Experimental Determination of Unwinding Temperature
Experimental Determination of Unwinding Temperature
The unlinking temperature for hybridization of oligonucleotides to target sequences can be calculated using the method described in the overview of this chapter (see the overview of this chapter on unlinking temperature and hybridization temperature), or the unlinking temperature can be determined by experimentally determining the temperature at which the irreversible dissociation of double-stranded DNA occurs (Ti) Tm. This experiment was derived from the last volume of the Experimental Guide to Molecular Cloning (3rd edition) by Peitang Huang.
Operation method
Experimental Determination of Unwinding Temperature
Materials and Instruments
Denaturing solution Neutralization buffer Oligonucleotide prehybridization solution Phenol/chloroform Sodium acetate SSC Appropriate restriction enzyme to enzymatically cleave double-stranded target DNA Control DNA Target DNA Oligonucleotide probe Move makings For more product details, please visit Aladdin Scientific website.
Boiling water bath apparatus Crosslinking apparatus, microwave or vacuum oven Flat-tip tweezers Glass test tubes Cellulose nitrate or nylon membranes Paper piercing punch Blinking cups Thermometer Heavy blotting paper Precision temperature-controlled circulating water bath apparatus
Solutions and buffers
Dilute the storage solution to the appropriate concentration.
Denaturing Solution (for double-stranded target DNA only)
1.5mol/L NaCl
0.5mol/L NaOH
Neutralization buffer (for double-stranded target DNA only)
0.5mol/LTris-Cl (pH 7.4)
1.5mol/LNaCl
Oligonucleotide prehybridization solution
6XSSC (or 6XSSPE)
0.01mol/L sodium phosphate (pH 6.8)
1 mmol/LEDTA (pH 8.0)
5xDenherdt's solution
0.5% (m/V) SDS
100ug/ml denatured, fragmented salmon DNA (see Scheme 10 in Chapter 6)
Phenol/chloroform (1:1, V/V) (for double-stranded target DNA only)
Sodium acetate (3 mol/L, pH 5.2) (double-stranded target DNA only)
2XSSC
Enzyme and Buffer
Appropriate restriction enzyme for cleavage of double-stranded target DNA
See step 3.
Nucleic acids and oligonucleotides
Control DNA
Single- or double-stranded DNA carrier unrelated to the target DNA sequence, see step 3.
Target DNA
Ideally, the target DNA should be cloned into the M13 phage vector and purified as single-stranded DNA (see Chapter 13). Double-stranded plasmid DNA or PCR products can also be denatured and used as described in Step 3 of this protocol.
Probes
Oligonucleotide probes
Unpurified oligonucleotides can also be used in this assay if the specific activity of the phosphorylation-labeled probe is greater than 106 cpm/ug.
Prior to synthesizing the probe, the probe should be checked for possible homologous and/or complementary sequences between the probe and the vector used to clone the target gene. Most commercially available DNA analysis software can be used for sequence matching studies of commonly used vectors to avoid problems during hybridization.
Specialized equipment
Boiling water bath unit
Cross-linking apparatus (Stratalinker, Stratagene; GS Gene Linker, Bio-Rad), microwave or vacuum oven Flat-tip tweezers
Glass test tubes (17X100 mm)
Nitrocellulose or nylon membrane
Paper piercing punch
Scintillation cups, each containing 10 ml of water-soluble scintillator
Thermometer
Thick absorbent paper ("Whatman3 MM, Schleicher&SchuellGB004, or Sigma QuickDraw)
Precision temperature controlled circulating water bath unit
See steps 7, 10, and 12.
Additional Reagents
The reagents required for Step 1 of this protocol are listed in Scenarios 2 and 4 of this chapter.
Methods
1. Phosphorylation method (Scheme 2) Radiolabeled oligonucleotides of 1-10 pmol are used as probes and excess undoped [ y-32P ]ATP is removed by CPB precipitation (Scheme 4).
2. Four small circular filter membranes (3-4 mm in diameter) were made by punching holes in nitrocellulose or nylon membranes with a paper piercer. Arrange the small membrane sheets on one Parafilm membrane and mark two of the filter membranes with a soft pencil.
3. Spot the target DNA and control DNA onto the filter membrane in the following manner:
Single-stranded target DNA
a. Dissolve approximately 100ng of target DNA in 1-3ul of 2XSSC and dispense on each labeled membrane.
b. Spot an equal amount of vector DNA (M13 phage single-stranded DNA without insertion sequence) on unlabeled membranes.
c. After the liquid dries, remove both sets of membranes from the Parafilm membrane with flat-tipped tweezers (e.g., Millipore tweezers) and place them between thick filter paper.
d. Bake in a vacuum oven at 80°C for 1-2 h to immobilize the DNA on the membranes.
