Concept of hybrid molecules: When double strand DNA is steamed to a temperature exceed the melting temperature (Tm), it will separate into 2 single strands DNA due to breaks of H bonds. If the reaction temperature is then decreased slowly plus other appropriate experimental conditions, these ssDNA will pair again. This phenomenon is called the hybridization of molecules. Characteristic of the hybrid molecules: Specificity, the re-pairing occurs only between two complementary sequences. | LAI PHAÂN TÖÛ (MOLECULAR HYBRIDIZATION) CHÖÔNG 4 Concept of hybrid molecules When double strand DNA is steamed to a temperature exceed the melting temperature (Tm), it will separate into 2 single strands DNA due to breaks of H bonds. If the reaction temperature is then decreased slowly plus other appropriate experimental conditions, these ssDNA will pair again. This phenomenon is called the hybridization of molecules. Characteristic of the hybrid molecules: Specificity, the re-pairing occurs only between two complementary sequences. These complementary sequences can be DNA or RNA, leading to the formations of DNA-DNA, RNA-RNA or hybrid DNA-RNA TYPES OF HYBRIDIZATION - Hybrid in liquid phase (Lai trong pha loûng) - Hybrid on solid phase (Lai treân pha raén) - in situ hybridization (Lai taïi choã) - Southern Blot - Northern Blot - Western Blot First described by E. M. Southern in 1975. Applications of Southern hybridization RFLP’s, VNTR’s (Variable Number Tandem Repeat) and DNA . | LAI PHAÂN TÖÛ (MOLECULAR HYBRIDIZATION) CHÖÔNG 4 Concept of hybrid molecules When double strand DNA is steamed to a temperature exceed the melting temperature (Tm), it will separate into 2 single strands DNA due to breaks of H bonds. If the reaction temperature is then decreased slowly plus other appropriate experimental conditions, these ssDNA will pair again. This phenomenon is called the hybridization of molecules. Characteristic of the hybrid molecules: Specificity, the re-pairing occurs only between two complementary sequences. These complementary sequences can be DNA or RNA, leading to the formations of DNA-DNA, RNA-RNA or hybrid DNA-RNA TYPES OF HYBRIDIZATION - Hybrid in liquid phase (Lai trong pha loûng) - Hybrid on solid phase (Lai treân pha raén) - in situ hybridization (Lai taïi choã) - Southern Blot - Northern Blot - Western Blot First described by E. M. Southern in 1975. Applications of Southern hybridization RFLP’s, VNTR’s (Variable Number Tandem Repeat) and DNA fingerprinting Checking of the gene knockout mice Southern Blot (1975) Northern Blot (1977) Technique for detecting specific RNAs separated by electrophoresis by hybridization to a labeled DNA probe. Transfer RNA onto membrane Hybridize with probe Detection Western blot -Immunoblot (1979) Technique for detecting specific proteins separated by electrophoresis by use of labeled antibodies. Transfer proteins in SDS-PAGE onto Nylon membrane Critical parameters Concentration of target DNA, RNA, protein Homology between the probe and the sequences being detected (specificity) Tm = 69,3 + 0,41 (% G + C) Factors can be changed: Hybridization temp. Washing temp. Salt concentration during washing High temp., low salt: high stringency Low temp., high salt: low stringency If 50 % formamide is used 42 oC for 95 ~ 100 % homology 37 oC for 90 ~ 95 % homology 32 oC for 85 ~ 90 % homology Southern hybridization Transfer buffer Flow chart of Southern hybridization Preparing the samples and running the gel .
