Week 3: DNA Extraction from Agarose Gel and Quantification; PCR Digoxigenin Probe Synthesis

Introduction

Southern blot analysis detects and quantifies specific sequences in DNA samples. It can confirm the presence of the cloned NBS sequence in the previously isolated recombinant plasmid obtained in Module 1. Steps of Southern blot analysis include separating DNA fragments by agarose gel electrophoresis, denaturing and transferring DNAs from the gel onto a membrane, labeling of a probe for the target DNA, which is used to hybridize the DNAs on the membrane, followed by washing and detecting the hybridized labeled DNA(s). Prior to synthesizing probes for Southern blot hybridization, DNAs for labeling must be purified. The band containing the known NBS sequence from pSCA7 (T1-T3-W6) was excised from the agarose gel after electrophoresis in Week 2. There are different ways to extract DNA from agarose gels. A rapid and effective method is transferring DNA from the gel onto the DEAE-cellulose membrane by electrophoresis, a classic method for DNA recovery, particularly for larger DNAs. A DEAE paper strip is inserted into the gel in front of a DNA band to trap all the DNA after electrophoresis. The DNA is then eluted from the membrane and precipitated with salt and ethanol. The other is to place the gel piece in a dialysis tube containing a buffer in a gel box filled with buffer and subject it to an electric current. The DNA moves into the solution inside the bag, followed by ethanol precipitation. Freeze-thaw is another method for DNA recovery from gel because it is simple and easy to perform with good yield. Lastly, commercially available purification kits, such as the GeneJET Gel Purification Kit, can extract DNA from the gel and will be used in this lab exercise.

The GeneJET Gel Purification Kit is designed to purify DNA fragments from standard or low-melting point agarose gels run in TAE or TBE buffer.¹⁶ It uses the silica-based membrane technology in the form of a spin column. The DNA-containing agarose is heated and dissolved in a solution to facilitate DNA binding to the column. After applying the melting agarose solution to the column, followed by centrifugation, the column is washed, and then the bound DNA is eluted. The kit can purify DNA fragments 25 bp–20 kb in size, with up to 95% recovery rates. Each column has a binding capacity of up to 25 µg of DNA and can process < 1 g of agarose gel. The entire procedure takes only 15 minutes, and the isolated DNA is ready for common downstream applications, i.e., ligation, restriction digestion, PCR, sequencing, and labeling.

A successful Southern blot analysis depends heavily on the success of probe synthesis, nucleic acid hybridization, and detection. Probes can be made with end¹⁷ or internally¹⁸ labeled radioactive or chemically modified nucleotides. After end labeling, an oligonucleotide is labeled with a single atom, and thus, it has a low specific activity. Two enzymes are commonly used to label each end; T4 polynucleotide kinase (T4 PNK) catalyzes the transfer of a radioactive phosphate from [γ-³²P]NTP or a nonradioactive label to the 5′-OH group of single- and double-stranded DNAs and RNAs, oligonucleotides, or nucleoside 3′-monophosphates,¹⁹ whereas terminal transferase or Terminal deoxynucleotidyl transferase (TdT), a DNA polymerase, labels its 3′-OH end with a [α-³²P] or nonradioactive dNTP.²⁰

For internal labeling, three methods are Nick Translation, Random Primed Labeling, and PCR Labeling. For “Nicked translation” labeling, DNA is treated with DNase I and Mg⁺² to produce single-stranded “nicks” followed by strand replacement in nicked sites by DNA polymerase I with 5′ → 3′ exonuclease activity to remove nucleotides in front, and the 5′ → 3′ polymerase activity adds nucleotides to the available 3′ ends.²¹ The “random primed” method relies on hybridizing oligonucleotides of all possible sequences to the denatured template DNA to be labeled.²² A “Klenow” fragment of DNA Polymerase I synthesizes the complementary DNA strand using random oligonucleotides as primers.

PCR labeling is another internal labeling that uses two primers, Taq DNA polymerase, a buffer containing labeling nucleotide, and a DNA template with a specific region for amplification for the labeling reaction.²³ All the newly synthesized complementary DNA is labeled radioactively or non-radioactively by putting a radioactive or nonradioactive-labeled nucleotide or their equivalents in the reaction mixture. The advantages of PCR-labeling include: i) requiring only a small amount of template DNA, 10–100 pg, and genomic DNA, 1–50 ng. ii) being able to label impure DNA. iii) requiring less optimization than other methods. iv) producing a large quantity of sensitive labeled probes and is recommended for truly short probes (< 100 bp). PCR labeling produces a highly labeled, specific, and extremely sensitive hybridization probe. Thus, among the different labeling methods, PCR labeling is preferred for making DIG-labeled probes when the template is short, limited in quantity, or partially purified since it requires less optimization than other methods and produces a high yield of sensitive probes with high specificity and sensitivity.²⁴

Probe labeling with³²P has been used for decades to detect a small number of target molecules in blots because of its strong signal. However, it is hazardous to handle and dispose of, requires long exposure, and has a short half-life with limiting probe stability. In contrast, nonradioactive nucleic acid probes, i.e., Digoxigenin (DIG), are stable for > one year and can be handled and disposed of without concern about radioactivity.²⁵ As a nonradioactive labeled probe, DIG, a small steroid hapten found in the flowers and leaves of the plants Digitalis purpurea, Digitalis orientalis, and Digitalis lanata (foxgloves), is safe and effective in probe labeling. It has high antigenicity and can be linked to the C-5 of dUTP via a spacer arm to form Digoxigenin-deoxyuridine triphosphate (DIG-11-dUTP) for nonradioactive DNA labeling (Figure 2-5).²⁵

