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 hybridizationRFLP’s, VNTR’s and DNA fingerprintingChecking of the gene knockout miceThe flow chart of Southern hybridization
6 Detection of the sickle-cell globin gene by Southern blotting 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.
7 Checking of the gene knockout mice Figure 1. Targeting strategy and confirmation of gene targeting event. (A) Map of the K5 gene locus, the targeting vector, and therecombinant K5 locus. The core promoter and the first two exons of the K5 gene up to the 59 EcoRI (R*) site were replaced by the HPRTminigene. The arrow in the HPRT minigene indicates the direction of transcription. In addition, an HSV/TK minigene was inserted as anegative selectable marker. The NotI restriction site was used to linearize the vector for transfection. Probes A and B mark the position of the59 and 39 probe used in Southern blotting. Arrows above the K5 gene locus and the recombinant allele indicate primer positions forPCR-based genotyping. Letters indicate restriction sites: A, ApaI; C, AccIII; H, HindIII; N, NotI; R, EcoRI; X, XbaI. (B) Example of Southern blotanalysis of ES cells. To confirm the correct targeting event, genomic DNA was digested with ApaI for detection with the 59 probe, which ledto 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 AccIII digest wasperformed 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 genotypesby PCR. Primers designed to identify wild-type and mutant alleles (A) were used for genotyping of the litters. The wild-type allele resultedin 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 almostcompletely lost contact with the dermis after the mechanical stress of birth. Paws were sometimes denuded (arrow). K5 2/2 animals diedwithin 1 h after birth.Molecular Biology of the CellVol. 12, 1775–1789, June 2001Complete Cytolysis and Neonatal Lethality in Keratin5 Knockout Mice Reveal Its Fundamental Role in SkinIntegrity and in Epidermolysis Bullosa SimplexBettina Peters,* † Jutta Kirfel,* † Heinrich Bu¨ ssow, ‡ Miguel Vidal, § andThomas M. Magin* †i
8 Flow chart of Southern hybridization Preparing the samples and running the gelSouthern transferProbe preparationPrehybridizationHybridizationPost-hybridization washingSignal detectionIsotopeNon-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 DNAGenomic DNAA single-copy of mammalian gene, 3 Kb average in length10 mg x 3 Kb/3 x 106 Kb = 10 mg x 1/106 = 10 pgPlasmid DNA or PCR products0.1 mg of a 3 Kb plasmid DNA 100 ng
11 Gel treatment Acid treatment Denaturation Neutralization 0.2 N HCl solutionDenaturationNaOH solutionNeutralizationTris-Cl buffer (pH8.0)
12 Southern transfer Measure gel and set up transfer assembly: Wick in tray with 20x SSCGelNitrocellulose or Nylon filters (soaked in H2O and 20x SSC)3MM Whatman filter paperPaper towelsWeight
13 After Southern transfer Dissemble transfer pyramid and rinse nitrocellulose in 2x SSCBake nitrocellulose at 80C for 2 hr or UV-crosslink Nylon membrane for seconds
14 Preparation of probesSynthesis of uniformly labeled double-stranded DNA probesPreparation of single-stranded probesLabeling the 5 and 3 termini of DNA
15 Synthesis of double-stranded DNA probes Nick translation of DNALabeled DNA probes using random oligonucleotide primers
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.
19 Labeling the 5 and 3 termini of DNA 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.
21 Non-isotope labeling Digoxigenin-11-dUTP (DIG-dUTP) labeling DNA labelingOligonucleotide labelingRNA labeling
22 PCR Labeling, Random Primed Labeling, and RNA Labeling
23 PrehybridizationAdd prehybridization solution and prehybridize at hybridization temperature for 2-4 hr
24 HybridizationRemove prehybridization solution and add hybridization solutionAdd 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 detectedTm = (log Ci) [% (G+C)] (% formamide)- 600/n (% mismatch)Factors can be changed:Hybridization temp.Washing temp.Salt concentration during washingHigh temp., low salt: high stringencyLow temp., high salt: low stringencyIf 50 % formamide is used42 oC for 95 ~ 100 % homology37 oC for 90 ~ 95 % homology32 oC for 85 ~ 90 % homology
27 Comparison of nitrocellulose and nylon membranes NCNylonHydrophobic bindingCovalent bindingFragileDurableProbe length > 200 ~ 300 bp< 200 ~ 300 bp is O.K.Lower backgroundHigher backgroundCannot be exposed to basic solutionCan be exposed to basic solutionNot easily reprobedCan be reprobed several times
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 gelNorthern transferProbe preparationPrehybridizationHybridizationPost-hybridization washingSignal detectionIsotopeNon-isotope
32 Preparation of agarose/formaldehyde gel E.g. Prepare a 350 ml 1.2% agarose/formaldehyde gel4.2 g agarose in g water. Microwave, then cool to 60C. 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 buffer8.75 l of 37% formaldehyde25 l of formamide.Prepare RNA samples:38.75 l of premixRNA (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 55C
34 Running the RNA gelAdd 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 RNA gel28 S18 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 sampleAssembling the Western blot sandwichTransferring proteins from gel to nitrocellulose paperStaining of transferred proteinsBlocking nonspecific antibody sites on the nitrocellulose paperProbing electroblotted proteins with primary antibodyWashing away nonspecifically bound primary antibodyDetecting bound antibody by horseradish peroxidase-anti-Ig conjugate and formation of a diaminobenzidine (DAB) precipitatePhotographing the immunoblot