The overall goal of this procedure is to accurately identify the sex of day 30 pig embryos, using phenol free DNA obtained from each embryo. And two pairs of sex-specific primers for a PCR reaction. This method has the potential to answer various questions related to reproductive physiology in livestock, such as the effect of maternal factors on sex ratio including uterine capacity and the metabolic state of the sow as well as various paternal factors.
The advantage of this technique involves no toxic chemicals such as phenol-chloroform, or expensive columns during DNA purification. So, let's get the show on the road. Visual demonstration of this method is as important as symptom preparation.
The steps are easy to learn but special care on the details is important, to avoid symptom cross contamination. Transfer embryo samples to a negative eighty degree celsius freezer, according to the text. Label sample tubes with the sample ID needed for the number of samples to be analyzed.
Use dry ice to half fill a thermal container and insert a pre-labled sample tube into the dry ice. While wearing winter gloves, with a pair of examination gloves on top, transfer pre-chilled mortars and pestles from the negative eighty degrees celsius freezer to a dry ice container. Next, place a frozen embryo inside the mortar on the dry ice, then, pour adequate liquid nitrogen to cover the embryo.
And use a pestle to grind it into a fine powder. Using a microspatula transfer the embryo powder to a pre-labled sample tube, and place the tube in the negative eighty degrees freezer. Repeat the grinding and freezing of each additional embryo, changing examination gloves in between samples to avoid cross contamination.
After labelling all microcentrifuged tubes needed for the number of samples to be analyzed, transfer the sample tubes containing embryo powder from the negative eighty degrees celsius freezer to a container of dry ice. Pipette 180 microlitres of 50 millimolar sodium hydroxide into each pre-labled microcentrifuged tube. Preheat an incubator to ninety-five degrees celsius.
With a toothpick, transfer approximately five to ten milligrams of embryo powder from a sample tube into a pre-labeled tube of sodium hydroxide. Slowly lift the toothpick from the solution to visualize the DNA lysate as a sticky white, transparent-like substance. Transfer the samples with DNA lysate to the ninety-five degrees celsius incubator for five minutes, then immediately transfer the tube to an insulated container filled with ice.
Next, add twenty microlitres of one molar tris-hydrochloride directly into the tubes, and tap the tubes to gently mix. With pH paper, ensure that the pH is approximately 8.0. Then, centrifuge the tubes at two thousand G, and room tempurature for two minutes to remove undissolved tissue debris.
Transfer 150 microlitres of the top clear supernatant into new tubes. Or, a 96 well plate. And store at four degrees celsius for up to two weeks.
Or, negative twenty degrees celsius for up to one year. The success of this protocol is rely on how specific you design your primer on X and Y chromosome. So how do you make sure they are on X and Y chromosome?
Using NCBI Map Viewer, is the best way to show that. To design sex-specific primers for PCR, use the NCBI website to obtain accession numbers for pore sign sex determining region Y.Or SRY and zinc finger protein X-linked, or ZFX. Copy and paste the accession numbers to an online primer design tool.
Use nucleotide blast or blast N to validate the specificity of the primers against the current pore sign genomic database 104. To enter the sequences, are only located on the X and Y chromosome for SRY and ZFX, respectively. To carry out PCR with the DNA lysates, use any hot start ready mix PCR enzyme, and prepare a master mix by adding two sex-specific primers at a final concentration of 0.3 micromolar in a 15 microlitre PCR reaction.
For the first time running the reactions prepare one PCR tube for the no template negative control, and two additional tubes for positive controls by adding one microlitre of commercially obtained sex pore sign genomic DNA. For subsequent rounds of PCR, include one microlitre of a sample from the last successful sexing analysis as a positive control. Set up the following PCR program in a thermocycler and carry out PCR.
To varify the samples after PCR prepare a 2%TBE agarose gel with cyber DNA gel stain or ethidium bromide. Add 1.5 microlitres of 10X loading dye to the samples before loading onto the gel and running. Observe and adjust the band intensities under fluorescent light and capture an image of the gel.
Identify embryos with one band as female and two bands as male. Shown here is a representative result for sex determination from 345 DNA lysates screened by PCR. The optimal primer's annealing temperature of 65 degrees celsius is sightly higher than the TM of the primers shown here for generating similar intensity and predicted ampilcon sizes.
Two amplified products of SRY and ZFX are presented here. Lysate sample from female embryos produce only a single band of 506 based pairs DNA fragment from the ZFX gene located on the X chromosome. All male embryos produce two DNA fragments with equal intensity of 400 base pairs for SRY and 506 base pairs for ZFX.
As shown here, DNA lysates did not show any PCR reaction inhibition after screening 345 embryos. Only three individuals were not sexed and the percentage of female embryos was slightly higher than males. Once this technique is mastered from grinding to carrying out PCR, it takes about one hour to process around ten embryos.
When doing this technique, its important to avoid cross contamination, especially when transferring embryo powder to the tubes. Following this procedure, we can use other methods such as, DNA methylation, microarray, and sequencing to investigate genomic and epigenetic effects of nutrition and infectious disease between sexes. The development of this technique will pave the way for researchers in the areas of reproductive physiology and animal science to conduct research related to sexual dimorphism in various livestock species.
After watching this video you should have a good understanding how to design sex specific primers, abstract DNA from embryos and perform PCR reaction using DNA lysates. Remember that working with dry ice is quite dangerous, so take proper precaution wear gloves and goggles.
