不同PCR法对牛性别鉴定的影响研究

为建立一种有效的牛早期胚胎PCR性别鉴定方法,试验以12头已知性别的牛全血为试验材料,提取基因组DNA,分别运用常规PCR法、多重PCR法和巢式PCR法进行牛性别鉴定研究,用2对SRY(Y染色体性别决定区)引物和1对牛特异性引物扩增牛核基因组DNA进行牛性别鉴定,建立3种牛性别鉴定的PCR反应体系。结果表明,3种方法准确率均为100%,其中巢式PCR法检测灵敏度可达13pg,多重PCR法整个鉴定过程约需3h。     

应用PCR技术鉴定牛早期胚胎性别方法优化的研究

研究的目的是优化胚胎PCR性别鉴定方法的条件，建立实用的鉴定方法。试验设计获得了牛种属和公牛特异的引物，利用聚合酶链反应（PCR）对模板DNA进行扩增，通过常规双重PCR法和巢式PCR法的比较研究，结果表明巢式PCR法大大提高了鉴定牛早期胚胎性别的灵敏度。对组织DNA样品、血样、颗粒细胞和公牛冷冻精子进行检测，结果全部与实际性别一致；对于胚胎样品，判定结果与产犊性别一致。通过批量胚胎现场的检测，证明建立的胚胎性别鉴定方法可用于生产。     

牛早期胚胎性别鉴定PCR反应体系的优化研究

根据牛SRY基因序列设计合成2对巢式PCR引物作为性别鉴定引物，根据牛酪蛋白基因序列设计了一对引物作为内标引物建立了牛胚胎性别鉴定的PCR反应体系。同时对常规PCR和巢式PCR在牛早期胚胎性别鉴定中的实用性进行比较。15头公牛、13头母牛的DNA样品检测结果表明：使用巢式PCR公牛可以扩增出205bp的SRY基因片段和403bp的酪蛋白基因片段，母牛只能扩增出403bp的酪蛋白基因片段；而使用常规PCR时公牛扩增出255bp的SRY基因片段和403bp的酪蛋白基因片段，母牛只能扩增出403bp的酪蛋白基因片段，其性别鉴定结果和实际完全一致。由于巢式PCR只需10个细胞就可以在紫外透射分析仪下看到扩增结果，而常规PCR则需要20～30个细胞，所以胚胎性别鉴定时使用巢式PCR效果更好。试验采用巢式PCR鉴定了10个奶牛胚胎的性别，同时还对血清是否会对试验结果产生影响进行了研究。     

牙釉质基因巢式扩增鉴定牛早期胚胎性别的研究

根据牙釉质基因在牛X染色体和Y染色体上存在的差异设计巢式引物,对牛静脉血基因组DNA样本以及10枚牛早期胚胎DNA样本进行巢式扩增和电泳分析,以鉴定性别。结果表明:利用此方法能够对牛10 pg量血液基因组DNA进行扩增鉴定性别,雌性产生1条片段长度为311 bp的源于X染色体的条带,雄性产生1条源于X染色体的条带(311 bp)和1条片段长度为251 bp的源于Y染色体的条带,对10枚胚胎进行鉴定,6枚为雄性,4枚为雌性。说明牙釉质基因巢式PCR扩增鉴定牛早期胚胎性别方法可靠,准确性和敏感性较高。     

哺乳动物胚胎性别鉴定试剂盒的研究

依据牛、山羊、兔、猪等哺乳动物SRY基因高度同源性设计一对22 bp的SRY引物,按照3×2×3因子组合,建立了PCR扩增胚胎DNA最适条件。采用此最适条件扩增了15个羊-兔异种克隆胚胎DNA,结果表明PCR法可以用来鉴别哺乳动物胚胎的性别。采用设计的性别鉴定引物,按照最优PCR扩增胚胎DNA条件配制了PCR性别鉴定试剂盒。     

