Development of a DNA microarray for detection of expressed equine classical MHC class I sequences in a defined population.
Development of a DNA microarray for detection of expressed equine classical MHC class I sequences in a defined population.
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DOI：
10.1007/s00251-010-0463-y
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发表时间：
2010-09
期刊：
影响因子：
3.2
通讯作者：
Mealey, Robert H.
中科院分区：
文献类型：
--
作者：
Ramsay, Joshua D.;Leib, Steven R.;Orfe, Lisa;Call, Douglas R.;Tallmadge, Rebecca L.;Fraser, Darrilyn G.;Mealey, Robert H.
建立一种准确、高效的马MHC I类分子分型方法，将有助于马T淋巴细胞免疫应答的研究。在这里，设计了一个DNA微阵列来检测表达的经典MHC I类基因，包括血清学定义的马白细胞抗原(ELA)-A单倍型，代表了一个封闭的阿拉伯马种马群。最初，利用RT-PCR产物的克隆和测序，在六匹马中鉴定与ELA- A1、A4和W11单倍型以及一个未定义单倍型相关的序列。随后，设计了序列特异性、保守性（阳性对照）和随机核苷酸（阴性对照）23- 27-mer寡核苷酸微阵列探针，并使用机器人阵列仪将其定位到环氧涂层掩膜载玻片上。每匹马的大量RT-PCR产物通过缺口翻译进行生物素化，杂交到阵列上，并使用酪酰胺信号扩增进行检测。该微阵列持续检测到9个经典MHC I类转录本中的8个，并允许ELA单倍型关联。然后在一组ELA不同的马和矮种马中进行RT-PCR产物的克隆和测序，其中鉴定了六个新序列。这组被用来确定阵列的特异性。总的来说，微阵列比克隆和测序更有效地检测了在这一定义的马群体中表达的经典MHC I类序列，并且比血清学更具特异性。这些结果证实了基于微阵列的方法在马的高分辨率MHC I类分型中的实用性。有了额外的探针，该阵列可以在更广泛的人群中使用。
Development of an accurate and efficient molecular-based equine MHC class I typing method would facilitate the study of T lymphocyte immune responses in horses. Here, a DNA microarray was designed to detect expressed classical MHC class I genes comprising serologically defined equine leukocyte antigen (ELA)-A haplotypes represented in a closed Arabian horse breeding herd. Initially, cloning and sequencing of RT-PCR products were used to identify sequences associated with the ELA- A1, A4, and W11 haplotypes, and one undefined haplotype, in six horses. Subsequently, sequence- specific, conserved (positive control), and random nucleotide (negative control) 23- to 27-mer oligonucleotide microarray probes were designed and spotted onto an epoxy-coated masked slide using a robotic arrayer. Bulk RT-PCR products from each horse were biotinylated by nick translation, hybridized to the array and detected using tyramide signal amplification. The microarray consistently detected eight of nine classical MHC class I transcripts and allowed ELA haplotypic associations to be made. Cloning and sequencing of RT-PCR products were then performed in a group of ELA disparate horses and ponies, in which six novel sequences were identified. This group was used to determine the specificity of the array. Overall, the microarray was more efficient than cloning and sequencing for detecting expressed classical MHC class I sequences in this defined population of horses, and was significantly more specific than serology. These results confirmed the utility of a microarray-based method for high resolution MHC class I typing in the horse. With additional probes the array could be useful in a broader population.
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影响因子：
3.2
作者：
Tallmadge RL;Campbell JA;Miller DC;Antczak DF
通讯作者：
Antczak DF
DOI：
10.1111/j.1744-313x.1995.tb00239.x
发表时间：
1995-06-01
期刊：
EUROPEAN JOURNAL OF IMMUNOGENETICS
影响因子：
--
作者：
ELLIS, SA;MARTIN, AJ;MORRISON, WI
通讯作者：
MORRISON, WI
影响因子：
3.2
作者：
Ellis, SA;Bontrop, RE;Marsh, SGE
通讯作者：
Marsh, SGE
影响因子：
3.2
作者：
BAILEY, E
通讯作者：
BAILEY, E
影响因子：
--
作者：
Elsner, HA;Blasczyk, R
通讯作者：
Blasczyk, R