Department of Clinical Physiopathology, University of Florence, Viale Pieraccini 6, 50139 Florence, Italy.
Clin Chim Acta. 2011 May 12;412(11-12):901-5. doi: 10.1016/j.cca.2011.01.014. Epub 2011 Jan 22.
The BRAF gene has been identified as an oncogene in human cancer and the V600E mutation has been shown to be associated with clinico pathological features of primary invasive melanomas. As BRAF may be an attractive therapeutic target, it is crucial to have a sensitive method for detecting mutated DNA in biological samples. Our aim was to investigate COLD-PCR (co-amplification at lower denaturation temperature-PCR) as a new approach for the pre-analytical enrichment of the BRAFV600E variant in formalin fixed paraffin embedded (FFPE) melanoma tissues.
COLD-PCR was used to selectively amplify BRAFV600E minority alleles from mixtures of wild-type and mutated sequences, and from biological samples. The method shows higher specificity than other conventional PCR-based methods in detecting somatic mutations.
We used COLD-PCR to increase the theoretical sensitivity of three different post-PCR methods: sequencing, pyrosequencing and HRMA. The gain in sensitivity seems to be more evident for HRMA, which allows the detection of 3.1% mutated alleles. More than 20% of patients initially classified negative for BRAFV600E were found positive after COLD-PCR.
COLD-PCR was confirmed as a suitable method for the enrichment of mutated alleles, particularly for samples in which the percentage of tumor cells is very low.
BRAF 基因已被鉴定为人类癌症中的致癌基因，V600E 突变已被证明与原发性浸润性黑色素瘤的临床病理特征相关。由于 BRAF 可能是一个有吸引力的治疗靶点，因此拥有一种用于检测生物样本中突变 DNA 的敏感方法至关重要。我们的目的是研究 COLD-PCR（低温共扩增 PCR）作为一种新方法，用于在福尔马林固定石蜡包埋（FFPE）黑色素瘤组织中预分析富集 BRAFV600E 变体。
COLD-PCR 用于从野生型和突变序列的混合物以及从生物样本中选择性扩增 BRAFV600E 少数等位基因。该方法在检测体细胞突变方面比其他基于常规 PCR 的方法具有更高的特异性。
我们使用 COLD-PCR 提高了三种不同的 post-PCR 方法的理论灵敏度：测序、焦磷酸测序和 HRMA。对于 HRMA 而言，灵敏度的提高似乎更为明显，其允许检测到 3.1%的突变等位基因。超过 20%的最初被归类为 BRAFV600E 阴性的患者在经过 COLD-PCR 后被发现为阳性。
COLD-PCR 被确认为富集突变等位基因的合适方法，特别是对于肿瘤细胞百分比非常低的样本。
