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Daily Research on Tuberculosis: PCR, Electrophoresis, and Extraction of Tuberculosis DNA
Indonesia (CIMSA-ISMKI) - Universitas Airlangga, Surabaya
Tuberculosis Study Group, Institute of Tropical Disease, Universitas Airlangga Universitas Airlangga, Campus C, Jalan Dr. Ir. H. Soekarno, Mulyorejo, Surabaya, Indonesia
Prof. Dr. Ni Made Mertaniasih, dr., MS., Sp.MK(K)
Agnes Dwi Sis Perwitasari, A.Md.K
Type of Research Project
- Basic science
What is the background of the project?
There are roughly 500.000 new cases of tuberculosis annually, which contributes to 175.000 deaths. This puts Indonesia in the second place, right after India, as the country with the most tuberculosis cases in the world. Hence, researcher must continuously do researches to find every important detail regarding the prevention and treatment of tuberculosis. In order to run researches, the TB study group needs a lot of Mycobacterium samples, so it is a must to do daily DNA extraction and electrophoresis to deliver the samples needed.
What is the aim of the project?
The aim is for the exchange students to learn how to use Polymerase Chain Reaction (PCR), do electrophoresis, and observe the making of agarose and extraction of Tuberculosis Deoxyribose Nucleic Acid (TB DNA)
What techniques and methods are used?
1. Extraction of TB DNA DNA extraction involves separating the nucleic acids in a cell away from proteins and other cellular materials. The extraction of DNA generally follows three basic steps: • Lyse (break open) the cells. • Separate the DNA from the other cell components. • Isolate the DNA. 2. PCR (Polymerase Chain Reaction) Polymerase chain reaction, or PCR, is a technique to make many copies of a specific DNA region in vitro (in a test tube rather than an organism). The basic steps are: • Denaturation (96°C): Heat the reaction strongly to separate, or denature, the DNA strands. This provides single-stranded template for the next step. • Annealing (55 - 65°C): Cool the reaction so the primers can bind to their complementary sequences on the single-stranded template DNA. • Extension (72°C): Raise the reaction temperatures so Taq polymerase extends the primers, synthesizing 3. Electrophoresis Gel electrophoresis is a technique commonly used in laboratories to separate charged molecules like DNA, RNA and proteins according to their size. An electric current is applied across the gel so that one end of the gel has a positive charge and the other end has a negative charge. The movement of charged molecules is called migration. Molecules migrate towards the opposite charge. A molecule with a negative charge will therefore be pulled towards the positive end. The gel consists of a permeable matrix, a bit like a sieve, through which molecules can travel when an electric current is passed across it. Smaller molecules migrate through the gel more quickly and therefore travel further than larger fragments that migrate more slowly and therefore will travel a shorter distance. As a result, the molecules are separated by size.
What is the role of the student?
- The student will mainly observe
- The tasks will be done under supervision
What are the tasks expected to be accomplished by the student?
In the beginning of the exchange programme, lectures from the expert would be given. Then, the students will mainly observe the steps and methods done by the study group. In this research, we would like to take the students to learn the daily research activities that is usually done daily by the TB team in the Institute of Tropical Diseases. Students will take part in the DNA isolation, PCR, and Gel Electrophoresis. By the end of the exchange program, the students are expected to make a presentation on the material given and learned throughout the month. The presentation will then be presented in front of the mentors. Aside from that, the students will be asked to make a scientific report and answering the questions given by the research mentors.
Will there be any theoretical teaching provided (preliminary readings, lectures, courses, seminars etc)
Yes. Before the students join the lab work, they will be given lectures from the tutors regarding the material they will be experimenting in the lab. The lectures that will be given are: 1) Epidemiology of Tuberculosis by Dr. Eko Budi Koendhori, dr., M.Kes., Sp.MK(K) 2) How to make a sample from sputum by Dr. Eko Budi Koendhori, dr., M.Kes., Sp.MK(K) 3) Blood culture, urine culture, and pus culture by Lindawati Alimsardjono, dr., M.Kes., Sp.MK(K) 4) Molecular Observation on PCR, Electrophoresis, Agarrose making, and the final product by Agnes Dwi Sis Perwitasari, S.Si 5) Microscopic Observation with Agnes Dwi Sis Perwitasari, S.Si 6) Observation of TB DNA Extraction by Agnes Dwi Sis Perwitasari, S.Si 7) Final Discussion on Tuberculosis with Deby Kusumaningrum, dr., M.Si
What is expected from the student at the end of the research exchange? What will be the general outcome of the student?
- The student will prepare a presentation - The student will prepare a scientific report
What skills are required of the student? Is there any special knowledge or a certain level of studies needed?
There are no required skills needed prior the internship
Are there any legal limitations in the student’s involvement
The students are not allowed to perform the lab work without supervision from the tutors
Type of students accepted
This project accepts: - Medical students - Graduated students (less than 6 months) - Pre-Medical students from the American-British system - Students in biomedical fields
- Mertaniasih; Ni Made et al. 2014. ‘THE ANTI-TB DRUG SENSITIVITY OF MYCOBACTERIUM TUBERCULOSIS FROM CEREBROSPINAL FLUID AND BONE TISSUE BIOPSY SPECIMENS OF PATIENTS SUSPECTED TUBERCULOUS MENINGITIS AND SPINAL TB IN DR SOETOMO HOSPITAL INDONESIA’. Indonesian Journal of Tropical and Infectious Disease. Vol 5 (3)
- Sapriadi and Syahridha. 2018. ‘FACTOR RELATED TO ANTI-TUBERCULOSIS DRUG RESISTENCY ON PULMONARY TUBERCULOSIS PATIENTS IN LABUANG BAJI HOSPITAL MAKASSAR’. Indonesian Journal of Tropical and Infectious Disease. Vol 7(2)
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