Name: accurate and phenol free dna sexing of day 30 porcine embryos by pcr
Uploaded: 2016-02-14T14:00:00
Description: Watch this Scientific Journal Video about Accurate and Phenol Free DNA Sexing of Day 30 Porcine Embryos by PCR at JoVE.com

The authors would like to acknowledge the cooperation and financial contributions of the following research funding agency: Alberta Livestock and Meat Agency Ltd., Pork CRC, Alberta Pork, Hypor A Hendrix Genetics Company and NSERC CRSNG.

In accordance with the Canadian Council on Animal Care guidelines and with approval of the Faculty Animal Policy and Welfare Committee - Livestock of the University of Alberta, pregnant sows were euthanized by trained staffs at approximately Day 30 of pregnancy and embryos were collected. Use examination gloves at all times during the procedures.
1. Sample Collection and Sample Storage
<ol>
	<li>Euthanize sow using captive bolt followed by exsanguination. Wear examination gloves to collect the reproductive tract...

<ol>
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Most of the existing protocols related to porcine embryos DNA sex typing are only suitable for early stage pre-implantation stage15,16. We have successfully developed a protocol suitable for porcine embryo screening during late gestation. Based on studies with similar developmental stages of embryos from previous studies6,18, the present protocol is considered to be safer and low cost.
This protocol is also suitable for a large number of samples for sex typing using PCR b...

The domestic pig has become a fundamental research subject in development, genetics and nutrition in both human and livestock sectors. The potential of pigs as biomedical models for human research can be attributed to their physiological similarities. In livestock, the manipulation of sex ratio can enhance the effectiveness of selection and genetic improvement programs1. Sexing individual embryos is a fundamental tool used in many experimental investigations including but not limited to genotype, epigenetics and X inactivation of sexual dimorphism during early embryo development2.
Studies in mice suggest that maternal diet and other factors may result in gender imbalance3. In pigs, causes of sex ratio imbalance include paternal breed4, uterine capacity5, and the sow&#39;s metabolic condition6. Since the differences observed in embryos and litters can be influenced by sexual dimorphism, researchers should be aware of embryo sex and sex ratios before drawing conclusions regarding their research. The development of efficient tools and protocols for sexing pig embryos at Day 30 of development will be discussed here.
Various methods of sex typing have been developed for genetic studies in model organisms and livestock. Particularly in livestock, identifying male and female early embryos is a very common practice to enhance genetic selection for breeding programs. Early embryos karyotyping in pig using Giemsa7 or the intense fluorescence8,9 techniques have been used for sex typing. However, these methods are time consuming and not suitable for screening large numbers of embryos quickly and accurately.
The most effective sex typing method is DNA amplification using a heat stable DNA polymerase and a pair of primers. DNA sexing by PCR method is more specific, rapid and sensitive, only requiring a minute amount of cellular materials. The first PCR-based embryo sexing was performed on humans10, and later in mice11, cattle12, buffalo13 and sheep14 pre-implantation embryos. In the pig, the earliest DNA sex typing method was established for pre-implantation embryos via a single pair of Y-chromosome specific DNA primers15. However, the most common PCR primers for sex determination were selected from the Y-chromosome of male specific SRY gene16 and the non-sex discriminative region of a zinc-finger gene located at both X and Y chromosome17. Subsequently, these primers have been applied to determine the sex of Day 30 embryos in this study with improved specificity of the primers to detect only the X- chromosome of a zinc-finger gene.
Genomic DNA from porcine pre-implantation embryos can be extracted by exposing an intact blastocyst to buffer with proteinase K16 or by taking a biopsy of a few cells from the individual early cleavage embryos15 and using them for direct PCR. However, release of DNA from porcine blood, hair, tissues or a large conceptus over a few centimeters in size is not effectively done using the proteinase K method. DNA extraction methods for these materials have been established using either time consuming phenol/chloroform protocols6 or expensive column based kits18. In order to avoid the use of potentially toxic chemicals, there is a trend to develop inexpensive, easy and phenol-free DNA extraction methods. This type of protocol for the isolation of PCR-quality genomic DNA from mouse19 and zebrafish20 tissues has been established using hot sodium hydroxide and Tris (HotSHOT). This study provides a protocol to obtain DNA with modified HotSHOT and redesigned duplex primer pairs for PCR sex typing directly from cell lysates of Day 30 porcine embryos with high accuracy.

