Hu J, van Eysden J, Quiros C F
Department of Vegetable Crops, University of California, Davis 95616, USA.
PCR Methods Appl. 1995 Jun;4(6):346-51. doi: 10.1101/gr.4.6.346.
Random amplified polymorphic DNA (RAPD) markers offer quick screening of different regions of the genome for genetic polymorphisms. The standard RAPD procedure uses a single 10-base-long random oligonucleotide as a primer to amplify short stretches of the genome by PCR. We modified the procedure by using two primers in each reaction in a Brassica napus mapping project. We found that the two-primer RAPD tends to amplify more and smaller fragments than the standard RAPD technique. These new bands were always amplified in the two-primer reactions, and Southern analysis revealed that they had no homology to the bands amplified in single-primer reactions involving the same primers. Furthermore, these new markers were not linked to markers amplified with the same primers in the standard RAPD reactions, suggesting that they were amplified from different genomic regions. The advantage of the two-primer RAPDs is that it allows more reactions to be carried out with a limited number of primers to generate more markers. Using a single primer, the number of reactions is equal to the number of primers (n), which in turn limits the total number of markers. When using two primers in all possible combinations, the total number of reactions increases to n x (n-1/2). This method could be useful in conjunction with bulked segregant analysis to develop high density maps of certain chromosomal regions. We used this approach to map a second marker linked to a gene governing low linolenic acid concentration in a B. napus F2 population.
随机扩增多态性DNA（RAPD）标记可快速筛选基因组的不同区域以寻找遗传多态性。标准的RAPD程序使用单个10个碱基长的随机寡核苷酸作为引物，通过聚合酶链反应（PCR）扩增基因组的短片段。在一个甘蓝型油菜作图项目中，我们对该程序进行了改进，即在每个反应中使用两个引物。我们发现，与标准RAPD技术相比，双引物RAPD倾向于扩增更多且更小的片段。这些新条带总是在双引物反应中被扩增出来，Southern分析表明它们与在涉及相同引物的单引物反应中扩增出的条带没有同源性。此外，这些新标记与标准RAPD反应中用相同引物扩增出的标记不连锁，这表明它们是从不同的基因组区域扩增出来的。双引物RAPD的优点在于，在引物数量有限的情况下，可以进行更多反应以产生更多标记。使用单个引物时，反应数量等于引物数量（n），这反过来限制了标记的总数。当以所有可能的组合使用两个引物时，反应总数增加到n×（n - 1/2）。该方法结合混合分离群体分析法，对于绘制某些染色体区域的高密度图谱可能会很有用。我们使用这种方法在一个甘蓝型油菜F2群体中定位了与控制低亚麻酸浓度的基因连锁的第二个标记。
