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Abbreviations 15
Abstract 16
PARTⅠ 18
Ⅰ. Introduction 18
Ⅱ. Materials and Methods 20
Ⅱ.1. Patients and Controls 20
Ⅱ.2. Cytogenetic and Y Chromosome Microdeletion Assay 21
Ⅱ.3. Pyrosequencing Assay for Screening of MTHFR C677T and A1298C 22
Ⅱ.4. Polymerase Chain Reaction Restriction Fragment Length Polymorphism for C677T and A1298C of MTHFR 22
Ⅱ.5. Statistical Analysis 23
Ⅲ. Results 24
Ⅳ. Discussion 27
Reference 38
Abstract in Korean 42
PARTⅡ 43
Ⅰ. Introduction 43
Ⅱ. Materials and Methods 45
Ⅱ. 1. Patients and Controls 45
Ⅱ. 2. Cytogenetic and Y Chromosome Microdeletion Assay 45
Ⅱ. 3. Cell and Its culture condition 45
Ⅱ. 4. RNA Extraction and DNAse treatment 46
Ⅱ. 5. Semi-quantitative Reverse Transcription Polymerase Chain Reaction (RT-PCR) 46
Ⅱ. 6. Northern Blot Analysis 48
Ⅱ. 7. Array Fabrication 48
Ⅱ. 8. Probe Preparation and Hybridization to cDNA microarray 49
Ⅱ. 9. Analysis of Gene Expression 49
Ⅲ. Results 51
Ⅲ. 1. The identification of X inactivation in a normal male and female, and KS patient by semi-quantitative RT-PCR and northern blot analysis 51
Ⅲ. 2. Differential expression of X chromosomal genes in a normal male vs KS patient and a normal female vs KS patient 51
Ⅲ. 3. The X chromosomal genes in KS patient (47, XXY) showed abnormal inactivation 52
Ⅳ. Discussion 54
Reference 65
Abstract in Korean 68
Table 1. Distribution of MTHFR C677T and A1298C genotypes in infertile
and fertile males 30
Table 2. The frequency of MTHFR 677 genotype according to the four
subtype of infertile male group 32
Table 3. The frequency of MTHFR 677 genotypeinfertile males according
to cytogenetic and chromosome deletion analysis 33
Table 4. Frequency of MTHFR 677 genotype in unexplained infertile male
group 34
Table 5. Genes that showed elevated expression levels over twice signal
intensity in cell of KS patient than cell of female and male 57
Table 6. Identification by semi-quantitative RT-PCR of randomly selected
genes with differential expression from male, female and KS by cDNA
microarray 59
Figure 1. The pyrosequencing and PCR-RFLP results from male control and
infertile patients 35
A. The pyrosequencing data for 677 C→T Polymorphism of MTHFR gene 35
B. The pyrosequencing data for 1298 A→C Polymorphism of MTHFR gene 35
C. The MTHFR C677T genotype by PCR-RFLP 36
D. The MTHFR A1298C genotype by PCR-RFLP 36
Figure 2. Detection of the XIST transcripts by RT-PCR and Northern blot
an alysis 61
A. Results by semi-quantitative RT-PCR in a KS, normal male and
female 61
B. Results by northern blot analysis in a KS, normal male and
Figure 3. The X chromosome-specific cDNA array hybridized to Cy3- and Cy5- labelled cDNA from lymphoblastoid cell line 63
A. Hybridization from cDNA of a normal female (Cy3-labelled cDNA) and KS (Cy5-labelled cDNA) on cDNA microarray 63
B. Hybridization of a normal male (Cy3-labelled cDNA) and KS (Cy5-labelled cDNA) on cDNA microarray 63
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