Olivier M, Meehl M A, Lust G
James A. Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA.
J Hered. 1999 Jan-Feb;90(1):78-82. doi: 10.1093/jhered/90.1.78.
Random amplified polymorphic DNA (RAPD) markers have been used in genetic studies of several plant and animal species. However, concerns exist about the reproducibility of RAPD-PCR reactions. Therefore, the use of specific 24mer primer pairs in standard PCR reactions has been suggested for reliable amplification of characterized polymorphic RAPD sequences. The purpose of this article is describe the application of the RAPD-PCR assay to genetic studies of dogs and to investigate the amplification of RAPD sequences with specific primer pairs. Of 240 decanucleotide primers tested by PCR, 34.6% resulted in amplification of at least one polymorphic fragment with samples of a Labrador retriever pedigree. We cloned and sequenced five of these RAPD fragments and synthesized specific 24mer primer pairs for each. Two primer pairs amplified a sequence exclusively from samples that were positive for the RAPD fragment, while three others amplified the respective sequence from all DNA samples. A new polymorphism was observed in the restriction digest products with Msel of one of the amplification products. None of the cloned sequences contains an open reading frame longer than 213 bases. Two sequences hybridized only to specific fragments of genomic DNA from samples that amplified the RAPD, the remaining three sequences hybridized to multiple sequences in all canine samples tested by Southern analysis. None of the five fragments hybridized to human or murine genomic DNA. Data suggested that RAPD sequences can be used as molecular markers in genetic studies of diseases in dogs. However, the use of specific primer pairs leads to loss of the RAPD polymorphism in three of five sequences tested.
随机扩增多态性DNA（RAPD）标记已用于多种动植物物种的遗传研究。然而，人们对RAPD-PCR反应的可重复性存在担忧。因此，有人建议在标准PCR反应中使用特定的24聚体引物对，以可靠地扩增已鉴定的多态性RAPD序列。本文的目的是描述RAPD-PCR分析在犬类遗传研究中的应用，并研究用特定引物对扩增RAPD序列。在通过PCR测试的240个十聚体引物中，34.6%的引物至少扩增出一个拉布拉多猎犬家系样本的多态性片段。我们克隆并测序了其中五个RAPD片段，并为每个片段合成了特定的24聚体引物对。两个引物对仅从RAPD片段呈阳性的样本中扩增出一个序列，而另外三个引物对则从所有DNA样本中扩增出相应序列。在其中一个扩增产物的Msel酶切产物中观察到一种新的多态性。所有克隆序列均未包含长度超过213个碱基的开放阅读框。两个序列仅与扩增RAPD的样本的基因组DNA特定片段杂交，其余三个序列在通过Southern分析测试的所有犬类样本中与多个序列杂交。五个片段均未与人或小鼠基因组DNA杂交。数据表明，RAPD序列可作为犬类疾病遗传研究中的分子标记。然而，在测试的五个序列中，有三个序列使用特定引物对导致RAPD多态性丧失。
