URBANA, ILLINOIS

University of Illinois

Chemiluminescent Southern detection of maize genomic single copy sequences

--Torbert Rocheford and *Nancy Wallace

* Present address: Dekalb Plant Genetics, Groton, CT

We have been increasingly working with nonisotopic methods of detecting maize single copy sequences on Southern blots for the past few months. We are pleased to report use of a digoxigenin based protocol that produces satisfactory results with UMC and BNL probes. All of our new RFLP work will use this method and we are in the process of phasing out our radioactive work as old membranes become used up through reprobing. The protocol we report here is a result of discussions with Frans Krens at Wageningen, Netherlands, and Joe Lanzillo at New England Medical Center, Boston. We have taken their protocols and slightly modified and integrated them so that the procedure is suitable and repeatable for maize single copy detection work. The procedure still needs some refinements, particularly in the area of repeatability and in background levels. Since we are committed to nonradioactive methods, we will continue to optimize various conditions.

We have used the protocol below with a rotating hybridization oven (Robbins Scientific) for hybridization steps and a glass tray on a shaker for the detection steps. We have recently shifted to the hybridization oven for both the hybridization and detection steps because it will help us to scale up the procedure to the level we desire for our RFLP investigations. Use of glass trays on shakers for detection is quite suitable for qualitative molecular biology work but may be unwieldy and cumbersome for large scale RFLP operations or for large labs performing many Southern hybridizations a day.

Southern Blotting
    1. Restricted maize genomic DNA samples are electrophoresed on 1% agarose 1X TAE gels. Currently, we load approximately 15 ug of DNA per lane. Loading this amount of DNA insures there will be enough target for initial detection and for reprobing. If there is not enough target DNA the procedure simply does not work.
    2. After staining, visualization, and rinsing, the gel is depurinated for 15 minutes, denatured for 30-60 minutes, and neutralized for 30 minutes.
    3. The nylon membrane we recommend is Hybond-N (Amersham). The membrane is saturated for 10 minutes with H20 and then 10 minutes with the transfer solution: 1M ammonium acetate, 20 mM sodium hydroxide. We transfer our blots on a Stratagene Plosiblotter, crosslink with a Stratagene UV crosslinker for approximately 30 seconds, and bake the membrane at 80 C for two hours.

DNA Probe Labelling
We have labelled PCR amplified inserts as well as inserts cut out of gels. We are currently labelling 200 ng at a time since this gives high levels of incorporation of digoxigenin. We use Boehringer Mannheim DNA labelling kit (Cat. No. 1175 033). We will try labelling larger quantities in the future, however due to the ability to reuse the Dig-labelled probe, the need to label up very large quantities of probe is reduced.

    1. Denature 200 ng of DNA in an H2O volume of 15 ul by heating for 10 min. at 95 C.
    2. On ice, add : 2 ul hexanucleotide mixture, 2 ul dNTP mixture. Mix and add: 1 ul of klenow. Mix and briefly spin down in microfuge.
    3. Incubate 37 C overnight. Stop reaction by adding 2 ul of 0.2 M EDTA (pH 8.0). Add 20 ug of tRNA and mix. Precipitate with 1.8 ul of 3M sodium acetate and 3 volumes ethanol (71.4 ul). Leave at -70 for >30 minutes.
    4. Centrifuge at 12,000 x g then wash with 50 ul ice-cold 70% ethanol. Dry thoroughly. Resuspend in 50 ul TE and 0.1% SDS. (5 ul of 1% SDS) at 37 C for 20 min with frequent vortexing.

Prehybridization and Hybridization
    1. Prewet membrane with H2O and then 2X SSC. Prehybridize membrane one hour or more at 65 C in hybridization oven with > 20 ml/100 cm2 with the following prehybridization solution: 5X SSC, 1.0% Tropix blocking reagent, 0.1% N-laurylsarcosine, 0.02% SDS (store hybridization solution in freezer in 50 ml aliquots).
    2. Hybridize with 3 ml of the above solution for a single membrane in a bottle. Denature the probe in 500 ul of the hybridization solution and then add to the other 2.5 ml, quickly mix, and add to the hybridization bottle. We use 12 ng/ml of hybridization solution, higher concentrations of probe may increase background. Hybridize at 65 C overnight (when we reuse probes, we simply boil the 3 ml and add to the hybridization bottle).

