Introduction
Non-isotopic Chemiluminescent Detection
Indirect Method: North2South (N2S) Biotin Systems
North2South Chemiluminescent Substrate
Data Imaging North2South Blots
Stripping and Reprobing North2South Blots
Tech Tips

Introduction to Southern and Northern Blotting

Nucleic acid blotting involves using labeled single-stranded DNA or RNA to hybridize and detect complementary single-stranded nucleic acid that has been affixed to a solid membrane surface. Traditionally, the target nucleic acid is presented in a complex mixture of nucleic acid fragments that has been electrophoresed to separate the fragments by relative size prior to their transfer and fixation to nylon or nitrocellulose membrane.

Southern blotting (named after its inventor, E.M. Southern) was developed as a method to separate and analyze digested genomic DNA in gels. After denaturing and electrophoresing the DNA, it is transferred to a membrane surface where DNA fragments of interest are identified by hybridization with labeled, complementary, gene-specific DNA probes. Southern blots are used in gene discovery and mapping studies to monitor gene incorporation and to assess evolutionary relationships among organisms.

Northern blotting (named by analogy to Southern blotting) is used to characterize one or more specific mRNA transcripts in a sample of total RNA. The RNA is denatured, size-separated by electrophoresis and transferred to nylon membrane, where it is hybridized and detected with labeled, complementary, target-specific DNA or RNA probes. Northern blots may be used to quantitate a target, determine mRNA transcript size, detect alternative splice variants of a gene and identify closely related species.

Factors that influence hybridization efficiency and specificity in Northern and Southern analyses include temperature, ionic strength, destabilizing agents, mismatched base pairs, duplex length, viscosity and base composition. High salt favors hybridization reactions (i.e., less specificity and greater background), while decreased salt and/or increased detergent or temperature increases hybridization specificity and reduces background.

Traditionally, the DNA or RNA probe in Southern and Northern blotting procedures is labeled with radioactive phosphorus (e.g., ³²P) or sulfur (e.g., ³⁵S) by incorporation of radiolabeled nucleotides during its synthesis. In recent years, alternative, non-isotopic methods have largely replaced radioactive systems. Pierce offers one of the leading systems for Northern and Southern blotting utilizing chemiluminescent detection that rivals sensitivity of isotopic methods (Figures 2 and 3).

Figure 2. Southern blot of yeast genomic DNA. HindIII digest (0.8, 0.4, 0.2, 0.1 µg/In) hybridized with 6 ng/ml HRP-TCM-1 (PCR) probe vs. 3 ng/ml (2.8µCi/ml) isotopic probe with A) North2South Direct Kit; 1-minute exposure or B) isotopic method; 24-hour exposure with intensifying screens.

Figure 3. Nothern blot of total yeast DNA. Total yeast RNA (2, 1, 0.5, 0.25 µg/In) hybridized with 4 ng/ml HRP-TCM-1 cRNA probe prepared and detected with A) North2South Direct Kit; 15-minute exposure, or B) isotopic method; 24-hour exposure with intensifying screens.

North2South Hybridization and Detection Systems

Non-isotopic Chemiluminescent Detection
Pierce North2South Hybridization and Detection Technology facilitates non-isotopic nucleic acid probe labeling, hybridization and detection by way of horseradish peroxidase enzyme activity and a patented, enhanced luminol-based chemiluminescent substrate detection system. Two methods have been developed utilizing this technology. In the direct method, a nucleic acid probe is covalently attached to a peroxidase label. In the indirect method, the nucleic acid probe is covalently modified with biotin that in turn is detected with a peroxidase labeled-streptavidin.
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Indirect Method: North2South (N2S) Biotin Systems
Biotin labeling techniques for nucleic acids were developed starting in the 1980s. There are many methods for incorporating biotin into nucleic acid probes. Random prime labeling (Product # 17075), in vitro transcription, end-labeling reactions (Product # 89818), photoactivation (Product # 29986), or incorporation via PCR or during oligonucleotide synthesis are some of the most popular techniques. Probes biotinylated by many methods work well for detection with the North2South Chemiluminescent Hybridization and Detection Kit (Product # 17097). Detection limits improve with increased number of biotin labels in the nucleic acid. Like isotopic probes, the biotinylated probe must be denatured (100°C for DNA or 80°C for RNA and oligonucleotides >60 bases) and snap-cooled prior to use.

