What are the 2 major approaches used for Embryo Sexing and which is the one used more often
- Polymerase Chain reaction (PCR)
* Fluorescence In Situ Hybridization (FISH)-used more
Describe PCR in general
- Embryo biopsy
- DNA extraction
- PCR assay
- Visualize PCR prducts as a gel or sequence PCR products as a readout
Describe how PCR works
- Small amount of DNA is targeted by primers (such as Sry)
- Heat is applied to denature the original DNA
- DNA is cooled to allow the primers to anneal to the target genes
- The DNA is returned to optimal temperatures to allow Taq Polymerase to bind primers and synthesize new strands of DNA via primer extension
- The process repeats for 25-40 cycles
- this allows amplification of the original DNA by 100K times
Describe the nested PCR used by Kuneida et al. in 1992 published in Bio Repro on Y-chromosome specific sequences
- Product 1: 404 bp long (SRY2, SRY4) and 617/599bp long (ZFY3, ZFY4)
- Product 2: 147bp long (SRY1, SRY3) and 217/199np long (ZFY11, ZFY12)
- Increased specificity by using the 2nd PCR round
Describe the procedural control done by the nested PCR used by Kuneida et al. in 1992 published in Bio Repro
- Control was X-chromosome detection
- Product 1: 244bp long (NDS3, NDS4)
- Product 2: 111bp long (NDS1, NDS2)
Describe the results of the Kuneida et al. experiment on Y-chromosome specific sequences using PCR on mouse premplantaiont eombryos in 1992 that was published in Bio Repro
*When compared to the karyotypes at the blastomere and morula PCR produced the same results
What does it mean when is said PCR is a dual primer set within one assay?
*One primer for male and one for female needed
Describe FISH in general
- Embryo biopsy
- Cells fixed to a microscope slide
- Gene/chromosome-specific probes are hybridized
- Gene/chromosome are identified visually
Describe how an embryo biopsy is performed for embryo sexing
- Embryo is held in place by a holding pipette that holds negative pressure to capture the zona pellucida (coat around embryo)
- acidic buffer is applied to make a small hole in the ZP so the embryo can be accessed via infusion pipette
- A suction pipette is then used to remove the blastomere
Describe how FISH works after the embryo biopsy is complete
- The blastomere is attached to a microscope slide
- Probe DNA that is identical to the DNA of interest is labeled with a fluorescent dye
- The DNA in the cells and the probe DNA are denatured via heat
- The heat is reduced so the probe DNA anneals complimentary to the DNA in the blastomeres
- After hybridization, the slide is placed under a microscope and exposed to UV light
- The probe marker can be visualized allowing for indentification
What are the different probe types that can be used in FISH and what colors are they?
- Y-chromosome: red
- X-chromosome: green
- Autosomal chromosome 18 (control): teal