Sry基因和Y染色体重复序列在牛早期胚胎性别鉴定中应用效果的比较

本试验以牛Sty基因和Y染色体重复序列作为雄性特异性基因,分别设计引物,建立多重巢式PCR体系,比较二者在牛早期胚胎性别鉴定中的应用效果。试验结果表明,当扩增体系中的模板量为一个胚胎细胞的DNA量时,以Y染色体重复序列构建的扩增体系比Sry基因具有更高的灵敏度和稳定性,更适合用于牛早期胚胎性别鉴定。     

兔早期胚胎PCR性别鉴定体系的建立

根据兔SRY基因序列设计两对引物作为兔雄性特异性引物,根据兔APP基因序列设计1对引物作为内标引物,分别建立了兔早期胚胎性别鉴定的双重PCR和巢式PCR反应体系,在不同浓度的基因组DNA和兔早期胚胎上进行性别鉴定应用,同时,对兔SRY巢式PCR引物特异性进行了分析。结果表明,多重PCR扩增兔基因组DNA可以准确判定其性别,扩增灵敏度为100pg基因组DNA;多重PCR鉴定24枚兔32细胞桑椹胚性别,只能对整胚成功鉴定。巢式PCR,公兔基因组DNA扩增出282bp的SRY基因片段,母兔没有扩增产物,扩增灵敏度为10pg;对24枚兔32细胞桑椹胚性别鉴定结果表明,巢式PCR可以对少至4个胚胎细胞进行准确鉴定,同一胚胎结果符合率为100%(24/24)。SRY引物只对兔雄性基因组DNA特异,而其他动物(人、牛、绵羊、小鼠)雄性DNA及兔的冲卵液,均无PCR产物。     

牦牛胚胎性别鉴定PCR反应体系的建立

研究旨在建立牦牛胚胎性别鉴定准确、可靠的PCR反应体系及研究切割取样对胚胎发育能力的影响.根据牦牛SRY和HSL基因序列分别设计合成2对巢式PCR引物作为性别鉴定引物和2对引物作为内标引物,通过优化PCR反应条件,对已知性别牦牛血液基因组DNA和牦牛杂种胚胎DNA采用巢式PCR进行性别鉴定.结果表明,血液样本性别鉴定与实际性别完全相符,准确率100%.从早期胚胎取8个细胞进行性别鉴定,结果雄性为45.8%(11/24),雌性为54.2%(13/24),取样后胚胎发育率为56.7%.结果说明已成功建立牦牛胚胎性别鉴定的PCR体系.     

应用AML基因进行牛性别鉴定的研究

本文以染色体牙釉蛋白基因(AML)对牛肌肉基因组进行性别鉴定，为应用此基因进行胚胎鉴定提供理论依据。本研究应用奶牛染色体牙釉蛋白基因的DNA序列，设计了牛染色体特异性引物P1和P2，并通过聚合酶链反应(PCR)扩增牛肌肉组织中的X和Y染色体上的牙釉蛋白基因内第五内含子。最后依据其分子量的差异对电泳后的性染色体所呈现的不同区带进行区分，从而达到性别鉴定的目的，具有很高的应用价值。     

牛胚胎性别鉴定荧光定量PCR方法的优化与应用

旨在建立一种可检测牛早期胚胎性别的复合探针体系,减少常规PCR方法电泳所带来的污染,提高鉴定准确率,降低鉴定成本.本研究以SRY为牛胚胎性别鉴定的目标基因,以进口YCD-PCR性别鉴定试剂盒为对照组(n=9543),荧光定量PCR的单引物分别扩增体系(荧光单扩,FSPSA,n=6570)和双引物混合扩增体系(荧光双扩,...     

