LM 6

Question 1

What are the 2 major approaches used for Embryo Sexing and which is the one used more often

Answer 1

• Polymerase Chain reaction (PCR)

* Fluorescence In Situ Hybridization (FISH)-used more

Question 2

Describe PCR in general

Answer 2

• Embryo biopsy
• DNA extraction
• PCR assay
• Visualize PCR prducts as a gel or sequence PCR products as a readout

Question 3

Describe how PCR works

Answer 3

• 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

Question 4

Describe the nested PCR used by Kuneida et al. in 1992 published in Bio Repro on Y-chromosome specific sequences

Answer 4

• 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

Question 5

Describe the procedural control done by the nested PCR used by Kuneida et al. in 1992 published in Bio Repro

Answer 5

• Control was X-chromosome detection
• Product 1: 244bp long (NDS3, NDS4)
• Product 2: 111bp long (NDS1, NDS2)

Question 6

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

Answer 6

*When compared to the karyotypes at the blastomere and morula PCR produced the same results

Question 7

What does it mean when is said PCR is a dual primer set within one assay?

Answer 7

*One primer for male and one for female needed

Question 8

Describe FISH in general

Answer 8

• Embryo biopsy
• Cells fixed to a microscope slide
• Gene/chromosome-specific probes are hybridized
• Gene/chromosome are identified visually

Question 9

Describe how an embryo biopsy is performed for embryo sexing

Answer 9

• 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

Question 10

Describe how FISH works after the embryo biopsy is complete

Answer 10

• 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

Question 11

What are the different probe types that can be used in FISH and what colors are they?

Answer 11

• Y-chromosome: red
• X-chromosome: green
• Autosomal chromosome 18 (control): teal