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The RNA of four species of Leishmania using the primer CTA involved: donovani, major, mexicana, and tarentolae. The RNA and organelles in the cells were broken down utilizing reverse transcriptase enzymes. The environment is free of RNase in order to maintain purity of the samples. Complementary DNA, cDNA, is created from the isolation of RNA. This ensures that the DNA is pure enough to further the investigation. A nanodrop is performed and the nanodrop of the primer CTA of Leishmania donovani had a measurement of 1.91. The nanodrop of the primer CTC of Leishmania major had a ratio of 1.87. The 260nm demonstrates the absorption of the DNA and the 280nm depicts the absorption of proteins and amino acids present in the sample. The restriction digestion of cDNA creates receptor sites utilizing EcoR1/Mse1 enzyme mix. The enzymes cut the cDNA in specific known sites on the cDNA that allow places for known sequences which are ligated later. Adapter ligation attaches sequences to sites that cut by the restriction enzymes. Adapter ligation is performed at a low temperature in order for the molecules of cDNA to move more slowly which allows them to ligate to each other. The pre-amplification number of cDNA strands with the known sequences will increase through three temperature processes composed of denaturing, annealing, and extending. The strands are denatured at 94° C in order for the DNA to separate and become denatured. This allows for the process of annealing to occur at ~50° C. The primers bind to the strands of DNA. Subsequently, the extension temperature is raised up to ~72°C. The nucleotides are added to the DNA and they increase the size of the molecule of DNA. Selective amplification selects a selective primer that will bind to the CTA primer of Leishmania. Three pieces of DNA will bind with the selective primers and will be amplified. Consequently, the DNA pool is narrowed down. Electrophoresis is performed on the CTA primers of the species and the molecules are separated through a current based on size.
Leishmania, also called Kala - Azar or Black Fever is of the genus trypanosome protozoa, which is responsible for the disease Leishmaniasis. Leishmania is intracellular with macrophage replicating amastigotes. There are over 300 million people at risk of becoming infected with Leishmaniasis, and 1.5- 2 million new cases a year. There are over 12 million active cases of Leishmaniasis which makes it an endemic in more than 60 countries, and the second most important parasitic disease after Malaria. There are three different stages of Leishmania, the visceral, muccocutaneous and Cutaneous, which are detailed below.
The visceral form of Leishmaniasis, caused by the donovani and shagase strands, is the most severe and can be deadly if a secondary bacterial infection occurs. There is a 3-8 month incubation period with symptoms later occurring as a fever, weight loss, anemia and hepatosplenomegaly (enlargement of the liver and spleen). The cutaneous form, caused by the major and mexicana strands, is the least severe form of leishmaniasis and is commonly self healing. The sand fly bite develops as a papule which later increases in size and ulcerates. This process can take up to eight months and it is possible for the papule to reoccur. The muccocutaneous form of Leishmaniasis, which is caused by the brazilanas strand, has a one to three month incubation period. It occurs first as a papule at the site of the sandfly bite and travels by metastasis and infects the nose, mouth and pharynx which create difficulty eating and a risk of infection. The severity of Leishmaniasis is determined by the infecting parasite species and immunological status of the host.
Leishmania occurs in two forms; the amastigote and promastigote form. The promastigote form of Leishmania occurs in the midgut of female Phlebotomus and Lutzomyia species of sand flies as 5-15um elongated flagellated cells. The amastigote form occurs in the mammalian host as 3-5um ovoid cells. The transmission of Leishmaniasis occurs through the bite of a sand fly into an infected host. Sand flies, which are the vector, ingest the Leishmania organism as an amastigote into its digestive tract when it feeds on an infected mammal. The amastigotes later develop into promastigotes in the sand fly's digestive tract. During the next feed the flagellated matacycid promastigotes are injected into a host. The promastigotes infect the macrophages of the mammalian host and become amastigotes. The cycle continues.
Leishmaniasis is detected by the examination of tissue, lymph nodes, or a liver biopsy under a microscope. Leishmaniasis is diagnosed by a direct agglutination test. The treatment of Leishmania comes in four commonly used forms. The treatment of Visceral Leishmanasis can be achieved through a Sodium Stibogluconate or Medlumine Antimonate intravenous or intramuscularly drip. Amphotericin B, a polyene antifungal drug, is the most commonly used treatment although it produces severe or lethal side effects. Miltefosine is a relatively new treatment, and the first to be orally active. By taking Miltefosine for three to four weeks there is a 95% cure rate. Cutaneous Leishmaniasis is not life threatening and the papule can be lasered off or treated with paromomycin ointment. As a barrier against Leishmania some governments have begun spraying houses with insecticide. A treatment for Visceral Leishmaniasis is important because without effective treatment Visceral Leishmaniasis has a morality rate of 75-95%.
cDNA is a complimentary copy of RNA which is created using reverse transcriptase enzymes. This enzyme works on a single strand of mRNA, generating its complementary DNA based on the pairing of RNA base pairs. Leishmania does not have intron which makes the mRNA look identical to the cDNA. cDNA is used to clone eukaryotic genes and to express specific proteins in cells that are usually not expressed. Consequently, they will transfer the cDNA that codes for the protein to the recipient cell.
RNA was isolated by adding 1ml TRIzol to the gDNA samples and incubating. The sample was then centrifuged for 5 minutes at 12,000xg and the aqueous layer was removed without disturbing the pellet. The pellet was then washed in .5ml Isopropanol and centrifuged for 10 minutes at 12,000xg. The supernatent was removed and the pellet was washed with 1ml of 70% ethanol. The pellet was then air dried and RNAse free water was added.
cDNA is a complementary copy of RNA which is created using reverse transcriptase enyzmes. A short double stranded overhang is created at the end of the DNA sequence so that the primers contained in the Vilo Reaction mix can bind and create target cited for transcription.
