Slot Blotting of Genomic Tail DNA David Murphy

1. Introduction

Slot or dot blotting is a technique whereby nucleic acids can be applied to a solid matrix, unfractionated, using a vacuum manifold. Slot blotting is the quickest, easiest, and, apart from polymerase chain reaction, probably the most sensitive assay of transgenic animal genotype (1). However, it can only be applied to animals that bear a transgene that has hybridizable segments with little or no homology to the host genomic DNA—for example, a hybrid gene with a viral or procary-otic reporter element, or a gene from another species with sufficient sequence divergence to allow the transgene to be distinguished from the host gene. Genomic slot blotting should not be used for the initial identification of a transgenic founder animal. For this, Southern blotting is preferable as it can give more information about the number of integration sites and the presence of transgene rearrangements, deletions, and so forth. However, genomic slot blotting is useful for the rapid screening of subsequent generations of transgenics when detailed information about the structure of the transgene is not needed.

2. Materials

2. 5MNH4 acetate.

3. Nylon hybridization matrix (e.g., Amersham [UK] Hybond-N).

4. 20X SSPE: 3.6M NaCl, 200 mM Na phosphate buffer, pH 6.8, 20 mM EDTA.

5. Slot-blot applicator (e.g., Schleicher and Schuell, Dassel, Germany) linked to a vacuum line.

6. UV light (312 nAf) transilluminator.

3. Methods

1. Adjust 5-15 (ig of genomic tail DNA (see Note 1) and copy number standards (see Note 2) to 0.3M NaOH in 200 pL. Include controls (see Note 3).

2. Incubate 10 min in a boiling water bath and quench on ice.

3. Add 400 (iL of 5M NH4 acetate. Apply to a nylon hybridization membrane (see Note 4), previously equilibrated with 2X SSPE, using a slot-blot applicator.

4. When the DNA solution has entirely passed through the filter, disassemble the slot-blot applicator. Rinse the filter in 2X SSPE. Air-dry, then bake at 80°C for 60 min.

5 Covalently crosslink the DNA to the matrix by exposure, DNA side down, on Saran wrap, to a UV light (312 nm) transilluminator for 2 min (see Note 5).

6. Proceed as described in Chapter 54 (Filter Hybridization).

4. Notes

1. Genomic DNA prepared using rapid techniques from tail tissue are severely contaminated and difficult to quantitate accurately by spectrophotometry. Semiquantitative slot-blot analyses should therefore be performed in duplicate with one filter being hybridized to a transgene probe, the other with a probe for a gene endogenous to the host animal. False negatives can thereby be avoided.

2. Transgene copy number can be determined by slot-blotting by comparing the level of hybridization to copy number standards (prepared by diluting a known quantity of the unlabeled transgene DNA) to the level of hybridization to dilutions of the transgenic mouse genomic DNAs. The latter figure should be corrected with respect to the hybridization of a probe to an endogenous standard host gene

3. Slot blotting is notoriously prone to give false positive signals. This can best be avoided by: (1) rigorous testing of the probe using Southern blots to ensure that it is specific for the transgene; (2) inclusion in all assays of adequate negative (i.e., nontransgenic) controls; (3) assiduous preparation of all probes, solutions, DNAs, and so forth in order to avoid contamination.

4. Note that these methodologies have been developed for neutral nylon membranes (e.g., Amersham Hybond-N) and have not been tested on positively charged membranes (e.g., Amersham Hybond-N+, Bio-Rad [Richmond, CA] Zeta-Probe, NEN-Du Pont [Boston, MA] Genescreen Plus).

5. The time needed to covalently UV crosslink nucleic acids to a nylon filter must be determined empirically. The energy output of UV transilluminators varies considerably. Too much exposure will damage the nucleic acids, and reduce the autoradiographic signal following hybridization. Too little exposure will not link the nucleic acids to the filter. During hybridization and washing the target nucleic acids will be lost, again resulting in a reduced autoradiographic signal. To determine the optimal exposure time, identical filters bearing a known nucleic acid are exposed to the UV transilluminator for differing lengths of time. Following hybridization to the same probe, the optimum time, corresponding to the strongest autoradiographic signal, can be determined. Note that the energy output of a transilluminator changes over a period of months and thus regular recalibration is required. Stratagene (La Jolla, CA) produces a system that emits a fixed, measured amount of UV energy.

Reference

1. Murphy, D. and Hanson, J. (1987) The production of transgenic mice by the microinjection of cloned DNA into fertilised one-cell eggs, in DNA Cloning, A Practical Approach, vol. 3 (Glover, D. M., ed.), IRL Press, Oxford, UK, pp 213-248.

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