Southern northern and western blotting
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Southern northern and western blotting
- 1. Southern, Northern and Western blotting
- 2. Comparison of Southern, Northern, and Western analyses of Gene X
- 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
- 16. Nick translation
- 20. T4 polynucleotide kinase activity
- 22. PCR Labeling, Random Primed Labeling, and RNA Labeling
- 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
- 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.
- 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
Hinweis der Redaktion
- 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