| A Polymerase Chain Reaction(PCR)-based method for sex determination of mammals embryos by amplification of mammals SRY-specific sequence was established. A pair of primers of 22 bases in length were designed based on regions of sequence homology among cattle, goats, rabbit and pigs to amplify a 163-bp region of cattle Sry. Genomic DNA extracted from male and female cattle, were directly used as template of PCR. The goat-rabbit interspecies clone embryos were collected washed twice with 0.145 mmol/L NaCl, then were transferred to 20uL lysate buffer(10 mmol/L Tris-HCl pH8.3, 50 mmol/L KC1, 2 mmol/L MgCl2, 0.45% NP-40, 0.45% Tween-20, 0.1μg/μL Proteinase K), and incubated for 60 min at 55℃. The lysates were directly used as embryos DNA template of PCR. Optimal PCR conditions developed using the factorial protocol for SRY primers using genomic cattle DNA and goat-rabbit interspecies clone embryos DNA were as follows: 100 ng of genomic DNA from a male cattle or 20 μL goat-rabbit interspecies clone embryos lysate were amplified, with 2 IU Taq DNA polymerase, supplied MgCb-free buffer and a 3×2×3 factorial combination of concentrations of SRY primers (0.1 μmol/L, 0.3 μmol/L, or 0.5μmol/L), dNTPs ( 0.1 mmol/L, or 0.2 mmol/L each), and MgCl2( 0.75 mmol/L, 1.5 mmol/L, or 2.25 mmol/L). The total volumes of PCR were 50μL. All reactions were carried out in Flexigene. After initial denaturation at 95℃ for 5 min, the amplification was performed by using 30 or 35 cycles of denaturation at 95℃ for 1 min, annealing at 58℃ for 1 min, and extension at 72℃ for 1 min. A final period of extension was carried out for 9 min and final holding at 4℃. The PCR products were resolved after electrophoresis in 2% agarose gel with ethidium bromide. The PCR products were visualized while the gel was exposed to ultraviolet light. Optimal conditions were defined as those that generated the strongest PCR products (thebrightest band) for SRY without non-specific banding. The results showed that optimal PCR condition for SRY primers using genomic cattle DNA as template were as following 0.1μmol/L SRY primers, 0.1 mmol/L dNTPs, 1.5 mmol/L MgCl2, annealing at 58℃ and 30 cycles; while optimal PCR conditions for SRY using embryo lysate as template were 0.5μmol/L SRY primers, 0.1 mmol/L dNTPs, 1.5 mmol/L MgCl2, annealing at 58℃ and 35 cycles.Using optimized genomic DNA PCR conditions for SRY primers, genomic DNA from nine black-white cattle, eleven Wuanbei yellow cattle, three Boer goats, four rabbit, and seven pigs were amplificated, The results showed that the sexing method was verified. All male samples had bands representing SRY-specific, whereas all female samples and control samples had no band. Using optimized embryo DNA PCR conditions for SRY primers, fifteen goat-rabbit interspecies clone embryos were amplificated, the sexing method was verified too. All male goat-rabbit interspecies clone embryos samples had bands representing SRY-specific, while female goat-rabbit interspecies clone embryos and control samples had no band. The result showed that PCR amplification of SRY-specific DNA sequences may be used to determine mammal's sex and mammal embryonic sex. The advantages of PCR-based embryo sexing are relatively simple, rapid, and inexpensive, but maintaining high degrees of accuracy.On basis of the optimal embryo DNA PCR conditions for SRY primers, the PCR sex determination kit was made, which could be used in practice and has adventages of convenience, less cost, more clear, and accurate.
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