Hwang, You-Jin
(Division of Biological Science, Gachon University of Medicine and Science)
,
Bae, Mun-Sook
(Division of Biological Science, Gachon University of Medicine and Science)
,
Yang, Jae-Hun
(Division of Biological Science, Gachon University of Medicine and Science)
,
Kim, Bo-Kyoung
(Division of Biological Science, Gachon University of Medicine and Science)
,
Kim, Sang-Ok
(Division of Biological Science, Gachon University of Medicine and Science)
,
Lee, Eun-Soo
(Division of Biological Science, Gachon University of Medicine and Science)
,
Choi, Sun-Gyu
(Division of Biological Science, Gachon University of Medicine and Science)
,
Kwon, Ye-Ri
(Division of Biological Science, Gachon University of Medicine and Science)
,
Seo, Min-Hae
(Division of Biological Science, Gachon University of Medicine and Science)
,
Park, Choon-Keun
(College of Animal Life Science, Kangwon National University)
,
Kim, Dae-Young
(Division of Biological Science, Gachon University of Medicine and Science)
Sex-sorting of sperm is an assisted reproductive technology (ART) used by the livestock industry for the mass production of animals of a desired sex. The standard method for sorting sperm is the detection of DNA content differences between X and Y chromosome-bearing sperm by flow cytometry. However,...
Sex-sorting of sperm is an assisted reproductive technology (ART) used by the livestock industry for the mass production of animals of a desired sex. The standard method for sorting sperm is the detection of DNA content differences between X and Y chromosome-bearing sperm by flow cytometry. However, this method has variable efficiency and therefore requires verification by a second method. We have developed a sex determination method based on quantitative real-time polymerase chain reaction (qPCR) of the porcine amelogenin (AMEL) gene. The AMEL gene is present on both the X and the Y chromosome, but the length and sequence of its noncoding regions differ between the X and Y chromosomes. By measuring the threshold cycle (Ct) of qPCR, we were able to calculate the relative frequency of X chromosome. Two sets of AMEL primers were used in these studies. One set (AME) targeted AMEL gene sequences present in both X and Y chromosome, but produced PCR products of different lengths for each chromosome. The other set (AXR) bound to AMEL gene sequences present on the X chromosome but absent esholthe Y-chromosome. Relative product levels were calculated by normalizing the AXR fluorescence to the AME fluorescence. The AMEL method accurately predicted the sex ratios of boar sperm, demonstrating that it has potential value as a sex determination method.
Sex-sorting of sperm is an assisted reproductive technology (ART) used by the livestock industry for the mass production of animals of a desired sex. The standard method for sorting sperm is the detection of DNA content differences between X and Y chromosome-bearing sperm by flow cytometry. However, this method has variable efficiency and therefore requires verification by a second method. We have developed a sex determination method based on quantitative real-time polymerase chain reaction (qPCR) of the porcine amelogenin (AMEL) gene. The AMEL gene is present on both the X and the Y chromosome, but the length and sequence of its noncoding regions differ between the X and Y chromosomes. By measuring the threshold cycle (Ct) of qPCR, we were able to calculate the relative frequency of X chromosome. Two sets of AMEL primers were used in these studies. One set (AME) targeted AMEL gene sequences present in both X and Y chromosome, but produced PCR products of different lengths for each chromosome. The other set (AXR) bound to AMEL gene sequences present on the X chromosome but absent esholthe Y-chromosome. Relative product levels were calculated by normalizing the AXR fluorescence to the AME fluorescence. The AMEL method accurately predicted the sex ratios of boar sperm, demonstrating that it has potential value as a sex determination method.
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제안 방법
In this study, qPCR of the AMELX and AMELY genes was used to determine the sperm sex ratios in boar ejaculates. Results were affirmed and corrected for primer efficiency by using positive control ratios determined for sow and boar tissue.
, 2008). In this study, we applied AMEL gene qPCR in the determination of porcine semen sex ratios. We used the relative method to demonstrate the utility of AMEL gene qPCR data for estimating the frequencies of X- and Y-chro- mosome-bearing spermatozoa.
Real-time qPCR was performed using an iQrM5 Multicolor Real-Time PCR Detection System (Bio-Rad Laboratories) which monitored the PCR reactions, for two set of primers simul- taoriously, and produced separate fluorescence amplification plots for each primer set product. The quantification was performed by raperimental determ fbtion of the thressetd etcle (Ct), defined as the PCR etcle number.
Results were affirmed and corrected for primer efficiency by using positive control ratios determined for sow and boar tissue. The AME primer set, amplified a portion of intron 2 which differed in length between the two AMEL alleles (Sembon et al, 2008).
We derived a mathematical model which was used to correct for the error introduced into the sex ratio value by a discrepancy between the PCR efficiencies of each primer set. The mathematical model was based on the efficiency of primer sets for amplification of each individual AMEX and AMEY gene (Table 1~3).
To assess sperm sex ratio in semen samples, we performed qPCR of the AMELX and AMELY specific gene sequences. The reference gene quantities could be estimated amplifying AMEL gene sequences on both the X and Y chromosome with one primer set (AME).
The sex ratio in this study was within this range. To confirm the accuracy of AMEL qPCR method, we could mix sow and boar DNA sample in 1:5 or 3:3 ratio and, then applied to this sexing method. We expected the sex ratio for '1F:5M' (sow DNA:boar DNA=1:5) and '3F:3M' (sow DNA:boar DNA=3:3) with the X-chromosome comprising 58.
36 for unsorted sperm. To determine the accuracy of this test, we could mix sow and boar DNA sample in 1:5 or 3:3 ratio and, then applied to AMEL qPCR method. The mean value of '1F:5M' (sow DNA:boar DNA=1:5) was 1.
The amplification of this nonspecific sequence may account for the difference between the efficiencies of the AME and AXR primer sets. To minimize the effect of this discrepancy, a mathematical model was developed to correct for the different amplification efficiencies when calculating sex ratios for samples.
이론/모형
The 2 MCT method was used to calculate the relative ratio of X-bearing sperm in semen samples using the real-time qPCR data. Normalized expression was calculated as the relative quantity of X-specific sequence normalized to the relative quantities of the X/Y common sequence (AMELX and AMELY).
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