NUCLEOTIDE SEQUENCE OF POLI-C HUMAN MITOCHONDRIAL DNA HVSI REGION

• Main Author: Puspitasari (NIM 105 03 041), Dea
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/5914
• Institution: Institut Teknologi Bandung

Abstract : <br /> <br /> <br /> <br /> <br /> Mitochondria has its own genome that is maternaly inherited. There is a region of Hypervariable Segment I (HVSI) that has high polymorphism rate on the D-loop area of human mitochondrial DNA (mtDNA). There are a lot of publication concerning heteroplasmy phenomena of human mtDNA and its suggested as the cause of the incompletion of sequence determination HVSI region which has poli-cytosin (poli-C) stretch through direct sequencing shown by the unread sequences after those stretch. The previous experiment has solved this problem by using cloning method. Heteroplasmy on sample proved as length variation of poli-C from several sequenced clones. The objective of this research is to prove a hypothetic that heteroplasmy of mtDNA as the cause of the problem through sequencing of DNA template clones mixture that have different poli-C by variable composition. This research started by preparation DNA template of bacterial single colony by lysis method, then was amplified using Polymerase Chain Reaction (PCR) method, mixed DNA clones that have length variation of poli-C, followed by sequencing of the amplified mtDNA using Dideoxy Sanger method along with nucleotides analysis using Seqman TM version 4.0.0. Analysis of PCR product showed 0.4 kb band on the agarose gel electrophoresis. Samples electrophoregraph along with HVSI nucleotide sequence for each clones and clones mixture was obtain through sequencing process. Analysis of HVSI nucleotide sequences for each clones and clones mixture to Cambridge Reference Sequence (CRS) shown significant indication that heteroplasmy caused unsuccessfully of direct sequencing the samples that have poli-C stretch. <br />