Use of SNPs to determine the breakpoints of complex deficiencies, facilitating gene mapping in Caenorhabditis elegans.
Use of SNPs to determine the breakpoints of complex deficiencies, facilitating gene mapping in Caenorhabditis elegans.
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DOI：
10.1186/1471-2156-6-28
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发表时间：
2005-05-26
期刊：
影响因子：
2.9
通讯作者：
Singson A
中科院分区：
文献类型：
--
作者：
Kadandale P;Geldziler B;Hoffmann M;Singson A
基因缺失或缺陷已被用于各种生物体的基因定位和发现，从线虫秀丽隐杆线虫一直到人类。大缺失的一个问题是确定其断点的位置。在缺失基因组的一个区域而保留一些间插序列的复杂缺陷的情况下，这会加剧。以前的方法，使用遗传互补或细胞学受到低标记密度的阻碍，因此在定位复杂缺陷的断点时不是很精确。越来越多的单核苷酸多态性（SNP）的鉴定已经导致这些作为遗传标记的使用，并且因此导致它们用于使用分子生物学方法定义缺失的断点。在这里，我们表明，单核苷酸多态性可以用来帮助定位一个复杂的缺陷，在C的断点。优美的该技术使用遗传杂交和分子生物学的组合，在试图确定缺陷的断点时，通过强有力的内部控制产生稳健且高度可重复的结果。这种技术和标准遗传图谱的结合使用使我们能够在克隆基因的尝试中迅速缩小感兴趣的区域。与以前用于定位缺陷断点的方法不同，我们的技术具有不受起始材料量限制的优点。它还包括内部控制，以消除假阳性和假阴性。该技术还可以很容易地适用于既存在遗传缺陷又存在SNP的其他生物体，从而有助于这些其他模型中的基因发现。
Genetic deletions or deficiencies have been used for gene mapping and discovery in various organisms, ranging from the nematode Caenorhabditis elegans all the way to humans. One problem with large deletions is the determination of the location of their breakpoints. This is exacerbated in the case of complex deficiencies that delete a region of the genome, while retaining some of the intervening sequence. Previous methods, using genetic complementation or cytology were hampered by low marker density and were consequently not very precise at positioning the breakpoints of complex deficiencies. The identification of increasing numbers of Single Nucleotide Polymorphisms (SNPs) has resulted in the use of these as genetic markers, and consequently in their utilization for defining the breakpoints of deletions using molecular biology methods. Here, we show that SNPs can be used to help position the breakpoints of a complex deficiency in C. elegans. The technique uses a combination of genetic crosses and molecular biology to generate robust and highly reproducible results with strong internal controls when trying to determine the breakpoints of deficiencies. The combined use of this technique and standard genetic mapping allowed us to rapidly narrow down the region of interest in our attempts to clone a gene. Unlike previous methods used to locate deficiency breakpoints, our technique has the advantage of not being limited by the amount of starting material. It also incorporates internal controls to eliminate false positives and negatives. The technique can also easily be adapted for use in other organisms in which both genetic deficiencies and SNPs are available, thereby aiding gene discovery in these other models.
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