The purpose of this is to allow the focus to be specifically on the transcribed regions of the gene.
cDNA was created from the isolated RNA by adding 4µl of 5X Vilo Reaction Mix, 2µl of 10X Superscript Enyzme Mix, 2ug of RNA and 12µl of DEPC treated H20. The samples were then thermocycled at 25° C for 10 minutes, 42° C for 60 minutes, 85° C for 5 minutes and 4° C for 1 minute. The sample was then nanodropped to ensure quality concentration and purity.
Restriction Digest of cDNA
Restriction Digest of cDNA allows for the formation of known receptor sites through the use of EcoR1/Mse1 enzyme mixes. These enzymes cut at specific, known locations, called restriction sites, allowing for the known sequences to be ligated. It is crucial that the restriction enyzmes (in this case EcoRI/Mse1 are kept on ice because as proteins, they denature as the temperature increases.
On ice, 2.5µl of 5X Reaction Buffer, 9µl of the template cDNA, 1µl of EcoRI/Mse1 enyzme mix were added to a PCR tube. The samples were then thermocycled for 2 hours at 37° C, 15 minutes for 70° C, and 4° C until ready to be ligated.
Adapter ligation is a crucial step where the known sequences are attached to the restriction sites which were created through the restriction digest. The T4 ligase used in this experiment was highly concentrated and therefore not much was needed. T4 ligase is used for creating cDNA by inserting the specified DNA molecµles into the vector.
Using the cDNA from the restriction digest (12.5µl), on ice 12µl of Adapter Mix and .5µl of T4 Ligase were added directly the cDNA sample. The samples were centrifuged and thermocycled at 20° C for two hours and 4° C until ready to use. A 1:10 dilution of the ligation mixture was performed and 10µl of the newly created ligation mixture and 90µl of low TE buffer were added to a new PCR tube. Both the adapter ligation product and the 1:10 dilution adapter ligation were stored at -20° C.
Pre-Amplification Polymerase Chain Reaction
Through Pre-Amplification PCR the cDNA with known sequences are mµltiplied by a three step process of denaturing, annealing, and extension. Denaturing allows the double stranded cDNA to separate while annealing allows for the primers to bind to the single stranded DNA. Extension allows the nucleotides which were added to the DNA to increase the size of the molecµles of the DNA.
In a clean PCR tube on ice add 2.5µl of the diluted 1:10 ligation mix, 20µl of AFLP pre-Amplification Primer mix, 2.5µl of PCR reaction buffer and .5µl of Taq Polymerase. The sample is then centrifuged and thermocycled at 94° C for 30 seconds (denaturing), 56° C for 1 minute (annealing), and 72° C for 1 minute (extension). This cycle is repeated for 20 cycles. Following the thermocycling, a 1:100 dilution of the pre-amp sample is performed (1µl of pre-amp samples, 99µl of distilled H20). The sample is stored at -20° C.
Selective AFLP Amplification
In selective AFLP a primer, CTA, was chosen as having a high probability of binding to our cDNA samples. Only pieces of cDNA that bind with the CTA primer are amplified allowing for a more narrow aray of cDNA to work with.
In a new PCR tube 6µl of Taq DNA Polymerase, which contains deonized water, 10x amplification buffer and Taq polymerase, is added along with 2µl of the diluted cDNA sample, 2µl of CTA Mse1 primer, and .5µl of both the IRDye 700 and 800 labled EcoR1primers.
Agarose Gel Electrophoresis
Gel electrophoresis was performed in order to separate the large molecules from a mix of similar molecµles, through the use of an electrical current. Large molecules are retained longer, allowing banding patterns to occur. The migration of the samples is inversely proportional to 10g/o of the size.
A .8x TBE buffer was created using 80ml of 10X buffer and 920ml of water. .5g of agarose, and .5µl of Ethinium Bromide were added and the buffer was heated until clear. This mixture was poured and allow to solidify on the gel plates. 5µl of ___ladder was added to the each of the cDNA samples. And 2µl were injected in to the corresponding wells of the gel and run at 125volts for 60 minutes.
Topo TA Cloning
The role of Topo Cloning is, through the use of heat shock, allow the plasmid (foreign DNA vector) to be inserted in to the ecoli cells. A blue-white screening test is performed to determine which colonies that form have the plasmid insert.
.5µl of PCR product is combined with 1µl of 10% buffer, 5µl of H2O, and 1µl of Ligase. And spread on Agar plates and allowed to grow overnight. In 6 separate PCR tubes, six white colonies that were picked off the plates and placed individually in each PCR tube boiled for 5 minutes. The samples were immediately placed on ice for five minutes and then centrifuged for 5 minutes.
Mini Prep Kit
Cells were re pelleted and then re suspended in 250ul of P1 buffer. 240Ul of P2 buffer and 350ul of the N3 buffer were added to the sample. The sample was then centrifuged at 13,000xg for 10 minutes. The supernatant was removed into a spin column. The sample was then centrifuged at 60 seconds and the flow through was removed. The column was then washed with .5ml of PB buffer. The sample was centrifuged for 60 seconds and washed with .75ml of buffer PE. The supernatant was then removed and a new microfuge tube was placed on the column. 50Ul of elution buffer to the column and let stand for 60 seconds. The sample was centrifuged for 60 seconds and then nanodropped.
Figure 2:Bar colors represent the derived value that reflects the degree of similarity between query sequence and the Blast search results.