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فصل اول کلیات تحقیق
مقدمه ................................................................................................................................................. 1
بیان مسئله .......................................................................................................................................... 3
اهمیت و ضرورت تحقیق ..................................................................................................................... 4
اهداف ................................................................................................................................................... 6
هدف کلی ........................................................................................................................................... 6
اهداف اختصاصی ............................................................................................................................... 6
اهداف کاربردی ....................................................................................................................... 6
سؤالات و فرضیه ها ............................................................................................................................. 7
فصل دوم پیشینه تحقیق
آناتومی گوش ....................................................................................................................................... 8
گوش خارجی ..................................................................................................................................... 8
گوش میانی ........................................................................................................................................ 9
گوش داخلی ...................................................................................................................................... 9
طبقه بندی ناشنوایی ......................................................................................................................... 11
طبقه بندی بر اساس سن شروع ..................................................................................................... 11
طبقه بندی بر اساس شدت ............................................................................................................. 12
طبقه بندی بر اساس علت فیزیولوژیکی ........................................................................................ 12
ژن های شناخته شده مرتبط با ناشنوایی......................................................................................... 13
بررسی ژن ها..................................................................................................................................... 22
GJB4 ............................................................................................................................ 22
GJC3 .............................................................................................................................. 25
ژن SLITRK6 ...................................................................................................................... 26
ژن SERPINB6 ................................................................................................................... 28
NESP4 ............................................................................................................................ 29
ژن CABP2 ......................................................................................................................... 30
ژن OTOGL ......................................................................................................................... 31
فصل سوم روش شناسی تحقیق
نوع مطالعه.......................................................................................................................................... 34
جامعه، نمونه آماری و روش نمونه گیری........................................................................................... 34
روش جمع آوری داده ها.................................................................................................................... 34
متغیره ها............................................................................................................................................ 34
روش شناسی تحقیق......................................................................................................................... 35
ملاحظات اخلاقی............................................................................................................................... 36
مواد و روش ها.................................................................................................................................37
استخراج DNA .................................................................................................................... 37 چگونگی استخراج DNA ..................................................................................................... 38
لیز سلول ................................................................................................................................ 39
هیدراسیون DNA............................................................................................................. 40
تعیین غلظت DNA استخراج شده ............................................................................................ 41
روش تعیین غلظت ......................................................................................................................... 42
نحوه خالص سازی نمونه های آلوده به پروتئین ......................................................................... 42
آنالیز پیوستگی برای بررسی 7 جایگاه ژنی مرتبط با ناشنوایی اتوزومی مغلوب غیرسندرومی .. 43
انتخاب مارکرهای STR .................................................................................................... 44
GJB4 ....................................................................................................................... 45
GJC3 ......................................................................................................................... 46
ژن SLITRK6 ................................................................................................................. 47
ژن SERPINB6 ............................................................................................................. 48
NESP4 ...................................................................................................................... 49
ژن CABP2 .................................................................................................................... 50
ژن OTOGL ................................................................................................................... 51
تعیین هتروزیگوسیتی مارکرهای STR در جمعیت ایران ............................................... 52
واکنش زنجیره ای پلیمراز .............................................................................................................. 53
مواد لازم برای واکنش PCR ......................................................................................................... 54
روش انجام PCR ....................................................................................................... 57
الکتروفورز ............................................................................................................................ 60
الکتروفورز با ژل پلی آکریل آمید .......................................................................... 61
مواد لازم جهت الکتروفورز ژل پلی آکریل امید .................................................... 62
طرز تهیه ژل اکریل امید 8 درصد .......................................................................... 63
رنگ آمیزی ژل به روش نیترات نقره ..................................................................... 65
مواد لازم جهت رنگ آمیزی نقره .............................................................. 65
روش رنگ آمیزی نیترات نقره .................................................................. 66
بررسی ژل های پلی آکریل آمید .............................................................. 67
پیدا کردن جهش به روش مستقیم تعیین توالی ............................................................... 68
الکتروفورز محصولات PCR بر روی ژل آگارز ..................................................... 70
مواد لازم جهت انجام الکتروفورز DNA برروی ژل آگارز ..................... 70
روش کار ..................................................................................................... 71
توالی پرایمرهای مورد استفاده برای تکثیر ........................................................... 72
روش آنالیز نتایج حاصل از Sequencing ............................................................ 74
فصل چهارم تجزیه و تحلیل و بیان نتایج حاصل از تحقیق
نتایج حاصل از مطالعات آنالیز پیوستگی ...................................................................................... 75
مارکرهای دارای فرکانس آللی بالاتر .................................................................................. 75
نتایج آنالیز پیوستگی در خانوادهای ناشنوا با توارث مغلوب ............................................ 77
خانواده L-3082 و ژن GJB4 ........................................................................................... 78
خانواده L-1902 و ژن GJC3 .......................................................................................... 81
خانواده L-346 و ژن SLITRK6 ..................................................................................... 84
خانواده L-346 و ژن NESP4 .......................................................................................... 86
خانواده L-1731 و ژن SERPINB6 ................................................................................ 88
خانواده L-346 و ژن CABP2 .......................................................................................... 91
خانواده L-346 و ژن OTOGL ........................................................................................ 94
فصل پنجم بحث ونتیجه گیری وپیشنهادات
بحث ونتیجه گیری ........................................................................................................................... 97
تحقیقیات اضافه ............................................................................................................................. 101
پیشنهادات ..................................................................................................................................... 103
منابع ............................................................................................................................................... 105
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