鉴别牛早期胚胎性别PCR方法引物的设计与筛选

根据牛Y-染色体特异重复序列、睾丸特异蛋白基因以及性别决定基因序列设计合成5对公牛Y-染色体特异引物，依据牛骨胳肌α肌动蛋白前体基因和微卫星DNA序列设计合成4对牛DNA特异引物(内标引物)。单重PcR扩增牛基因组DNA，筛选出4对牛Y-染色体特异引物和1对牛DNA特异内标引物。将不同的Y-染色体特异引物与内标引物组合，多重PCR扩增牛基因组DNA、已知性别的牛成纤维细胞和克隆胚胎，筛选出2个可用于牛早期胚胎性别鉴别的PCR引物组合：B34／A12和B78／A12。     

Development of Y‐chromosome‐Specific SCAR Markers Conserved in Taurine,Zebu and Bubaline Cattle

Sex pre‐selection of bovine offsprings has commercial relevance for cattle breeders and several methods have been used for embryo sex determination. Polymerase chain reaction (PCR) has proven to be a reliable procedure for accomplishing embryo sexing. To date, most of the PCR‐specific primers are derived from the few single‐copy Y‐chromosome‐specific gene sequences already identified in bovines. Their detection demands higher amounts of embryonic genomic material or a nested amplification reaction. In order to circumvent this, limitation we searched for new male‐specific sequences potentially useful in embryo sexing using random amplified polymorphic DNA (RAPD) analysis. Random amplified polymorphic DNA (RAPD) assay reproducibility problems can be overcome by its conversion into Sequence Characterized Amplified Region (SCAR) markers. In this work, we describe the identification of two bovine male‐specific markers (OPC16₃₂₃ and OPF10₁₁₆₈) by means of RAPD. These markers were successfully converted into SCARs (OPC16₇₂₆ and OPF10₉₈₄) using two pairs of specific primers.Furthermore, inverse PCR (iPCR) methodology was successfully applied to elongate OPC16₃₂₃ marker in 159% (from 323 to 837 bp). Both markers are shown to be highly conserved (similarity ≥95%) among bovine zebu and taurine cattle; OPC16₃₂₃ is also highly similar to a bubaline Y‐chromosome‐specific sequence. The primers derived from the two Y‐chromosome‐specific conserved sequences described in this article showed 100% accuracy when used for identifying male and female bovine genomic DNA, thereby proving their potential usefulness for bovine embryo sexing.     

Sex identification of Japanese black bear,Ursus thibetanus japonicus,by PCR based on amelogenin gene

A method for sex identification of the Japanese black bear was examined using a polymerase chain reaction (PCR) and sequencing of a part of the amelogenin gene. This gene is located on the X and Y chromosomes, and there are 54 nucleotide deletions on the Y chromosome-specific gene. Forty-seven (26 male and 21 female) DNA samples and 23 (13 male and 10 female) DNA samples, respectively extracted from white blood cells and hairs of Japanese black bears were analyzed. The primers SE47 and SE48 from this X-Y homologous region were used in sex identification by PCR amplification. These primers amplified X- and Y-specific bands, which could be used to discriminate between sexes by a length polymorphism in all samples. We suggest that PCR amplification using the primers SE47 and SE48 is useful for sex determination of the Japanese black bear and could be applied to DNA analysis of small samples such as hairs.     

滩羊公羊特异性RAPD标记的研究

利用混合样本分析方法对滩羊进行了RAPD分析 ,筛选了 6 0种随机引物 ,结果发现OPB17 32 0这一扩增片段只出现在公羊的混合样中 ,进一步对 5 0只滩羊进行逐个检测 ,发现 2 1只公羊均出现了OPB17 32 0这一DNA片段 ,而 2 9只母羊无一出现此条片段 ,据此推断OPB17 32 0为滩羊公羊的特异性RAPD标记。这一发现为滩羊胚胎性别鉴定和Y染色体上基因的研究提供了新的方法和标记参考座位。     

A novel repeated sequence located on the bovine Y chromosome: its application to rapid and precise embryo sexing by PCR

A novel repeated sequence specific to male cattle was identified and named S4. S4 is a highly repetitive sequence and is a 1.5 kb repeating unit that contains various internal repeated sequences. FISH analysis showed that S4 is localized on the whole long arm and the proximal region of the short arm of the Y chromosome. We found that a PCR primer set for S4 amplified a male-specific 178 bp product in addition to a 145 bp product common to both male and female cells. Although the origin of the 145 bp product is unknown, it acts as a positive internal control in practical embryo sexing. Due to the high copy number of S4, PCR required only 0.5 pg purified DNA for accurate amplification. This made it possible to reduce the amount of biopsy sample required for embryo sexing and thus result in less damage to embryos manipulated. These studies indicate that embryo sexing based on the S4 sequence is accurate and sensitive.     

