DNA fingerprinting is a common tool in biological sciences and applied in paternity testing, as well as forensic or phylogenetic studies. This protocol is designed to provide undergraduate students the basic principles of DNA fingerprinting in laboratory classes. It is based on the human D1S80 locus, a variable number of tandem repeats region, which alleles can differ in length between individuals.
DNA extraction, PCR, gel electrophoresis, and subsequent analysis can be tricky to perform, especially if done for the first time. The visual demonstration of all steps allows the viewers to observe general practice, details, and precautions that must be taken while carrying out this very robust protocol. To extract DNA from buccal mucosa epithelial cells, start by harvesting the cells using a sterile oral swab.
Use a swab to rub vigorously on the inside of the cheek for 30 to 40 seconds. Place the tip of the collection swab into the labeled sterile micro-centrifuge tube and break off the length of plastic that extends beyond the edge, and close the tube. Preheat a heating block to 65 degrees Celsius and prepare all required buffers and solutions.
Add 500 microliters of lyse solution to the swab, making sure that the sample is completely immersed in the lyse solution and vortex the sample for at least five seconds. Incubate the sample for 10 minutes at 65 degrees Celsius and vortex occasionally. After incubation, remove the swab by pressing it against the tube wall to obtain a maximal sample volume.
Add 100 microliter denaturation buffer to lyse cells and mix thoroughly by inverting the tube until a white precipitate becomes visible. Incubate for five minutes at room temperature, then centrifuge the sample for five minutes at 17, 950 G.Transfer 450 microliters of the supernatant into a clean, 1.5 milliliter micro-centrifuge tube. Then add 450 microliter of iso 2-propanol and mix thoroughly by inverting the tube to precipitate the DNA.
Incubate for five minutes at room temperature and centrifuge for five minutes. Discard the supernatant and invert the tube on a clean paper towel and dry the pellet. Wash the DNA with 500 microliter of 70%ethanol, centrifuge shortly for one minute, and discard the supernatant.
To dry the pellet completely, incubate the sample for five minutes at 65 degrees Celsius in a heating block. Finally, add 30 microliters of elution buffer and incubate for 10 minutes at 65 degrees Celsius to inactivate the DNA sys. Prepare the master mix by adding the green PCR buffer, DNTPs, primers, water, and polymerase to the micro-centrifuge tube.
Label the PCR tubes with sample numbers and aliquot 45 microliters of the PCR master mix to each PCR tube. Next, add five microliters DNA, or, for the no-template control, add water. Close the PCR tubes and centrifuge them briefly.
Place the tubes in the thermal cycler and start the PCR program. When the program is complete, store the samples at four degrees Celsius until further processing. Prepare a 1.5%agarose gel by weighing 1 1/2 grams of agarose powder and mixing it with 100 milliliters of TAE buffer solution in a bottle.
Heat the agarose mixture carefully in a microwave and swirl the mixture in between until the agarose has become dissolved completely. Be aware that boiling delays may occur. After a short cooling of the agarose mixture, add two microliters of Pec green.
Pour the gel and use a comb with enough wells for the samples. After complete polymerization of the agarose gel, load the wells with 10 microliter of each of the PCR samples and add a molecular weight standard to the flanking wells. Run the gel at 150 volt for approximately 40 minutes.
To visualize the PCR products, image the gel using ultraviolet light. For the analysis of the amplified D1S80 alleles, place a ruler next to the molecular weight standard starting at the wells on the photograph gel image. Measure the distance for each band of the weight standard and record the lengths in a table calculation program.
Next, determine the logarithm of each fragment size of the weight standard. Insert a scatterplot using log-transformed, weight-standard fragment sizes, and the measured distances from your gel. Fit a trend line and display the regression equation and the R-square value.
In a new table, insert the measures distances from the PCR amplicons to the wells. To determine the size of these amplicons, use the regression equation from the linear regression and calculate the antilog to obtain the fragment sizes in base pairs from the amplicons. Finally assign the repeat units of 16 base pairs to each measured amplicon.
The extraction of DNA and amplification of the D1S80 locus was successful for all studied individuals, and the no-template control in lane 10 did not show any amplicons. From the fragment length analysis, the individuals were classified in being homozygous or heterozygous with two alleles. The number of repeat units of the tandem repeats was clearly relatable, and can now serve for further analysis.
Here we describe a simple and cost-effective method for implementing molecular fingerprinting in undergraduate practical classes. The entire procedure can be completed within a single workday, and comprises of four different steps. In the first step, the DNA template is prepared.
Buccal epithelial cells are harvested by swab sampling. Oral swabs are a reliable tool to provide sufficient quantity and quality of cells for DNA extraction. Furthermore, the DNA extraction protocol does not use organic solvents, which is advantageous in undergraduate laboratory classes.
Step one can be completed in 90 minutes or less. In the second step, the D1S80 locus is amplified by PCR. The PCR conditions presented here are robust even in case of poor quality of the DNA template.
This step can be completed in 2 1/2 hours. The PCR products are then analyzed by agarose gel electrophoresis. Agarose gel electrophoresis has many advantages compared to other techniques, such as the avoidance of hazardous components and a simple preparation technique.
This analysis can be completed in 90 minutes. After the electrophoresis, the fragment length results can be analyzed within 90 minutes by linear regression analysis, either performed by using a table calculation program, or by using a simple calculator. In summary, the presented fingerprinting method can be utilized in hands-on practicals to teach the use of molecular markers and its application in biological science.
