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使用多重竞争性等位基因特异性PCR(mxKASP)基因分型法确定合子性

Determining Zygosity With Multiplex Kompetitive Allele-Specific PCR (mxKASP) Genotyping.

作者信息

Meilink Willem R M, de Visser Manon C, Theodoropoulos Anagnostis, Fahrbach Michael, Wielstra Ben

机构信息

Institute of Biology Leiden Leiden University Leiden the Netherlands.

Naturalis Biodiversity Center Leiden the Netherlands.

出版信息

Ecol Evol. 2025 Jun 21;15(6):e71642. doi: 10.1002/ece3.71642. eCollection 2025 Jun.

Abstract

We introduce multiplex Kompetitive Allele-Specific PCR (mxKASP): a modification of "classical" KASP genotyping that allows zygosity to be determined in diploid organisms. Rather than targeting a SNP associated with a single marker, mxKASP targets two non-homologous markers. We show proof of concept by applying mxKASP to the balanced lethal system in newts, in which individuals are known to possess either: (1) zero copies of the 1A version of chromosome 1 and two copies of the 1B version; (2) one copy of 1A and one copy of 1B; or (3) two copies of 1A and zero copies of 1B. mxKASP is successful in amplifying both a 1A and a 1B marker in a single reaction (if present), allowing the zygosity of individuals to be inferred. We independently confirm our mxKASP results with a multiplex PCR approach. We argue that mxKASP can be applied to rapidly and economically determine zygostity in diploid organisms, for a large number of samples at once.

摘要

我们介绍了多重竞争性等位基因特异性PCR(mxKASP):这是对“经典”KASP基因分型的一种改进,可用于确定二倍体生物的纯合性。mxKASP不是针对与单个标记相关的单核苷酸多态性(SNP),而是针对两个非同源标记。我们通过将mxKASP应用于蝾螈的平衡致死系统来证明其概念,在该系统中,已知个体要么具有:(1)1号染色体1A版本的零拷贝和1B版本的两个拷贝;(2)1A的一个拷贝和1B的一个拷贝;要么(3)1A的两个拷贝和1B的零拷贝。mxKASP能够在单个反应中成功扩增1A和1B标记(如果存在),从而推断个体的纯合性。我们用多重PCR方法独立验证了mxKASP的结果。我们认为mxKASP可用于快速、经济地确定二倍体生物中大量样本的纯合性。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0c50/12181748/355cb875dace/ECE3-15-e71642-g001.jpg

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