• Full Name Full Name Comment goes here.
    Are you sure you want to
    Your message goes here
    Be the first to comment
No Downloads

Views

Total Views
10,980
On Slideshare
0
From Embeds
0
Number of Embeds
0

Actions

Shares
Downloads
367
Comments
0
Likes
5

Embeds 0

No embeds

Report content

Flagged as inappropriate Flag as inappropriate
Flag as inappropriate

Select your reason for flagging this presentation as inappropriate.

Cancel
    No notes for slide
  • More than 3000 human genetic diseases are attributable to single-gene defects. In most of these the mutation is recessive: that is, it shows its effect only when an individual inherits two defective copies of the gene, one from each parent. One goal of modern medicine is to identify those fetuses that carry two copies of the defective gene long before birth so that the mother, if she wishes, can have the pregnancy terminated. In sickle-cell anemia, for example, the exact nucleotide change in the mutant gene is known (the sequence GAG is changed to GTG at a specific point in the DNA strand that codes for the β chain of hemoglobin). For prenatal diagnosis, two DNA oligonucleotides are synthesized - one corresponding to the normal gene sequence in the region of the mutation and the other corresponding to the mutant sequence. If the oligonucleotides are kept short (about 20 nucleotides), they can be hybridized with DNA at a temperature selected so that only the perfectly matched helix will be stable. Such oligonucleotides can thus be used as labeled probes to distinguish between the two forms of the gene by Southern blotting on DNA isolated from fetal cells collected by amniocentesis. A fetus carrying two copies of the mutant β-chain gene can be readily recognized because its DNA will hybridize only with the oligonucleotide that is complementary to the mutant DNA sequence.
  • Figure 1. Targeting strategy and confirmation of gene targeting event. (A) Map of the K5 gene locus, the targeting vector, and the recombinant K5 locus. The core promoter and the first two exons of the K5 gene up to the 59 Ec oRI (R*) site were replaced by the HPRT minigene. The arrow in the HPRT minigene indicates the direction of transcription. In addition, an HSV/TK minigene was inserted as a negative selectable marker. The No tI restriction site was used to linearize the vector for transfection. Probes A and B mark the position of the 59 and 39 probe used in Southern blotting. Arrows above the K5 gene locus and the recombinant allele indicate primer positions for PCR-based genotyping. Letters indicate restriction sites: A, Ap aI; C, Ac cIII; H, Hi ndIII; N, No tI; R, Ec oRI; X, Xb aI. (B) Example of Southern blot analysis of ES cells. To confirm the correct targeting event, genomic DNA was digested with Ap aI for detection with the 59 probe, which led to a 5.6-kb band for the targeted allele and a 4.4-kb band for the wild-type allele. For detection with the 39 probe, an Ac cIII digest was performed resulting in a 6.6-kb fragment for the targeted allele and a 7.8-kb fragment for the wild-type allele. (C) Identification of genotypes by PCR. Primers designed to identify wild-type and mutant alleles (A) were used for genotyping of the litters. The wild-type allele resulted in a product of ;1.8-kb size, the targeted allele in a 1.4-kb product. (D) Neonatal homozygous K5 2/ 2 mouse. The fragile epidermis almost completely lost contact with the dermis after the mechanical stress of birth. Paws were sometimes denuded (arrow). K5 2/ 2 animals died within 1 h after birth. Molecular Biology of the Cell Vol. 12, 1775–1789, June 2001 Complete Cytolysis and Neonatal Lethality in Keratin 5 Knockout Mice Reveal Its Fundamental Role in Skin Integrity and in Epidermolysis Bullosa Simplex Bettina Peters,* † Jutta Kirfel,* † Heinrich Bu¨ ssow, ‡ Miguel Vidal, § and Thomas M. Magin* †i

