Week 4: Southern Blot Hybridization and Detection

Introduction

There are different techniques for detecting specific sequences in nucleic acid samples. The procedure often involves nucleic acid hybridization, which can be used to (i) screen complementary DNA (cDNA) or genomic libraries, (ii) analyze gene expressions in cells (Northern blot analysis), and (iii) study the organization of specific regions of the genome (Southern blot analysis). The hybridization relies on the chemical properties of nucleic acids, meaning the complementarity of the DNA bases (A/T, G/C) and RNA (A/U, G/C) and the reversibility of DNA denaturation and reannealing. Suppose the two hybridizing strands contain a complementary sequence. In that case, the hybrid is stable under high temperature and high stringency conditions, i.e. a solution with an extremely low salt concentration close to water. In contrast, if the two do not have complementary sequences, hybridization does not occur. With partial complementarity, the hybrid is unstable under high stringency conditions (low salt) but stable under low stringency (high salt). The Melting Temperature (Tm) at which the two nucleic acid strands separate is unique to their specific sequence. The double-stranded nucleic acid with high GC content is more stable compared to those with high AT pairs.

The DIG nonradioactive system is a sensitive nucleic acid labeling and detection method. It has been used in various hybridization techniques for Southern blot, Northern blot, Dot blot, and colony and plaque screening. Its detection depends primarily on 1) the chemical property of the molecule used for probe labeling, 2) hybridization conditions, such as temperature and salt concentration, and 3) the detection method, in which the hybridized labeled probes can be detected with an antibody-conjugated to an enzyme, such as alkaline phosphatase.²⁶ Enzymatic activity of direct or indirect labeling is detected as chemiluminescent signals captured on an X-ray film or a color precipitate on the membrane after reacting with the substrate. For example, labeled probes hybridized to a target sequence are detected with streptavidin (biotin) or anti-DIG monoclonal antibody conjugated to an enzyme, often peroxidase or phosphatase (Figure 2-6, top).²⁶ The enzymatic activity can be detected through a chemiluminescent reaction captured on X-ray film or through a chromogenic reaction by incubating the membrane with the alkaline phosphatase substrate, BCIP (5-bromo-4-chloro-3-indolyl phosphate, toluidine salt) and NBT (nitroblue tetrazolium salt), to produce a color precipitate deposited directly on the membrane (Figure 2-6, bottom).

After confirming a successful labeling reaction by agarose gel electrophoresis prior to the lab, the probe is hybridized to the DNA on the membrane corresponding to the NBS sequence, followed by immunodetection. Briefly, 5 µl of PCR-amplified DIG-labeled probe is separated in a 2% TAE agarose gel. The size of the probe should be larger than the original unlabeled template as revealed in the gel. The prehybridization and hybridization must start the night before, followed by washing and detection in the lab the following day. A summary of the Southern blot analysis is shown in Figure 2-7.

Procedure

Please see Appendix A for solution preparation.

A. Prehybridization

Pre-warm DIG EasyHyb solution in 50 ml Falcon tube to 42°C.

1. Calculate your membrane’s size (cm²) and determine the needed volume of DIG EasyHyb buffer (20 ml/100 cm²).
2. Place the membrane in a glass tube for the HYBAID Mini Oven. The DNA side of the membrane faces inward in the tube.
3. Prewet the membrane in 2XSSC slowly and evenly, and then discard the solution.
4. Add a proper amount (20 ml/100 cm² filter or a minimum of 5 ml) of the prewarmed DIG EasyHyb solution. The membrane is incubated for ≥ 30 minutes in the Hybaid Mini Oven at 42°C. The solution should move freely. Avoid bubbles trapped between the membrane and glass wall.
5. While waiting, make 50 µl sterile H₂O containing a proper amount of DIG-labeled DNA probe: 2.5 µl probe/ml of prehybridization solution (3.5 ml/100 cm²) into a 0.2 ml PCR tube. Too much probe results in a remarkably high background.
6. Denature the probe at 95°C for 5 minutes in a PCR machine and then quickly cool on slushy ice.

B. Hybridization Overnight

1. Discard the initial 5 ml of DIG EasyHyb solution in the glass tube after ≥ 30 minutes.
2. Add a proper amount of fresh DIG EasyHyb solution (3.5 ml/100 cm² membrane).
3. Add the 50 µl denatured probe (step 5 in section A) into the solution without touching the membrane and pipette up and down several times, but avoid making bubbles that may lead to a high background.
4. Incubate the glass tube in the HYBAID oven with rotation overnight.

C. Post-hybridization washes conducted in the lab the next day

The amount of solution used in the following steps depends on container/membrane size.

The 0.5XSSC, 0.1% SDS washing solution is preheated in a water bath set at 60°C.

1. Pour off the overnight hybridization solution with DNA probe or save it at -20°C for re-use if needed. Probes are typically good for 2–4 uses.
2. Wash the membrane twice for 5 minutes each in 2XSSC, 0.1% SDS (50 ml/100 cm²) at room temperature.
3. Discard the solution.
4. Wash the membrane twice with preheated 0.5XSSC, 0.1% SDS at 60°C for 15 minutes each.
5. Swirl the tube and try to make the membrane move near the top of the tube.

D. Detection

Ensure that a sufficient volume of each solution covers the membrane in the container slightly larger than the membrane, and follow the steps below for detection.

1. Carefully remove the membrane and place it in a square box containing 25 ml of Washing Buffer for 5 minutes on a shaker at a low speed.
2. After discarding the solution, add sufficient blocking solution (~25 ml) into the box, which is put back on the shaker at room temperature for 30 minutes. While waiting, you should make an alkaline phosphatase conjugate anti-DIG antibody solution (Anti-DIG-AP), such as adding 4 µl of antibody (1:5000) to 20 ml of blocking solution.
3. Discard the blocking solution, add sufficient Anti-DIG-AP antibody solution, and shake for 30 minutes.
4. Wash membranes twice for 15 minutes in 30 ml of washing buffer. Equilibrate membranes for 2–5 minutes in Detection Buffer.
5. Incubate the membrane in freshly prepared color substrate solution and store it in the cabinet beneath your bench. Do not shake during color development. The bands will appear in about 10 minutes.
6. After 15–20 minutes, stop the reaction by rinsing the membrane with dH₂O. If needed, you can take pictures multiple times along the way to get the best results before stopping the reaction with dH₂O.

Data Analysis and Discussion

1. Based on the results of the agarose gel electrophoresis from Week 2 and the Southern blot hybridization and detection from this week, did you successfully clone the NBS sequence into the pSC-A-amp/kan? How do you know? Explain.