K.HARITHA
Comparison of Southern, Northern, and Western analyses of Gene X
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
Southern hybridization
Transfer buffer
Detection of an RFLP by Southern blotting
Flow chart of Southern hybridization
Preparing the samples and running the gel
Southern transfer
Probe preparation
Prehybridization
Hybridization
Post-hybridization washing
Signal detection
IsotopeNon-isotope
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).
Critical parameters (I) Note the complexity of DNA
Genomic DNAA single-copy of mammalian gene, 3 Kb average in length
10 g x 3 Kb/3 x 106 Kb = 10 g x 1/106 = 10 pg
Plasmid DNA or PCR products 0.1 g of a 3 Kb plasmid DNA 100 ng
Gel treatment
Acid treatment 0.2 N HCl solution
Denaturation NaOH solution
Neutralization Tris-Cl buffer (pH8.0)
Southern transfer Measure gel and set up
transfer assembly: Wick in tray with 20x SSC Gel Nitrocellulose or Nylon filters
(soaked in H2O and 20x SSC)
3MM Whatman filter paper Paper towels Weight
After Southern transfer
Dissemble transfer pyramid and rinse nitrocellulose in 2x SSC
Bake nitrocellulose at 80C for 2 hr or UV-crosslink Nylon membrane for seconds
Preparation of probes
Synthesis of uniformly labeled double-stranded DNA probes
Preparation of single-stranded probes
Labeling the 5 and 3 termini of DNA
Synthesis of double-stranded DNA probes
- Nick translation of DNA- Labeled DNA probes using random
oligonucleotide primers
Nick translation
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.
In vitro transcription
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
T4 polynucleotide kinase activity
Non-isotope labeling Digoxigenin-11-dUTP (DIG-dUTP) labeling
- DNA labeling- Oligonucleotide labeling- RNA labeling
PCR Labeling, Random Primed Labeling, and RNA Labeling
Prehybridization
Add prehybridization solution and prehybridize at hybridization temperature for 2-4 hr
Hybridization
Remove prehybridization solution and add hybridization solution
Add 500,000 cpm of the probe/ml hybridization solution.
Hybridize overnight at appropriate temperature.
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
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 washingHigh temp., low salt: high stringencyLow 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
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
Signals detection Autoradioragraphy Non-isotope detection system
- Chemiluminescent detection- Colorimetric detection- Multicolor detection
Autoradiography
Exposure to x-ray film
Northern blotting or Northern hybridization
Technique for detecting specific RNAs separated by electrophoresis by hybridization to a labeled DNA probe.
The flow chart of Northern hybridizationPrepare RNA samples and run RNA gel
Northern transfer
Probe preparation
Prehybridization
Hybridization
Post-hybridization washing
Signal detection
IsotopeNon-isotope
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 60C. Add 35 ml 10x MOPS running buffer and 10.5 ml 37% formaldehyde
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 55C
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
An example of Northern blotting
Northern blot
RNA gel 28 S
18 S
Western blotting, or immunoblotting
Technique for detecting specific proteins separated by electrophoresis by use of labeled antibodies.
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
SDS polyacrylamide-gel electrophoresis (SDS-PAGE)
Analysis of protein samples by SDS polyacrylamide-gel electrophoresis and Western blotting
Protein bands detected by specific antibody
SDS-PAGE Western blot
Comparison of Southern, Northern, and Western blotting techniques
Southern blotting Northern blotting Western blotting Molecule detected
DNA (ds) mRNA (ss) Protein
Gel electrophoresis
Agarose gel Formaldehyde agarose gel
Polyacrylamide gel
Gel pretreatment
Depurination, denaturation, and
neutralization
- -
Blotting method Capillary transfer Capillary transfer Electric transfer Probes DNA
Radioactive or nonradioactive
cDNA, cRNA Radioactive or nonradioactive
primary antibody
Detection system
Autoradiography Chemiluminescent
Colorimetric
Autoradiography Chemiluminescent
Colorimetric
Chemiluminescent Colorimetric
DNA FINGER PRINTING
The basic methodology of DNA profiling in plants involve first the extraction of DNA from plant cells, quantification and quality assessment of extract. The further steps are of two types,
1) PCR based. - RAPD, ISSR, SSR 2) Non PCR based. – RFLP
PCR based techniques diluted DNA is mixed with a master mix comprising the PCR buffer, DNTPS, primer, water and the Taq polymerase enzyme in a PCR eppendorf tube .
The mixture is loaded into the PCR. The PCR is pre-programmed for appropriate number of cycles and temperature variations depending on the technique.
After required cycles, the samples are subjected to electrophoresis, either AGE or PAGE, depending on the technique. The staining is done for revealing the banding pattern.
Restriction Fragment Length Polymorphisms (RFLPs)In this method, unequal lengths of DNA fragments are obtained by cutting Variable Number of Tandem Repeat (VNTRs) sequences up to 30 sequences long with restriction enzymes at specific sites.
There are different VNTRs, as there are different plant species, number and location of restriction enzyme-recognition sites.
PCR amplification of DNA is not required for this method. The routine southern blot experiment can be used.
The complimentary DNA sequences are radiolabeled on agarose gel for visualization in this method. This method is used to identify the origins of a particular plant species.
This method is not much favored for DNA fingerprinting, as it has many drawbacks. The results cannot indicate the chance of match between two organisms.
The other drawback of RFLPs is a costly process which involves lot of labor and money.
Randomly Amplified Polymorphic DNAs (RAPDs)This method is most commonly used for primary assay.
This method helps in screening the differences in DNA sequences of two species of plants.
This method is used to search the sequences required for random amplification. In this method, using short single primers at low annealing tempratures, DNA is cut and amplified.
Using electrophoresis and superimposing the gels, a banding pattern is identified. The gel is cut where the target band is found and the DNA is isolated and sequenced.
This target is used to assess DNA from other cultivars. This technique is more cost-effective than RFLPs. The drawback for this method is that RAPDs lack specificity due to low annealing temperatures and easier reaction conditions.
Simple Sequence Repeats (SSR)Simple sequence repeats are microsarellites. They show high degree of polymorphism.
They are isolated using hybridized probes followed by their sequences. They are detected by gel electrophoresis using specific dyes or radiolabelling.
The advantage of SSRs is that the amount of DNA required is less than RFLPs. The assays involving SSRs are more robust, making them more efficient than RAPDs.
The drawback of this method is that seperate SSR primers are needed for each species.
Amplified Fragment Length Polymorphism (AFLP)This method is a PCR based derivative of RFLP. Here sequences are selectively amplified using the primers. This method is more useful than RFLP or RAPD as more loci can be evaluated. AFLP helps in determining a large number of polymorphism. This method is also cost effective.
ADVANTAGES OF DNA FINGERPRINTING IN PLANTS ARE AS FOLLOWS:DNA fingerprinting is used for the identification of genetic diversity within a breeding population. It is used to identify a gene of interest. In the United States, it is also used to detect a genetically modified organisms in agriculture.
RFLP markers are used to detect the genetic distance in wheat.
RAPD markers are used for characterization, estimation of genetic relatedness and determination of genetic diversity of tea germplasm. It is also used to find genetic relatedness and difference in figs.
AFLP markers help in assessing genetic diversity among cultivars such as wheat. It also helps detect higher level of polymorphism.
DNA fingerprinting of herbal drugs can be useful in authenticating the various claims of medical uses related to the plants.