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- 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
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