Chapter 4: Hybrid molecules

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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.

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Nội dung Text: Chapter 4: Hybrid molecules

CHÖÔNG 4 LAI PHAÂN TÖÛ (MOLECULAR HYBRIDIZATION)
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
Southern Blot (1975) • 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
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 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).
Transfer of DNA, RNA from agarose gel onto membrane by capillary
• Transfer methods: • Capillary: overnight • Electric: 2-3 h • Vacuum: 30 min • Note: • Sequences > 5 kb: Low transfering efficiency  hydrolyse DNA partly by: • - weak acid  to break purins partly • - strong base  to break
• Dissemble transfer pyramid and rinse nitrocellulose in 2x SSC • Bake nitrocellulose at 80°C for 2 hr or UV- crosslink Nylon membrane for seconds After Southern transfer
Preparation of isotope probes • Synthesis of uniformly labeled double- stranded DNA probes • Preparation of single-stranded probes • Labeling the 5′ and 3′ termini of DNA
Ñaùnh daáu baèng caùc ñoàng vò phoùng xaï Radioactively Labeled (dATP)
Ñaùnh daáu baèng phöông phaùp hoaù hoïc NonRadioactively Labeled Precursors
Synthesis of double-stranded DNA probes - Nick translation of DNA - Labeled DNA probes using random oligonucleotide primers
Preparation of single-stranded probes • Synthesis of single-stranded DNA probes using bacterio-phage M13 vectors. • Synthesis of RNA probes by in vitro transcription by bacteriophage DNA-dependent RNA polymerase.
In vitro transcription