Washes
    1. 2 X 5 minutes at room temp. 2X SSC; 0.1% SDS
    2. 2 X 5 minutes at room temp. 0.2X SSC, 0.1% SDS
    3. 2 X 15 minutes at 65 C 0.2X SSC, 0.1% SDS

(Some of the room temperature washes may be holdovers from radioactive protocols; we are in the process of reducing or eliminating the room temperature washes and changing simply to three high stringency 65 C washes).

Signal Detection

The following steps have the volumes used when signal detection is performed in a glass tray on a shaker. For the hybridization oven, we are currently reducing the volumes used.

    1. Rinse membrane in 50-100 ml of buffer 1 for 5 min.
    2. Incubate with 100 ml Buffer 2 for 30 min.
    3. Incubate with 15 ml of fresh, filtered anti-Dig (Boehringer Mannheim) solution for 30 min. We add anti-Dig to 2-3 ml of solution, filter just that amount with .45 um millipore syringe filter, and add to the remainder of solution.
    4. Wash 3X in 50-100 ml Buffer 2 for 10 min each.
    5. Wash 3X in 50-100 ml Buffer 1 for 10 min each.
    6. Incubate 2X 5 min in Tropix assay buffer.
    7. Incubate in 15 ml of Tropix AMPPD solution for 10 min.
    8. Drain excess solution and seal membrane in seal-a-meal bag. Do not allow membrane to dry at this time or until probe is stripped.
    9. Expose film for 2-3 hours at room temperature. Develop film and determine length of subsequent exposure, if necessary. The blot can be put down the following day. Overnight exposures are usually too long and produce a lot of background. There is no need to worry about loss of signal. Sometimes the signal to noise ratio is better when the membrane is exposed a day or two later, but not always.

Solutions
Buffer 1: 0.1 M Tris-HCl (pH 7.5); 0.15 M NaCl

Buffer 2: Buffer 1 + 0.5% Tropix blocking reagent (Cat No. AB 100)

Anti-Dig: 3 ul Boehringer Mannheim anti-Dig solution (Cat no. 1093-274) per 15 ml Buffer 2. This gives a 1:5,000 dilution. (We have successfully used 1:10,000 and 1:15,000 dilutions. We are currently using 1:10,000 and we will continue to investigate modification of dilutions since they result in cost savings and higher dilutions may reduce background in the hybridization oven.

Assay Buffer: to make 250 ml - 2.4 ml diethanolamine, 0.5 g MgCl2, .05g sodium azide, adjust pH to 10.0 with HCl.

AMPPD Substrate Solution: to make 15 ml - 165ul Tropix AMPPD (Cat. no. PD 100-B) added to 15 ml Assay Buffer.

Stripping Blots
Nonstripped membranes are stored in their seal-a-meal bags in the freezer.

    1. Rinse membrane in 2X SSC.
    2. Incubate for 10 min. at 37 C in 0.2 M NaOH, 0.1% SDS.
    3. Rinse membrane in TE, membrane can then be stored at room temperature.

Comments
We have successfully been able to probe membranes 3-4 times and we have not exhausted the usefulness of these membranes. We will continue to reprobe these blots. The one major disadvantage of this system is that background can be a problem at times. Presently, we have less background problems than when we first started working with this type of detection system. Although background can be a problem, in general it does not prevent us from scoring the exposed films.

We are currently working on optimizing conditions for detection in bottles in the rotating hybridization oven. Our initial experiments revealed more background with detection in hybridization ovens than in glass trays on shakers, but the background has been reduced recently. We have increased the length of some of our detection incubations and washes and we are currently working with other reagent concentrations such as the anti-Dig concentration. The hybridization oven should enable scaling down the volumes used and probing more than one membrane per bottle, resulting in considerable time and cost savings. We have just started to probe two membranes per bottle.

A major advantage of this system is that reagents can be reused. We have not experimented with this very much since we were very concerned with reducing background levels upon reprobing. Since we have now reduced background levels, we are currently addressing reuse of solutions. This will also result in considerable cost savings. We have reused our Dig-labelled DNA probe hybridization solutions quite successfully. We greatly enjoy not having to use radioactive isotopes and I will be happy to discuss this procedure or send out updated modifications to this protocol to interested researchers. Finally, this technology has teaching attractiveness, for example undergraduate students can be integrated into lab operations more rapidly, calmly and safely than with radioactive procedures.


Please Note: Notes submitted to the Maize Genetics Cooperation Newsletter may be cited only with consent of the authors

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