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.