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PCR strategy for genotyping of pre-implantation embryos. Due to the scarcity of extractable genetic material, a nested PCR strategy was developed in order to genotype pre-implantation embryos. Separate reactions were used to amplify a 429 bp fragment from the wild-type allele and a 389 bp segment from the mutant allele, both spanning the 3' end of the short (upstream) arm of the targeting vector. In both reactions, a 213 bp sequence located within the short arm was co-amplified with the 'diagnostic' products as an internal control. The same sense primers were used to amplify 'diagnostic' sequences in both reactions, whereas the antisense primers were allele-specific. The primers, represented by triangles, are depicted in two (nested) sets for each of the three reactions. Exons are represented by open, vertical rectangles, the PGK-Neo cassette by an open, horizontal rectangle, while thin vertical lines denote the boundaries of the short arm and the 5' end of the long (downstream) arm. A grey, horizontal rectangle delineates the segment of the wild-type allele that is replaced by the PGK-Neo cassette in the mutant allele."}

    craft-ca-core-dev

    {"project":"craft-ca-core-dev","denotations":[{"id":"T21822","span":{"begin":1137,"end":1143},"obj":"SO:0001023"},{"id":"T21821","span":{"begin":1114,"end":1122},"obj":"SO:0005853"},{"id":"T21820","span":{"begin":1075,"end":1081},"obj":"SO:0001023"},{"id":"T21819","span":{"begin":853,"end":861},"obj":"SO:0005853"},{"id":"T21818","span":{"begin":789,"end":794},"obj":"SO:0000147"},{"id":"T21817","span":{"begin":687,"end":694},"obj":"SO:0000112"},{"id":"T21816","span":{"begin":666,"end":672},"obj":"SO:0001023"},{"id":"T21815","span":{"begin":653,"end":660},"obj":"SO:0000112"},{"id":"T21814","span":{"begin":643,"end":652},"obj":"SO:0000077"},{"id":"T21813","span":{"begin":560,"end":567},"obj":"SO:0000112"},{"id":"T21812","span":{"begin":432,"end":434},"obj":"SO:0000028"},{"id":"T21811","span":{"begin":389,"end":405},"obj":"SO:0001644"},{"id":"T21810","span":{"begin":321,"end":327},"obj":"SO:0001023"},{"id":"T21809","span":{"begin":294,"end":296},"obj":"SO:0000028"},{"id":"T21808","span":{"begin":277,"end":283},"obj":"SO:0001023"},{"id":"T21807","span":{"begin":246,"end":248},"obj":"SO:0000028"},{"id":"T21806","span":{"begin":191,"end":198},"obj":"UBERON:0000922"},{"id":"T21805","span":{"begin":178,"end":190},"obj":"GO:0007566"},{"id":"T21804","span":{"begin":99,"end":106},"obj":"SO:0000704"},{"id":"T21803","span":{"begin":55,"end":62},"obj":"UBERON:0000922"},{"id":"T21802","span":{"begin":42,"end":54},"obj":"GO:0007566"}],"text":"Nested PCR strategy for genotyping of pre-implantation embryos. Due to the scarcity of extractable genetic material, a nested PCR strategy was developed in order to genotype pre-implantation embryos. Separate reactions were used to amplify a 429 bp fragment from the wild-type allele and a 389 bp segment from the mutant allele, both spanning the 3' end of the short (upstream) arm of the targeting vector. In both reactions, a 213 bp sequence located within the short arm was co-amplified with the 'diagnostic' products as an internal control. The same sense primers were used to amplify 'diagnostic' sequences in both reactions, whereas the antisense primers were allele-specific. The primers, represented by triangles, are depicted in two (nested) sets for each of the three reactions. Exons are represented by open, vertical rectangles, the PGK-Neo cassette by an open, horizontal rectangle, while thin vertical lines denote the boundaries of the short arm and the 5' end of the long (downstream) arm. A grey, horizontal rectangle delineates the segment of the wild-type allele that is replaced by the PGK-Neo cassette in the mutant allele."}

    craft-ca-core-ex-dev

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