The overall goal of this video is to demonstrate three examples of PCR amplification directly from small amounts of un purified samples, including plant and animal tissues. Direct PCR can be performed using two alternative protocols. In the direct protocol, a tiny amount of un purified sample is added directly to the PCR reaction, whereas the dilution protocol requires a brief pre incubation step before PCR to release DNA from the sample material.
In the first example, genotyping of arabidopsis is achieved using the direct protocol. An allele specific restriction site is introduced into target DNA, and then allele specific single nucleotide polymorphisms are identified following restriction endonuclease digestion. Next, the dilution protocol is employed to first amplify specific DNA fragments from 15-year-old oak leaves, and then to genotype transgenic mice directly from mouse ear tissue.
In all three examples, DNA amplification is achieved without any sample purification steps prior to PCR through the direct PCR method. The main advantage of direct PCR over the traditional methods is that no DNA extraction or purification steps are required. This translates to savings in both time and expenses.
Begin the derived cleaved, amplified polymorphic sequence analysis or decaps genotyping assay by first formulating 20 or 50 microliter PCR reactions using the plant direct PCR kit as described in the written protocol. Next, cut a 0.50 millimeter punch from an Arabidopsis plant leaf using the Harris Uni unicorn and cutting mat, holding the puncher firmly. Press the cutting edge into the tissue and rotate the puncher back and forth.
Press the plunger to eject the punch disc into a PCR reaction mixture. Ensure that the sample drops into the PCR solution and does not stick to the two walls. To avoid cross-contamination in the direct protocol, it is critical to use the correct method for cleaning the sampling tool.
Clean the cutting edge of the puncher between each sample by dipping it into a 2%sodium hypochlorite solution. Press the plunger up and down a few times and wipe the cutting edge with a clean paper towel. The cutting mat should also be rinsed between samples.
Next, employ a Pico 24 well thermal cycler and PCR plate to perform the PCR reactions using the cycling conditions described in the text accompanying this video After PCR, spin the plant material. Transfer five microliters of the supernatant to a new micro centrifuge tube following edition of four microliters of water. Add one microliter of the restriction endonuclease SSP one mix gently and briefly centrifuge to collect the contents in the bottom of the tube.
Incubate the reaction mixture for one hour at 37 degrees Celsius. Then inactivate the restriction enzyme by incubating at 65 degrees Celsius for 20 minutes. Following restriction enzyme digestion, add 2.5 microliters of five x loading buffer to the reaction.
Perform aros gel electrophoresis to analyze the resulting fragments by loading 10 microliters of the resulting mixture into the gel. Well, to begin, cut a two millimeter punch of oak leaf. Place the sample into 20 microliters of dilution buffer supplied with the plant direct PCR kit.
Clean the cutting edge of the puncture and cutting mat as before, crush the leaf sample with a 100 microliter pipette tip by pressing it briefly against the tube wall. Spin the samples briefly in a micro centrifuge tube until the reactions are in the bottom of tubes. The supernatant can be used directly in the PCR or it can be diluted one to 10 or one to 100 in sterile water.
Depending on the sample type, use 0.5 microliters to one microliter of the supernatant or dilution as a template for a 20 microliter PCR reaction. Set up the reactions as described in the written protocol. Once the reactions are prepared, use the PICO 24 well thermal cycler and PCR plate to perform the PCR reactions using the cycling conditions described in the text.
After PCR, add five microliters of five x loading buffer to the reaction and perform aros gel electrophoresis with 15 microliters of the resulting mixture. Begin the dilution protocol for genotyping transgenic mice by placing a two millimeter punch of mouse ear tissue in 20 microliters of dilution buffer containing 0.5 microliters of DNA release additive supplied with the animal tissue. Direct PCR kit incubate the samples at room temperature for two minutes, followed by a two minute incubation at 98 degrees Celsius.
After centrifuging the samples, use one microliter of supernatant as the template for a 20 microliter PCR reaction. Any supernatant that is not to be used right away can be transferred to a new tube and stored at minus 20 degrees Celsius As a final step, the 24 well thermal cycler and PCR plate are used to perform the PCR reactions using the cycling conditions listed in the written procedure Following PCR, add five microliters of five x loading buffer to the reaction and perform aros gel electrophoresis with 15 microliters of the resulting mixture in the D caps technique. The restriction site within the snip of interest is either introduced or destroyed using a PCR primer with one or more mismatches to the target DNA.
In this example, the 160 base pair of PCR products containing the snip site of interest g or a allele in the arabidopsis genome were amplified from plant leaf punches with the fire plant direct PCR kit. The forward primer contained one mismatch at the three prime end creating an SS P one specific restriction site in the target DNA, which included the A allele but not in the other allele of the snip. The un purified PCR products were digested with SS P one restriction enzyme resulting fragments were analyzed on an aros gel lane's g and a correspond to the snip alleles of each sample.
In this image, the intact PCR products are on the left and the digested fragments are on the right. Dilution protocols are necessary for challenging plant material such as the oak leaf due to its high concentration of phenolic compounds using the dilution protocol described here. The 297 base pair fragment of chloro plastic DNA was amplified with a three-step protocol.
Oak leaf specimens with different dilution are shown in addition to positive and negative controls. The fire animal tissue direct PCR kit was employed for amplification of two fragments with a large size difference using only one primer set and resulted in abundant yields of both correctly sized products of 1500 base pairs for transgenic mice and 200 base pairs for wild type mice. The weaker upper band with heterozygote mice is due to the competition of both templates for the same primer repair.
However, the genotyping results are unambiguous. The stability of the samples prepared using the dilution protocol was also tested. The results show that diluted samples subjected to repeated freeze thaw cycles and those stored at minus 20 degrees Celsius for at least one year were comparable to samples that were used immediately for PCR To further test the performance of this procedure, four Amplicons were amplified from mouse ear tissues using the dilution protocol of the fire animal tissue direct PCR kit.
For comparison, DNA was first purified using a commercial DNA extraction kit and the same fragments were amplified using a hot start tac DNA polymerase according to manufacturer's recommendations only with the fire animal tissue direct PCR kit were all four amplicons successfully Amplified Direct PCR is especially useful in genotyping studies requiring amplification of large numbers of samples. Skipping the DNA purification step can significantly reduce the time needed to perform the experiment. The technique provides additional benefits by reducing contamination risk and saving valuable sample material.