Nonradioactive probes are labeled directly or indirectly. The former directly attaches fluorescent tags or cross-linking enzymes to nucleic acid. Indirect labeling involves incorporating nucleotides tagged with a hapten, like DIG, during probe synthesis.²⁵ DIG has several advantages, including high sensitivity, like that of radioactive probes, fast detection, low handling and disposal hazards, and long-term stability. Using the alkali-labile form of DIG-11-dUPT makes it easier and more efficient to strip blots for hybridization with a 2^nd DIG-labeled probe (Figure 2-5). The label could also be a fluorophore probe that is detected through excitation of the probe and emission of light from it, so-called chemiluminescent detection, whose signal is obtained by capturing it with X-ray film or digital imaging systems. Signals of the DIG labeling and detection that are produced through an enzymatic reaction with a chemiluminescent or chromogenic substrate have become popular. For this lab, the known NBS-containing DNA insert from the pSCA7 (T1-T3-W6) is extracted from the excised gel piece using the GeneJET Gel Extraction Kit,¹⁴ followed by PCR DIG labeling^7,8,23 (Figure 2-1). The labeled probe is then used to hybridize and detect the cloned DNA on the Southern blot (see Week 4 for details). The results can confirm the presence and location of the NBS sequence in the cloned recombinant plasmid.

Procedure

A. DNA Purification from Gel

The protocol is adapted from the manual of GeneJET PCR Purification Kit (Thermo Scientific).⁷ Steps 1–3 were conducted in the last lab exercise.

1. Weigh one 1.5 ml Eppendorf tube. Record the weight.
2. With a clean and sharp scalpel, excise the agarose gel piece containing the DNA fragment and place it into the tube from step 1.
3. Weigh the gel slice in the tube again and subtract the tube weight (step 1) to obtain gel weight. Note: We already did steps 1–3 in Week 2.
4. Add a 1:1 volume of Binding Buffer to the gel slice (volume: weight), e.g., add 100 µl of Binding Buffer for every 100 mg of agarose gel. Note: The color of the Binding Buffer should be yellow at the optimal pH. If the color of the solution is orange or violet, add 10 µl of 3 M sodium acetate, pH 5.2, and mix.
5. Incubate the tube at 50–60°C for 10 min or until the gel slice is completely dissolved. Mix by inverting every few minutes to facilitate the melting process. Ensure that the gel is completely dissolved.
6. Vortex briefly before transferring up to 800 µl of the solubilized gel solution to the GeneJET purification column.
7. Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube. Note: If the total volume exceeds 800 µl, the solution can be added to the column in stages. After each application, centrifuge the column for 30–60 seconds and discard the flow-through after each spin. Repeat until the entire volume has been applied to the column membrane. Do not exceed 1g of total agarose gel per column.
8. Add 700 µl of Wash Buffer (diluted with ethanol as described in the manufacturer’s protocol) to the GeneJET purification column. Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube.
9. This step is essential to avoid residual ethanol in the purified DNA solution as the presence of ethanol in the DNA sample may inhibit downstream enzymatic reactions: Centrifuge the empty column for one more minute to remove any residual buffer.
10. Transfer the column into a clean 1.5 ml microcentrifuge tube not provided in the kit.
11. Add 25 µl of Elution Buffer to the center of the purification column membrane. Allow the column to stand for 1 min at room temperature before centrifuging for 1 min.
12. Discard the GeneJET purification column, quantify the eluted DNA with a UV spectrophotometer and record the results in Table 2-3. The DNA is ready for PCR Digoxigenin probe synthesis. The rest of the DNA sample will be collected and stored at -4°C.

B. PCR DIG DNA Labeling

1. Add 4 µl of your purified DNA into a 0.2 ml PCR tube.
2. Add 16 µl of the pre-made PCR mix below to set up the PCR labeling reaction.
   

   Pre-made PCR mix	(16 µl)
   Sterile water	10.6 µl
   PCR Buffer with MgCl2  (10x) (vial 3)	2 µl
   PCR DIG Probe Synthesis Mix (vial 2) containing 2 mM each of dATP, dCTP, dGTP; 1.3 mM dTTP, 0.7 mM DIG-11-dUTP	2 µl
   Each of LM638 and LM 637 Primers (25 pmole each)	2 x 0.5 µl
   Enzyme Mix (vial 1)	0.4 µl

3. Place the tube in a thermocycler with a heated lid amplify the DNA by heating at:
   • 94°C for 1 minute
   • 35 cycles of 94°C for 30 sec, 50°C for 30 sec, 72°C for 30 sec
4. Store the tube at -20°C. (It will be used as a probe for Southern hybridization after being validated for successful DIG-labeling by agarose gel electrophoresis.)

Data Analysis and Discussion

1. Instead of using the DNA insert, why can we not use the whole plasmid for probe labeling since we can use the same LM638 and LM637 primers for making the probe by PCR? (Hint: Consider how PCR works and what happens using the circular recombinant plasmid instead of the linear DNA insert, particularly amplifying the short 500 bp DNA insert.)
2. If you run the DIG-labeled probe in an agarose gel, what would you expect regarding the size of your newly synthesized probe? (Will it be located in the same location as the original PCR product without DIG?)  Why or why not?