High resolution melting for mutation scanning of TP53 exons 5-8.
High resolution melting for mutation scanning of TP53 exons 5-8.
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高分辨率熔化用于TP53外显子5-8的突变扫描。
DOI：
10.1186/1471-2407-7-168
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发表时间：
2007-08-31
期刊：
影响因子：
3.8
通讯作者：
Dobrovic, Alexander
中科院分区：
文献类型：
--
作者：
Krypuy, Michael;Ahmed, Ahmed Ashour;Etemadmoghadam, Dariush;Hyland, Sarah J.;deFazio, Anna;Fox, Stephen B.;Brenton, James D.;Bowtell, David D.;Dobrovic, Alexander
p53通常在广泛的癌症中通过DNA结合结构域中的突变而失活。由于突变型p53经常影响对治疗的反应，因此有效和快速扫描TP53突变的方法可能具有临床价值。因此，我们评估了使用高分辨率熔解（HRM）作为肿瘤样本中TP53的快速突变扫描工具。我们设计了用于TP53外显子5至8的HRM突变扫描的PCR扩增子，并用来自已知突变的半合子或纯合子细胞系的DNA对其进行测试。我们使用正常野生型DNA中细胞系DNA的稀释液评估每个PCR扩增子的灵敏度。然后，我们对先前已测序TP53突变的卵巢肿瘤DNA样本进行了盲法评估，以评估HRM技术的敏感性和阳性预测值。我们还对TP53突变状态未知的乳腺肿瘤DNA样本进行了HRM分析。当外显子5扩增时，一个细胞系突变不容易观察到。由于外显子5含有多个解链结构域，我们将外显子分成两个扩增子用于进一步筛选。还将序列改变引入到一些引物中以改善扩增子的解链特性。在卵巢肿瘤DNA组的每个样本中观察到指示TP53突变的异常HRM曲线。HRM结果与测序结果的比较显示，每个突变都在正确的外显子中被HRM检测到。对于乳腺肿瘤组，我们通过HRM检测到7个异常熔解曲线，随后的测序证实了这些突变的存在，并且在预测的外显子中没有其他突变。HRM是一种简单快速扫描TP53突变的有效技术，可以显着减少TP53突变研究中所需的测序量。
p53 is commonly inactivated by mutations in the DNA-binding domain in a wide range of cancers. As mutant p53 often influences response to therapy, effective and rapid methods to scan for mutations in TP53 are likely to be of clinical value. We therefore evaluated the use of high resolution melting (HRM) as a rapid mutation scanning tool for TP53 in tumour samples. We designed PCR amplicons for HRM mutation scanning of TP53 exons 5 to 8 and tested them with DNA from cell lines hemizygous or homozygous for known mutations. We assessed the sensitivity of each PCR amplicon using dilutions of cell line DNA in normal wild-type DNA. We then performed a blinded assessment on ovarian tumour DNA samples that had been previously sequenced for mutations in TP53 to assess the sensitivity and positive predictive value of the HRM technique. We also performed HRM analysis on breast tumour DNA samples with unknown TP53 mutation status. One cell line mutation was not readily observed when exon 5 was amplified. As exon 5 contained multiple melting domains, we divided the exon into two amplicons for further screening. Sequence changes were also introduced into some of the primers to improve the melting characteristics of the amplicon. Aberrant HRM curves indicative of TP53 mutations were observed for each of the samples in the ovarian tumour DNA panel. Comparison of the HRM results with the sequencing results revealed that each mutation was detected by HRM in the correct exon. For the breast tumour panel, we detected seven aberrant melt profiles by HRM and subsequent sequencing confirmed the presence of these and no other mutations in the predicted exons. HRM is an effective technique for simple and rapid scanning of TP53 mutations that can markedly reduce the amount of sequencing required in mutational studies of TP53.
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影响因子：
4.7
作者：
Amikura, T;Sekine, M;Tanaka, K
通讯作者：
Tanaka, K
DOI：
10.1073/pnas.88.19.8405
发表时间：
1991-10-01
影响因子：
11.1
作者：
BORRESEN, AL;HOVIG, E;FRIEND, SH
通讯作者：
FRIEND, SH
影响因子：
5
作者：
Keller, G;Hartmann, A;Höfler, H
通讯作者：
Höfler, H
影响因子：
3.8
作者：
Krypuy M;Newnham GM;Thomas DM;Conron M;Dobrovic A
通讯作者：
Dobrovic A
DOI：
10.1111/j.1349-7006.1994.tb02937.x
发表时间：
1994-12-01
期刊：
JAPANESE JOURNAL OF CANCER RESEARCH
影响因子：
--
作者：
FUJITA, M;ENOMOTO, T;NOMURA, T
通讯作者：
NOMURA, T