NUCLEOTIDE SEQUENCES OF D-LOOP MITOCHONDRIAL DNA IN HUMAN MESODERM LAYER

• Main Author: Unwakoly, Semuel
• Format: Theses
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/35488
• Institution: Institut Teknologi Bandung

Adult individuals originate from a single cell as the fusion of a sperm and ovum forming a zygote. Human embryo posses organelle such as mitochondria with the main function as producers of energy in cells. Mitochondria has its own genetic material, called mitochondrial DNA (mtDNA). Cell growth into many cells during embryogenesis. When the numbers of cells reached a certain level, these cells differentiated into three embryonic layers; ectoderm, mesoderm, and endoderm respectively. There are cases in which mutations in the mitochondrial genome would cause genetic. Diseases affecting all human tissues, despite the fact that some diseases only occur in certain tissues. For example, the Myoclonic Epilepsy with Ragged Red Fibers (MERRF) disease that occurs in the muscle tissue, Leber’s Hereditary Optic Neuropathy (LHON) in optic nerve tissue, and the Kaerns-Sayre Syndrome occurs in the eye tissue respectively. Recently, it is not clear whether there is a polymorphisms in mtDNA nucleotide sequence in tissues originated from each of that embryonic layer. Therefore, the purpose of this study was to determine whether there are differences in the nucleotide sequences of the human mtDNA D-loop fragment in tissues derived from the mesoderm layer. Lymph tissue (L-1 and L-2) and, kidney tissue (G-1 and G-2) from two individuals were used in this study. Each tissue sample was lysed to obtain a DNA templates followed by amplification using Polymerase Chain Reaction (PCR) method. Nucleotide sequences of amplified DNA fragments of tissue samples were determined using Dideoxi Sanger method. Then, the in silico analysis was done by employing the SeqManTMII, EditSeqTM, and MegAlignTM DNASTAR software. PCR primers of M1 (from 15.978 to 15.997) and HV2R (429 to 409), which each size are 20 nt and 21 nt respectively, were used to amplify DNA fragment of 0.9 kb in size. Agarose gel electrophoresis were employed to confirm the PCR result, in which pUC19/HinfI was used as DNA size marker. Electrophoresis analysis of PCR results showed that there was a band of DNA fragment of each of the samples G-1, L-1, G-2, and L-2 with the size of about 0.9 kb. Electrophoregraph of sequencing results from all samples showed that there were 947 base pairs of nucleotides seqeunces which were able to be compared. The alignment results of G-1 and L-1 tissue samples showed that there were differences in their nucleotides sequence. The deletion of C was found at the nucleotide positions of 310. On the other hand, the result of G-2 and L-2 sample showed no differences in their nucleotides sequence. The nucleotides sequence difference of the sample-1 were also found in previous studies, they were deletion of C 16183 in the endoderm layer, and bases substitution of C16197T, C16270G, and C16444T of the ectoderm layers. These indicate that mutation occured during the process of embryogenesis in the tissue development on those three layers. These results suggest that it is necessary to do further research in the polymorphisms of mtDNA nucleotides sequence in various tissues from a single individual.