Sexing of newly-hatched chicks using DNA isolated from chorio-allantoic membrane samples by polymerase chain reaction in Denizli chicken

1. The aim of the present study was to determine the sex of newly-hatched chicks of Denizli chicken, a local Turkish breed, by polymerase chain reaction (PCR) using DNA extracted from the chorioallantoic membrane (CAM). 2. Fertilised eggs were incubated individually and a total of 20 CAM samples were collected following the hatching process. DNA was isolated from the CAM samples and PCR was performed using W-repeat (W) and 18 S ribosomal gene (R) primers. 3. Screening of the PCR products by agarose gel electrophoresis revealed that males have a single band (256 bp) and females have an extra second band (415 bp) as expected. 4. The present study describes a reliable, rapid, and simple multiplex PCR protocol that can be put into use to sex local breeds of chicken in which phenotypic sexing is impossible, using DNA isolated from the CAM that is discarded and remains attached to the egg shell following the hatching process.     

Cryopreservation and sexing of in vivo- and in vitro-produced bovine embryos for their practical use

My research awarded includes contributions to cryopreservation and sexing of bovine embryos produced in vitro and in vivo, as follows; (1) In vivo-derived morulae and blastocysts were cryopreserved in the presence of 10% glycerol, and the embryos were transferred into recipients after two-step dilution of glycerol in straw, with a practically acceptable pregnancy rate. (2) The survival rate of 16-cell stage embryos frozen in the medium with ethylene glycol was higher than that with DMSO or 1,2-propanediol. Addition of linoleic acid-albumin to culture medium enhanced the survival rate of post-thaw bovine 16-cell stage in vitro-produced (IVP) embryos. (3) Polarization of cytoplasmic lipid droplets by centrifugation of 2-cell stage embryos was found effective to increase freezing tolerance in 16-cell stage embryos developed from the centrifuged embryos, because blastomeres of 16-cell stage embryos were mostly lipid-free. (4) The usefulness of gel-loading tip (GL-Tip) as a container for ultra-rapid vitrification was demonstrated in IVP embryos from 2-cell to blastocyst stages, with a higher in vitro survival than the conventional two-step freezing. (5) PCR analysis for sexing of in vivo-derived Day-7 embryos indicated that male embryos developed faster and graded higher than female embryos. But such correlation between genetic sex and embryonic development was not found in IVP embryos obtained from individual cows. (6) Addition of 0.1-1.0% deproteinized hemodialysate product from calf blood to culture medium increased the producing efficiency of demi-embryos with good quality. Female embryos rather than male embryos required a longer time to repair after bisection. (7) In vivo-derived bovine embryos after biopsy for sexing by PCR analysis and subsequent vitrification using GL-Tips are available to practical use in the field. (8) Introduction of primer extension preamplification-PCR and purification of DNA product before standard sexing PCR of biopsy samples from Day 3-4 in vitro-derived embryos allowed accurate sex determination, and Day-7 blastocysts developed from Day 3-4 embryos were cryopreserved by GL-Tip vitrification without a loss of their viability. Thus the field application of bovine embryo transfer is in part supported by improvements of technologies in embryo cryopreservation and sex pre-determination.     

秦岭羚牛性别鉴定的初步研究

根据牛的Sry基因核心序列设计合成1对引物F1、R1。应用PCR技术对羚牛(TAKIN)Sry基因进行扩增,结果在羚牛雄性样本扩增出1条带(230 bp),而在雌性样本未见扩增带,显示了Sry基因的性别特异性,应用这1对引物对60只未知性别的羚牛组织样本进行了性别鉴定,结果雄性14个,雌性46个。该试验为羚牛种群性别比率及其种群动态变化机制研究提供了资料。