Transcript

  • 1. Southern, Northern and Western blotting
  • 2. Comparison of Southern, Northern, and Western analyses of Gene X
  • 3. Southern hybridization
    • First described by E. M. Southern in 1975.
    • Applications of Southern hybridization
      • RFLP ’ s, VNTR ’ s and DNA fingerprinting
      • Checking of the gene knockout mice
    • The flow chart of Southern hybridization
  • 4. Southern hybridization Transfer buffer
  • 5. Detection of an RFLP by Southern blotting
  • 6. Detection of the sickle-cell globin gene by Southern blotting
  • 7. Checking of the gene knockout mice
  • 8. Flow chart of Southern hybridization
    • Preparing the samples and running the gel
    • Southern transfer
    • Probe preparation
    • Prehybridization
    • Hybridization
    • Post-hybridization washing
    • Signal detection
    Isotope Non-isotope
  • 9. Preparing the samples and running the gel
    • Digest 10 pg to 10  g of desired DNA samples to completion.
    • Prepare an agarose gel, load samples (remember marker), and electrophorese.
    • Stain gel ethidium bromide solution (0.5  g/ml).
    • Photograph gel (with ruler).
  • 10. Critical parameters (I)
    • Note the complexity of DNA
      • Genomic DNA
        • A single-copy of mammalian gene, 3 Kb average in length
        • 10  g x 3 Kb/3 x 10 6 Kb = 10  g x 1/10 6 = 10 pg
      • Plasmid DNA or PCR products
      • 0.1  g of a 3 Kb plasmid DNA  100 ng
  • 11. Gel treatment
    • Acid treatment
      • 0.2 N HCl solution
    • Denaturation
      • NaOH solution
    • Neutralization
      • Tris-Cl buffer (pH8.0)
  • 12. Southern transfer
    • Measure gel and set up transfer assembly:
      • Wick in tray with 20x SSC
      • Gel
      • Nitrocellulose or Nylon filters (soaked in H 2 O and 20x SSC)
      • 3MM Whatman filter paper
      • Paper towels
      • Weight
  • 13. After Southern transfer
    • Dissemble transfer pyramid and rinse nitrocellulose in 2x SSC
    • Bake nitrocellulose at 80  C for 2 hr or UV-crosslink Nylon membrane for seconds
  • 14. Preparation of probes
    • Synthesis of uniformly labeled double-stranded DNA probes
    • Preparation of single-stranded probes
    • Labeling the 5  and 3  termini of DNA
  • 15. Synthesis of double-stranded DNA probes
    • Nick translation of DNA
    • Labeled DNA probes using random oligonucleotide primers
  • 16. Nick translation
  • 17. Preparation of single-stranded probes
    • Synthesis of single-stranded DNA probes using bacteriophage M13 vectors.
    • Synthesis of RNA probes by in vitro transcription by bacteriophage DNA-dependent RNA polymerase.
  • 18. In vitro transcription
  • 19.
    • Labeling the 3  termini of double-stranded DNA using the Klenow fragment of E. coli DNA polymerase I. (lack of 5 ’  3 ’ exonuclease activity)
    • Labeling the 3  termini of double-stranded DNA using bacteriophage T4 DNA polymerase.
    • Labeling the 5  termini of DNA with bacteriophage T4 polynucleotide kinase.
    Labeling the 5  and 3  termini of DNA
  • 20. T4 polynucleotide kinase activity
  • 21. Non-isotope labeling
    • Digoxigenin-11-dUTP (DIG-dUTP) labeling
      • DNA labeling
      • Oligonucleotide labeling
      • RNA labeling
  • 22. PCR Labeling, Random Primed Labeling, and RNA Labeling
  • 23. Prehybridization
    • Add prehybridization solution and prehybridize at hybridization temperature for 2-4 hr
  • 24. Hybridization
    • Remove prehybridization solution and add hybridization solution
    • Add 500,000 cpm of the probe/ml hybridization solution.
    • Hybridize overnight at appropriate temperature.
  • 25. 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
  • 26. Critical parameters (II)
    • Homology between the probe and the sequences being detected
      • Tm = 81 +16.6 (log Ci) + 0.4 [% (G+C)] - 0.6 (% formamide)- 600/n - 1.5 (% mismatch)
      • 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
  • 27. Comparison of nitrocellulose and nylon membranes NC Nylon Hydrophobic binding Covalent binding Fragile Durable Probe length > 200 ~ 300 bp < 200 ~ 300 bp is O.K. Lower background Higher background Cannot be exposed to basic solution Can be exposed to basic solution Not easily reprobed Can be reprobed several times
  • 28. Signals detection
    • Autoradioragraphy
    • Non-isotope detection system
      • Chemiluminescent detection
      • Colorimetric detection
      • Multicolor detection
  • 29. Autoradiography
    • Exposure to x-ray film
  • 30. Northern blotting or Northern hybridization
    • Technique for detecting specific RNAs separated by electrophoresis by hybridization to a labeled DNA probe.
  • 31. The flow chart of Northern hybridization
    • Prepare RNA samples and run RNA gel
    • Northern transfer
    • Probe preparation
    • Prehybridization
    • Hybridization
    • Post-hybridization washing
    • Signal detection
    Isotope Non-isotope
  • 32. Preparation of agarose/formaldehyde gel
    • E.g. Prepare a 350 ml 1.2% agarose/formaldehyde gel
      • 4.2 g agarose in 304.5 g water. Microwave, then cool to 60  C. Add 35 ml 10x MOPS running buffer and 10.5 ml 37% formaldehyde
  • 33. Preparation of RNA samples
    • Prepare a premix:
      • 5  l of 10x MOPS running buffer
      • 8.75  l of 37% formaldehyde
      • 25  l of formamide.
    • Prepare RNA samples:
      • 38.75  l of premix
      • RNA (0.5 to 10  g)*
      • water to 50  l
    • *If the mRNA species of interest makes up a relatively high percentage of the mRNA in the cell (>0.05% of the message), total cellular RNA can be used. If the mRNA species of interest is relatively rare, however, it is advisable to use poly(A) + RNA.
    • Incubate 15 min at 55  C
  • 34. Running the RNA gel
    • Add 10  l formaldehyde loading buffer to each sample and load gel. Run gel at 100 to 120 V for ~3hr.
    • Remove gel from the running tank and rinse several times in water. Place gel in 10x SSC for 45 min.
    • Do not need post-transferring gel treatment
  • 35. An example of Northern blotting Northern blot RNA gel 28 S 18 S
  • 36. Western blotting, or immunoblotting Technique for detecting specific proteins separated by electrophoresis by use of labeled antibodies.
  • 37. Flow chart of Western blotting
    • Electrophoresing the protein sample
    • Assembling the Western blot sandwich
    • Transferring proteins from gel to nitrocellulose paper
    • Staining of transferred proteins
    • Blocking nonspecific antibody sites on the nitrocellulose paper
    • Probing electroblotted proteins with primary antibody
    • Washing away nonspecifically bound primary antibody
    • Detecting bound antibody by horseradish peroxidase-anti-Ig conjugate and formation of a diaminobenzidine (DAB) precipitate
    • Photographing the immunoblot
  • 38. SDS polyacrylamide-gel electrophoresis (SDS-PAGE)
  • 39. Analysis of protein samples by SDS polyacrylamide-gel electrophoresis and Western blotting Protein bands detected by specific antibody SDS-PAGE Western blot
  • 40. Comparison of Southern, Northern, and Western blotting techniques