Chapter 4: Hybrid molecules pptx

LAI PHÂN TỬ (MOLECULAR HYBRIDIZATION) CH NG 4ƯƠ 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 loỷng) - Hybrid on solid phase (Lai treõn pha raộn) - in situ hybridization (Lai taùi choó) - 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 o C for 95 ~ 100 % homology • 37 o C for 90 ~ 95 % homology • 32 o C for 85 ~ 90 % homology Southern hybridization Transfer buffer Flow chart of Southern hybridization Preparing the samples and running the gel Southern transfer Probe preparation Prehybridization Hybridization Post-hybridization washing Signal detection Preparing the samples and running the gel • Digest 10 pg to 10 µg of desired DNA samples to completion. • Prepare an agarose gel, load samples (remember marker), and electrophorese. • Stain gel with ethidium bromide solution (0.5 µg/ml). • Photograph gel (with ruler). [...]... Labeling, Random Primed Labeling, and RNA Labeling Prehybridization Add prehybridization solution and prehybridize at hybridization temperature for 2-4 hr Hybridization • Remove prehybridization solution and add hybridization solution • Add 500,000 cpm of the probe/ml hybridization solution • Hybridize overnight at appropriate temperature Post-hybridization washing • Wash twice, 15 min each, in 1x... solution • Hybridize overnight at appropriate temperature Post-hybridization washing • Wash twice, 15 min each, in 1x SSC, 0.1% SDS at room temperature • Wash twice, 15 min each, in 0.25x SSC, 0.1%SDS at hybridization temp Comparison of nitrocellulose and nylon membranes NC Nylon Hydrophobic binding Covalent binding Fragile Durable Probe length > 200 ~ < 200 ~ 300 bp is 300 bp O.K Lower background Higher . of DNA-DNA, RNA-RNA or hybrid DNA-RNA TYPES OF HYBRIDIZATION - Hybrid in liquid phase (Lai trong pha loỷng) - Hybrid on solid phase (Lai treõn pha raộn) - in situ hybridization (Lai taùi. homology Southern hybridization Transfer buffer Flow chart of Southern hybridization Preparing the samples and running the gel Southern transfer Probe preparation Prehybridization Hybridization Post-hybridization. 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