The membranes can also be placed on filter paper and coupled with UV light to immobilize the DNA.
Double-stranded target DNA
a. If the target DNA has been cloned into a plasmid, digest the recombinant plasmid and vector with a restriction endonuclease that does not have a breakpoint in the target sequence to linearize it.
b. Purify the resulting double-stranded DNA or PCR product by phenol/chloroform extraction and standard ethanol precipitation. The DNA is solubilized into a 50-100 ng/ul solution using 2XSSC.
c. Spot the target and control DNAs onto the prepared filter membranes as described above. Transfer the membranes with flat-tip tweezers to thick filter paper saturated with denaturing solution and incubate for 10 min at room temperature.
d. Transfer the membrane to a thick filter paper impregnated with neutralization solution and incubate at room temperature for 10 min.
e. Transfer the membrane to a new dry piece of heavy filter paper and leave at room temperature until all liquid has evaporated. Bake in a vacuum oven at 80°C for 1~2 h, or irradiate the coupling with UV light to immobilize the DNA on the membrane.
4. Transfer all membranes with flat-tip tweezers to a polypropylene reaction tube containing 2 ml of oligonucleotide prehybridization solution, seal the tube, and incubate at 25°C below the Tm value of the solution used, with occasional motions.
The above protocol applies to hybridization solutions containing sodium salts, which can also be replaced by other solutions such as TMAC1 or TEAC1 if the Ti value in the solvent is to be determined.
After 5.2 h, radiolabeled oligonucleotides are added to the prehybridization solution. The final concentration of the oligonucleotide should be approximately 1 pmol/ml. Continue to incubate at 25°C or below for 2 to 4 h with occasional shaking.
6. Remove the membrane from the hybridization solution and immediately immerse it in 2XSSC at room temperature, shaking the solution constantly. Replace the solution every 5 min until the radioactivity of the membrane no longer changes (detected with a hand-held detector).
7. Adjust the temperature of the circulating water bath to 25°C below the Tm value and add 5 ml of 2XSSC solution to each of 20 glass test tubes (17xl00 mm). Measure the temperature inside the tubes with a thermometer and place all tubes in the water bath until the 2XSSC solution reaches a temperature of 25°C below the Tm value.
8. Place the membranes in 4 empty glass test tubes, add 1 ml of 2XSSC solution (prepared in step 7 and pre-warmed to below 25°C Tm) to each tube, and incubate in a circulating water bath for 5 min.
9. Remove the tubes from the water bath, transfer the liquid from the tubes to the scintillation vials, wash the glass tubes and the membranes in the tubes with lml of 2XSSC solution at room temperature and add the washings to the corresponding scintillation vials.
10. Increase the temperature of the water bath by 3°C and wait for the 2xSSC solution prepared in step 7 to equilibrate.
11. Add 1 ml of the 2XSSC solution at this temperature to the 4 test tubes containing the membranes and place the tubes in the water bath for 5 minutes.
12. Increase the temperature, repeating steps 9, 10, and 11, until 30°C above the Tm value is reached.
13. Place each of the 4 membranes in 4 glass test tubes (17x100 mm) containing 1 ml of 2XSSC solution and boil for 5 min to remove any residual radioactivity. The solution was cooled on ice and the liquid was transferred to a scintillation vial. The boiled tubes and membranes are washed with lml of 2XSSC solution and the washings are added to the corresponding scintillation vials.
14. Measure the activity of all scintillation vials with a liquid scintillometer (by Cherenkov counting, see Appendix 8). Calculate the percentage of total activity eluted at different temperatures using the following formula. 
where Xj is the fraction of total radioactivity eluted at temperature, ni is the activity eluted at i=Tj, and Tj denotes temperatures ranging from 25°C below the Tm value to 30°C above the Tm value.
If the experiment is well performed, the radioactivity of the filter membrane containing only carrier DNA should be very low. At temperatures well below the estimated Tm, this small amount of radioactivity should be completely released from the membrane. A large amount of radioactivity should be bound to the filter membrane containing the target DNA, and the elution of radioactivity should be steeply temperature dependent. Until the critical temperature is reached, almost no radioactivity is released from the membrane, whereas about 90% of the radioactivity should be released at 6-9°C above the critical temperature.
The temperature at which 50% of the radioactivity elutes from the membrane containing the target sequence is defined as the Ti of the probe-target sequence implicant.