The North2South Hybridization System requires pre-hybridization of the target-containing membrane for 30 minutes at 55°C (65°C for RNA:RNA). After pre-hybridization, the biotinylated probe is added at 3-5 ng/ml for RNA or ~30 ng/ml for DNA probes and incubated for 3-24 hours at the same temperature used for pre-hybridization. After hybridization, the membrane is washed 3 x 15 minutes with stringency wash buffer (2X SSC/0.1% SDS).

Following the stringency wash, the membrane is incubated with a protein blocker for 15 minutes. Then, a special stabilized preparation of horseradish peroxidase-conjugated streptavidin (streptavidin- HRP) is added in blocker at 1:300 dilution and incubated for 15 minutes at room temperature. The blot is washed 4 x 5 minutes with wash buffer, then 5 minutes in equilibration buffer. Finally, the blot is incubated in prepared chemiluminescent substrate and exposed to film.
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North2South Chemiluminescent Substrate
North2South and North2South Direct Products use a formulation of Pierce’s patented Super- Signal Chemiluminescent Substrate Technology to achieve exceptionally sensitive detection of peroxidase-labeled or bound hybridization complexes in Northern or Southern analyses. SuperSignal Technology utilizes luminol and a novel enhancer to provide unsurpassed intensity, sensitivity and signal duration. Horseradish peroxidase (HRP) is oxidized in the presence of a free radical scavenger. This intermediate interacts with luminol to generate weak light that is intensified by the presence of an enhancer. Maximum light emission is at 425 nm with instantaneous light output that lasts for hours.

The North2South SuperSignal Working Solution (WS) is prepared by mixing equal volumes of the luminol/enhancer and stable peroxide solutions. WS is applied to fully cover the target-bound side of a blot (~0.1 ml/cm² ). After incubating for 5 minutes at room temperature and draining off excess WS, the wet blot is carefully placed target side up in plastic wrap or a sheet protector to avoid trapped air, static charge or overflow of the substrate out of the protector. Finally, the blot is exposed to X-ray film or CCD camera.
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Data Imaging North2South Blots
Cooled CCD cameras offer advantages of high sensitivity, instant image manipulation and data analysis as well as the elimination of darkroom processing equipment and reagents. There is a wide range of CCD equipment and suppliers. Older model CCD cameras may require five-fold exposure times compared to film in order to produce equal sensitivity. Newer models have equal or greater sensitivity relative to film.

Although digital image capture is growing in popularity, X-ray film techniques are still widely-practiced, efficient and cost-effective methods for chemiluminescent or isotopic imaging. In order to achieve maximum sensitivity with film methods, it is very important to use fresh chemicals and good processing techniques. Obtaining the optimal exposure is often accomplished by trial and error with different exposure times. When used correctly, SuperSignal Substrates have good signal duration to allow for repeated film exposures (e.g., 1, 5 and 15 minutes; see Figures 2 and 3). Longer or shorter exposure times may be necessary. Films that have been overexposed (too dark from too long of exposure) may be corrected using Erase-It Background Eliminator (Product # 21065). This product can be used to effectively reduce background without altering the integrity of the data. The product is an inexpensive alternative to correcting film images, providing a cost savings on film and re-assay time.
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Stripping and Reprobing North2South Blots
Nucleic acids fixed on nylon can usually survive up to five stripping and re-probing cycles. The most common procedure for stripping DNA blots is washing the blot 3 x 5 minutes with boiling (95-100°C) 0.1% SDS. Alkaline wash treatment of 3 x 5 minutes with 0.4 N NaOH, 0.1% SDS may also be effective for DNA blots. Blots stripped by the alkaline method must be neutralized in Tris-EDTA buffer (TE). RNA blots are best washed with 0.1% SDS for 3 x 5 minutes, at 75-80°C. Stripped blots are best stored wet in TE at 4°C.

Recommended Reading
Jackson, J., et al. (2002). Detection of Nucleic Acids Using Chemiluminescence: From Northerns to Southerns and Beyond, In Luminescence Biotechnology: Instruments and Applications, Van Dyke, K., et al. , Ed., CRC Press, pp. 223-230.

References

1. Adilakshmi, T. and Laine, R.O. (2002). J. Biol. Chem. 277(6), 4147-4151.
2. Bach, S., et al. (2002). Infect. Immun. 70(2), 988-992.
3. Borst, E-M., et al. (1999). J. Virology 73(10), 8320-8329.
4. Haertel-Wiesmann, M., et al. (2000). J. Biol. Chem. 275(41), 32046-32051.
5. Campbell, F.M., et al. (2002). J. Biol. Chem. 277(6), 4098-4103.

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