Excess of non-parental bands in offspring from known primate pedigrees assayed using RAPD PCR.
Excess of non-parental bands in offspring from known primate pedigrees assayed using RAPD PCR.
复制标题
使用 RAPD PCR 检测已知灵长类谱系后代的非亲本条带过多。
DOI：
10.1093/nar/20.4.918
复制
发表时间：
1992
影响因子：
14.9
通讯作者：
Aquadro,CF
中科院分区：
文献类型：
--
作者：
Riedy,MF;Hamilton3rd,WJ;Aquadro,CF
随机扩增多态性DNA（RAPD）方法允许在聚合酶链反应中使用任意序列的单一引物检测DNA序列多态性（1）。我们曾试图使用这种技术来评估在一个队的chacma狒狒（Papio cynocephalus ursinus）的亲子关系。我们还检查了来自该物种和人类的已知谱系的个体。我们的研究结果表明，这种技术会导致一个不可接受的数量的非父母带内的系谱，从而提出了一个严重的问题，其使用的亲子关系分析。从Operon Ltd获得的20种不同的RAPD寡聚体用于初步筛选，选择5个最大的变量用于进一步分析：A16、A17、A18、A19和A20。共检测了18个野外采集的狒狒样本和24个家系个体，其中包括来自CEPH家系1468的10个狒狒和14个人。如（1）所述，使用5-10 ng基因组DNA，用0.4 mM MgCl 2进行PCR，并在琼脂糖凝胶上分离PCR产物。对照品在无Taq聚合酶或无模板DNA的情况下运行，未产生可见条带（图1）。在人和狒狒中，每个RAPD引物产生由3-18条大小在0.25 kb和6 kb之间的条带组成的扩增产物的独特模式（图1）。所有引物在一些子代中可重复地扩增产物，而在任一亲本中均未发现（即，非亲本条带）。我们研究了两个核心家庭的橄榄狒狒（Papio cynocephalus anubis），其中每一个都包括父母和三个后代（两个女性，一个男性）。每个家庭的父亲和母亲都是不相关的。在两个狒狒家系中，每个亲子组合的平均新带数为4.4，范围为1-9。同样，在CEPH家系中发现了高频率的非父母带，其中没有亲子关系的问题。在人类谱系中，每个亲本-后代组合的新条带的平均数量为2.7，范围为2-4。这些结果的解释包括PCR伪影和基因组突变。鉴于我们的质控结果和新条带的重复性，样品或试剂不太可能受到污染。复制过程中的聚合酶滑动、Taq聚合酶的非模板定向核苷酸添加以及体外重组体的扩增也可能产生人工产物条带（4）。基因组突变也可以产生新的条带，但要产生我们观察到的非亲本片段的数量，需要非常高的突变率（每代每条条带7-9%）。据报道，人类小卫星基因座D1 S7（5）的突变率高达每配子代每个基因座5%，但大多数基因座的突变率至少低一个数量级。我们在推测不相关的人类和狒狒中观察到的高平均条带共享概率（分别为62.8%和75.9%）与突变是新条带的唯一来源不一致。RAPD技术已被证明是有用的，在构建连锁图谱和检测遗传标记在各种生物（1-3）。然而，我们的研究结果，提出了严重的关注使用目前的RAPD技术进行亲子鉴定。无论是由于突变还是PCR伪影，非亲本条带的高频率出现使得这些遗传标记不适合用于亲子鉴定分析，因为它们将导致错误排除。有可能即将对这种方法进行修改，以消除我们遇到的问题。
The random amplified polymorphic DNA (RAPD) method allows the detection of DNA sequence polymorphisms using single primers of arbitrary sequence in the polymerase chain reaction (1). We have attempted to use this technique to assess paternity in a troop of chacma baboons (Papio cynocephalus ursinus). We have also examined individuals from known pedigrees of this species and humans. Our results demonstrate that this technique leads to an unacceptable number of non-parental bands within a pedigree, thus raising a serious concern regarding its use in paternity analysis. Twenty different RAPD oligomers obtained from Operon Ltd were used in the initial screening with the five most variable chosen for further analysis: A16, A17, A18, A19, and A20. A total of 18 field-collected baboon samples and 24 pedigree individuals, including10 baboons and 14 humans from CEPH pedigree 1468 were examined. PCR was carried out as described by (1) with 0.4 mM MgCl2 using 5-10 ng of genomic DNA and the PCR products separated on agarose gels. Controls were run with either no Taq Polymerase or with no template DNA and produced no visible bands (Figure 1). Each RAPD primer produced a distinct pattern of amplification products consisting of 3-18 bands ranging in size between 0.25 kb and 6 kb in both humans and baboons (Figure 1). All primers reproducibly amplified products in some offspring which were not found in either parent (ie, non-parental bands). We examined two nuclearfamilies of olive baboons (Papio cynocephalus anubis) each of which included both parents and threeoffspring (two females, one male). The sires and dams for each family were unrelated. In the two baboon pedigrees, the average number of novel bands per parent-offspring combination was 4.4 and ranged from 1-9 for the five primers. Similarly, a high frequency of non-parental bands was found in the CEPH pedigree for which there is no question of parentage. The average number of novel bands per parent-offspring combination in the human pedigree was 2.7 with a range of 2-4. Explanations for these results include PCR artifact and genomic mutation. Contamination of samples or reagents is unlikely given our control results and repeatability of novel bands. Polymerase slippage during replication, non-template directed addition of nucleotides by Taq polymerase and the amplification of in vitro recombinants may also generate artifactual product bands (4). Genomic mutation could also produce novel bands, but a very high rate of mutation (7-9% per band per generation) would be necessary to generate the number of non-parental fragments we observed. A mutation rate as high as 5% per locus per gamete generation has been reported for the human minisatellitelocusD1S7 (5) but most loci show mutation rates at least an order of magnitude lower. The high average band-sharing probabilities we observed for both presumably unrelated humans and baboons (62.8% and 75.9%, respectively) are not consistent with mutation being the sole source of novel bands. The RAPD technique has proven to be useful in constructing linkage maps and detecting genetic markers in a variety of organisms (1-3). Our results, however, raise serious concerns about the use of the current RAPD technique for paternity assessment. Whether due to mutation or PCR artifact, the high frequency of occurrence of non-parental bands make these genetic markers unsuitable for paternity analysis as they will lead to false exclusions. It is possible that modifications to this method may be forthcoming that will eliminate the problems we have encountered.