined “line” of DNA on a gel is called a band. Each band contains a large number of DNA fragments of the same size that have all travelled as a group to the same position
      One well is reserved for a DNA ladder, a standard reference that contains DNA fragments of known lengths. By comparing the bands in a sample to the DNA ladder, we can determine their approximate sizes. Helps give estimates of how long the other base pairs are when run alongside it.

Gel electrophoresis has many uses ranging from criminal investigations to paternity tests. It is used in conjunction with other techniques to determine if someone has a particular allele of a gene, such as screening for sickle cell anaemia.  

.


Malaria Infectious Diseases


Malaria

Malaria is a deadly infectious disease caused by the protozoa Plasmodium. An infectious disease is caused by a pathogen and is referred to as communicable as it is transferred from an infected to uninfected person. Malaria has four pathogens: Plasmodium falciparum, P. vivax, P. ovale, P. malariae, which are all transferred by the by the vector (a vector is an organism which carries a disease from one person to another or from an animal to a human): the female Anopheles mosquito. Malaria affects 500 million and kills more than 1 million people each year. It is endemic in more than 100 third world countries in tropical and subtropical regions such as Sub-Saharan Africa, Asia and Latin America. It is maintained there by transmission between people.
NB: Endemic means native or confined to a certain place or constantly present to a greater or lesser extent in a particular locality.

Transmission of Malaria
There are three ways in which malaria can be transmitted:
1.    The female anopheles mosquito
2.    Blood transfusion and when unsterile needles are re-used
3.    Movement from the placenta into the fetus
When a mosquito bites a person, blood taken by mosquitoes provides protein and iron needed for formation of eggs. If the person is a carrier for malaria or is infected with Plasmodium, the mosquito takes some of the gametes from the blood meal. These male and female gametes fuse in the mosquito’s gut to form infective stages, which move to the mosquito’s salivary glands. When she feeds again, she injects an anticoagulant from her salivary glands that prevents the blood meal from clotting so blood can flow freely from the human to the mosquito. The infective stages pass from the mosquito’s salivary glands into the human’s blood flow including the anticoagulant and enter the red blood cells where they multiply.
NB: “anti”- against, opposite “coagulant” causes coagulation which is to thicken or to change from liquid to a solid state. Hence, they are chemical substances that prevent or reduce coagulation of blood
Plasmodium is carried in the bloodstream to the liver, where they invade liver cells and reproduce asexually. At about eight days after infection, the liver cells burst, releasing millions of Plasmodium into the bloodstream where they invade the red blood cells.

Plasmodium reproduces asexually inside the red blood cells. Within the red cells the parasites grow in a membrane-bound digestive vacuole, hydrolyzing haemoglobin through secreted enzymes. Some parasites develop into sexual forms called Gametocytes, which infect the mosquito. Plasmodium makes you ill about 10-15 days after infection, when they burst out of the red blood cells and release a chemical that causes fever. The Plasmodium is then free to invade more red blood cells. A person may have many bouts of malaria fever.
At each stage, there is a huge increase the number of parasites, increasing the chances of infecting another mosquito or human.

Symptoms of Malaria
Typically, victims who are bitten by malaria-carrying mosquitoes experience no symptoms until 10 to 28 days after infection. The first clinical signs may be any combination of chills, fever, headache, muscle ache, nausea, vomiting, diarrhea, and abdominal cramps. Chills and fever occur in periodic attacks; these last 4 to 10 hours and consist first of a stage of shaking and chills, then a stage of fever and severe headache, and finally a stage of profuse sweating during which the temperature drops back to normal. Persons with malaria commonly have anaemia (owing to the destruction of red blood cells by the parasites), enlargement of the spleen (the organ responsible for ridding the body of degenerate red blood cells), and general weakness and debility.


Treatment of Malaria
This is through the use of anti-malarial drugs including quinine and chloroquine. These can be used as prophylactic (preventative) drugs, stopping an infection from occurring if the person is bitten. This is taken before or after visiting an area where malaria is endemic. Chloroquine inhibits protein synthesis and prevents the parasite from spreading. Proguanil inhibits sexual reproduction of Plasmodium within the biting mosquito.
When these drugs are used widely, Plasmodium has become resistant.  An example is chloroquine in parts of South Africa, Africa and New Guinea, leading to the introduction of mefloquine, which is rather expensive and has some unpleasant side effects such as restlessness, dizziness, vomiting and disturbed sleep.

Prevention of Malaria
The way in which a pathogen passes from one host to another is called a transmission cycle. Control methods try to break this cycle by removing conditions that are favourable for the spread of the pathogen. Prevention and control methods include:
§  Stocking ponds, irrigation and drainage of ditches and other permanent bodies of water with fish with which feed on mosquito larvae.
§  Spraying a preparation containing bacteria that kills mosquito larvae.
§  Sleeping under mosquito nets to avoid being bitten by mosquitoes.
§  Spraying oil or paraffin on stagnant bodies of water.
§  Use of drugs to kill the parasite
§  Killing adult mosquitoes with insecticides such as DDT

Worldwide Concerns
Malaria prevention programs are unsuccessful as:
§  Plasmodium became resistant to drugs used to control it.
§   Mosquitoes become resistant to DDT and other insecticides.
The reason for worldwide concern over malaria stems from:
§  An increase in drug-resistant forms of Plasmodium
§  An increase in the proportion of cases caused by P. falciparum, the form that causes severe, often fatal malaria.
§  Difficulties in developing vaccines against malaria
§  An increase in the number of epidemics, caused by climatic and environmental changes that favour the spread of mosquitoes.
§  Migration of people from areas where malaria is endemic, for political and economic reasons
Control methods now focus on working within health systems to improve diagnosis, improve the supply of effective drugs and promote appropriate methods to prevent transmission. Plasmodium genome has been sequenced, which may lead to the development of effective vaccines. Drugs are used in combinations to reduce the chances of drug resistance arising.
Three factors may lead to improvements in the control of malaria:
§  Use of modern techniques in gene sequencing and drug design
§  Development of vaccines targeted against different stages of the parasites life cycle.
§  A renewed international will to remove the burden of disease from the poorest parts of the world, allied to generous donations from wealthy individuals and foundations.