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title page
Contents
(Abstract) 5
Introduction 8
Materials and methods 13
Clinical characteristics of patients 13
PCR amplification and sequencing of the mtDNA control region and Cytb 13
Gene scan analysis for the determination of HV region length heteroplasmies 15
TA cloning 16
Determination of mtDNA copy number 17
Detection of mtDNA 4977 bp large deletion 18
Measurement of ROS level 19
Statistical analysis 20
Results 22
Sequencing data interpretatation for mtDNA nucleotide changes in peptic ulcer tissue from control subjects (heteroplasmic mutation in peptic ulcer) 22
Mitochondrial genome instability in peptic ulcer tissue from patients 22
mtDNA copy number 24
Discussion 26
References 32
(국문초록) 37
Table 1. Primer sets for the PCR and direct sequencing of the mtDNA control region and Cytb genes 39
Table 2. Sequence variations of the mtDNA control region and Cytb from 25 peptic ulcer patients 40
Table 3. Mitochondrial genome instability of peptic ulcer tissues and corresponding bloods 42
Table 4. Amino acid changes among polymorphism and mutation in Cytb gene 46
Fig. 1. PCR products of mtDNA control region (A) between nucleotide position (np) 16024 and np 576 (1.12kb) and cytochrome b gene (B) at np 14909-15396 (480bp) for direct sequencing.... 48
Fig. 2. Sequencing chromatograms disclose peptic ulcer tissue-specific heteroplasmic mutations from HV1 (patient No 9) (I), HV2 (patient No 9) (II) and Cytb gene (patient No 2) having nonsynonimous mutation (III).... 49
Fig. 3. Gene scan analysis of poly C-stretch region at np 303-315 (left panel) (A), np16184-16193 (center panel) (B) and CA repeat starting at np 514 (right panel) (C) revealed typical length heteroplasmic mutations from patients No 25, 22 and 9. 50
Fig. 4. Amplification plot (I), standard curves (II) obtained with serial 10-fold dilution from 5.8x108 copies to 5.8x105 copies/μl of mtDNA CYTB standard, and melting curve analysis (III) for quantitative mtDNA PCR....(이미지참조) 51
Fig. 5. mtDNA copies from peptic ulcer tissues (2.2x106 ± 1.1x106 molecules/μL, mean ± SD) had about 2.5-fold increase in the mtDNA copy No compared with corresponding those from blood (8.6x105 ± 0.8x106 molecules/μL)(이미지참조) 52
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