<table border="0" cellpadding="0" cellspacing="0" width="1184">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="26">
			<td height="26" style="height:27px;width:331px;">KAPA HiFi HotStart Ready Mix PCR kit&#160;</td>
			<td style="width:203px;">KAPABiosystems</td>
			<td style="width:189px;">KR0370</td>
			<td style="width:463px;">other Hot Start Taq polymerase can be used after optimization</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:331px;">SYBR Safe DNA Gel Stain</td>
			<td style="width:203px;">Life Technologies</td>
			<td style="width:189px;">S33102</td>
			<td>Ethidium bromide can be substituted for SYBR Safe</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:331px;">Pig female and male genomic DNA</td>
			<td style="width:203px;">Zyagen</td>
			<td style="width:189px;">GP-160-F1 &#38; GP-160-M5</td>
			<td>Postive controls from the tissues of known sex DNA can be used.</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:331px;">Typhoon FLA 9500 laser scanner</td>
			<td style="width:203px;">GE Healthcare Life Sciences</td>
			<td style="width:189px;">28-9969-43</td>
			<td>other imaging system can be used</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:331px;">Free Soft Nitrile Examination Gloves</td>
			<td style="width:203px;">WWR</td>
			<td>89038-270</td>
			<td>any other examination glove can be used</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:331px;">Sodium hydroxide, solid&#160;</td>
			<td style="width:203px;">Fisher</td>
			<td style="width:189px;">BP 359 -212</td>
			<td>Molecular Biology Grade</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:331px;">Eppendorff DNA LoBind Tubes, 1.5 ml, PCR clean</td>
			<td style="width:203px;">Eppendorff</td>
			<td style="width:189px;">0030 108.051</td>
			<td>heat resistant</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:331px;">ThermoStat plus</td>
			<td style="width:203px;">Eppendorff</td>
			<td style="width:189px;">22670204</td>
			<td>Use as a incubator for 95C, don&#39;t need to use the heater</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:331px;">Toothpick</td>
			<td>Bunzl Plc</td>
			<td style="width:189px;">75200815</td>
			<td style="width:463px;">Any round wooden toothpicks can be used - quality wood</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:331px;">Microcentrifuge 5417R/5417C</td>
			<td style="width:203px;">Eppendorff</td>
			<td style="width:189px;">22621807</td>
			<td>This model was discontinued. But another newer model can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Microspatula</td>
			<td style="width:203px;">Fisher</td>
			<td style="width:189px;">SDI28540115</td>
			<td>Autoclaved before use each time.</td>
		</tr>
	</tbody>
</table>

accurate and phenol free dna sexing of day 30 porcine embryos by pcr

Research into prenatal programming in the pig has shown that the sex of the developing embryo or fetus can influence the developmental outcome. Therefore, the ability to determine an embryo&#39;s sex is necessary in many experiments particularly regarding early development. The present protocol demonstrates an inexpensive, rapid and non-toxic preparation of pig genomic DNA for use with PCR. Day 30 embryos must be humanely collected according to the guidelines established by Institutional Animal Policy and Welfare Committees for the present protocol. The preparation of the whole embryo for this PCR based sexing technique simply involves grinding the frozen embryo to a fine powder using a pre-chilled mortar and pestle. PCR-quality DNA is released from a small amount of embryo powder by applying a hot incubation in an alkaline lysis reagent. Next, the DNA solution is mixed with neutralization buffer and used directly for PCR. Two primer pairs are generated to detect specific sex determining region of the Y- chromosome (SRY) and ZFX region of the X- chromosome with high accuracy and specificity. The same protocol can be applied to other elongated embryos (Day 10 to Day 14) earlier than Day 30. Also, this protocol can be carried with 96-welled plates when screening a large number of embryos, making it feasible for automation and high-throughput sex typing.

A representative result of sex determination from 345 DNA lysates screening by PCR is shown in Figure 7 and summarized in Table 1.
As can be seen in Figure 7, the primers annealing temperature at 65 &#176;C is the optimal condition in this PCR protocol generating similar intensity and predicated amplicon sizes (Figure 5) among different samples.
<p class="jo...

Watch this Scientific Journal Video about Accurate and Phenol Free DNA Sexing of Day 30 Porcine Embryos by PCR at JoVE.com

Accurate and Phenol Free DNA Sexing of Day 30 Porcine Embryos by PCR

This protocol describes an accurate, inexpensive, rapid and non-toxic method to determine the sex of Day 30 porcine embryo using PCR method after grinding an embryo into powder without phenol chloroform extraction and DNA column purification.

Research into prenatal programming in the pig has shown that the sex of the developing embryo or fetus can influence the developmental outcome. Therefore, ...

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.

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