TITLE: PCR Protocol for chicken sex identification.
AUTHORS: Liyan He, Priscila Martins, Joris Huguenin, Thi-Nhu-Ngoc Van, Taciana Manso, Therese Galindo, Lise Catherinot, Franck Molina, Julien Espeut

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is an invasive PCR based chicken gender determination protocol to identify the sex of fertilized egg at the early chicken’s embryo states. It is based on the differences between males and females sexual chromosomes: ZZ for males and ZW for females.</div><div class = "text-block">Two set of primers have been developped for this method: SWIM and 12S. They amplify 2 amplicons for female samples and 1 amplicon for male samples.</div></div>

[STEPS]
?. [Prepare the Primers Ddilutions]
Two set of primers (12S & SWIM) are used in this protocol for positive control (12S) and female indication (SWIM).  
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							SWIM Primers
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							SWIM Forward: GAG ATC ACG AAC TCA ACC AG
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							SWIM Reverse: CCA GAC CTA ATA CGG TTT TAC AG
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							12S Primers
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							12S Forward: CTA TAA TCG ATA ATC CAC GAT TCA
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							12S Reverse: CTT GAC CTG TCT TAT TAG CGA GG
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							Prepare a 10 µM primer working solution from 100 μM primer stock.
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?. [PCR Setup]
1. Assemble all reaction components on ice in this order:
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							  ABCD1Component25 µl reaction50 µl reactionFinal Concentration2Nuclease Free Waterqsp 25 µlqsp 50 µl-3Platinium Green Hot Start PCR 2X Master Mix12.5 µl25 µl1X410 µM Forward Primer SWIM-F0.5 µl1 µl0.2 µM510 µM Forward Primer 12S-F0.5 µl1 µl0.2 µM610 µM Reverse Primer SWIM-R0.5 µl1 µl0.2 µM710 µM Reverse Primer 12S-R0.5 µl1 µl0.2 µM8DNA extract0.5 - 1 µl1 µlThe final concentration of MgCl2 is 1.5mM and each dNTP is at 0.2mM final. 2. Seal the plate, vortex and centrifuge briefly. 3. Transfer the PCR plate into the thermocycler.
ABCD1Component25 µl reaction50 µl reactionFinal Concentration2Nuclease Free Waterqsp 25 µlqsp 50 µl-3Platinium Green Hot Start PCR 2X Master Mix12.5 µl25 µl1X410 µM Forward Primer SWIM-F0.5 µl1 µl0.2 µM510 µM Forward Primer 12S-F0.5 µl1 µl0.2 µM610 µM Reverse Primer SWIM-R0.5 µl1 µl0.2 µM710 µM Reverse Primer 12S-R0.5 µl1 µl0.2 µM8DNA extract0.5 - 1 µl1 µl
?. [Thermo-cycling conditions]
ABCD1StepTemperatureTime2Preheat the lid110°C-3Initial Denaturation94°C2 min435 CyclesDenature94°C30 sec5Anneal55°C30 sec6Extend72°C30 sec7Final Extension72°C5 min8Hold4°C-
ABCD1StepTemperatureTime2Preheat the lid110°C-3Initial Denaturation94°C2 min435 CyclesDenature94°C30 sec5Anneal55°C30 sec6Extend72°C30 sec7Final Extension72°C5 min8Hold4°C-
?. [Results analysis]
PCR amplifications can be analyzed by different manners:
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							- By electrophoretic migration on agarose gel:
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							For each sample, mix 10 μl of PCR product with 2 μl of 6X loading dye. Load them on a 2.5 % Gelgreen (Biotium, California USA) stained agarose gel in 0.5X TAE buffer and migrate DNA by electrophoresis at 100 Volts. 1 μg of DNA ladder (GeneRuler 50bp, Fisher Scientific, Illkirch France) can serve as a reference for the migration. Reveal the gel by Blue LED GelPicBox at 430nm (Nippongenetics, Düren Germany) or on a regular UV lamp.-
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							- By capillary electrophoresis:
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							Load 10 μl of the PCR reactions on a microfluidic capillary electrophoresis systems (Capillary LifeSciences, France) controlled by the Labchip GX version 4.1.1619.0 SP1 software. Capillary electrophoresis is the high throughput format of the traditional gel electrophoresis. It allows a rapid size-based separation and sensitive detection of specific DNA fragments via UV absorption or fluorescent labelling. The CE system is more advantageous than the conventional slab gel electrophoresis in terms of speed, high-throughput applicability, automated workflow, resolution, and sensitivity.
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							Results for males: When there is only one band at 131 bp appearing on the agarose gel, sample gender is male.
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							Results for females: When two bands at 131bp and 212bp are appearing on the agarose gel, sample gender is female.
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							Invalid results: If no band appear on the gel, it is invalidated. In this case, repeat the experience from PCR setup.
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							For more detail please look at our publication "Simple, sensitive and robust chicken specific sexing assays, compliant with large scale analysis" by Lyan He et al.
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?. [Lysis of chicken embryo's tissue and DNA extraction]
1. Prepare the Lysis Buffer:  ABC1Composition for 100 ml BufferQuantity (Volume)Final Concentration2Chelex Resin (Biorad Chelex 100)10g10%3SDS 20 %1 ml0.2%4Tris 1M pH81 ml10 mM5Nuclease Free Waterqsp 100 ml2. Put 150 µl of lysis buffer in each 1.5 ml tube or well of 96-well plate for each sample to analyze.
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							3. Pipet 10~20 mg of embryo tissues from the embryo head (brain), membrane, chorion or others into the tube/well containing the lysis buffer.
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							4. Add 1.5 µl proteinase K at 20 mg/ml stock into each tube/well and vortex 3 seconds.
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							5. Incubate the tubes/plate in the thermocycler. Program:
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							· Heat lid at 60°C
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							· Incubate 3h at 55°C 
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							· Incubate 15 min at 95°C
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							· Hold at 4°C
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							6. Centrifuge 5 minutes at 13000 rpm (or 15 mins at 8000 rpm) at room temperature. 7. Pipet 50 µl-100 µl of the supernatant and transfer to a new 96-well plate (this primary stock solution contains the template DNA at a concentration between 500 and 1000 ng/µl per tube) and store it:
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							- at 4°C if you are doing the PCR the same day.
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							- at -20°C if you are going to do the PCR another day (DNA is degraded with time in biological extracts).
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							8. Dilute the supernatant 10 times into a new 96-well plate (For example add 10 µl of the supernatant to 90 µl of nuclease free water). It will give a final concentration of prepared template DNA between 50 and 100 ng/µl.
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ABC1Composition for 100 ml BufferQuantity (Volume)Final Concentration2Chelex Resin (Biorad Chelex 100)10g10%3SDS 20 %1 ml0.2%4Tris 1M pH81 ml10 mM5Nuclease Free Waterqsp 100 ml