來(lái)自加州大學(xué)San Diego分校Moores癌癥中心的科學(xué)家們表示,，他們找到了一種診斷癌癥新技術(shù),，該技術(shù)可以在癌癥發(fā)展的最早期也就是只有數(shù)個(gè)癌細(xì)胞的階段就發(fā)現(xiàn)它們,。而目前最好的方法也只能在腫瘤發(fā)展到包含約100萬(wàn)個(gè)細(xì)胞時(shí)作出診斷,。
科學(xué)家在4月18日的在線《PLoS ONE》上的文章中描述了他們的一系列驗(yàn)證試驗(yàn),。他們可以在99.9%的正常DNA中尋找并放大微量導(dǎo)致癌癥的DNA,。目前的方法無(wú)法用于臨床設(shè)備,，因?yàn)樗鼈冃枰鄬?duì)較純的癌細(xì)胞群,，這對(duì)臨床樣品而言很困難。
Moores中心主任Dennis A. Carson說(shuō)：“我們找到了在很早期發(fā)現(xiàn)各種由于DNA損傷引起的癌癥的新技術(shù),。目前實(shí)驗(yàn)室正在與工程師合作,，以研制用于臨床的設(shè)備，這將廣泛用于病人,。”
Carson表示,，盡管還需要數(shù)年時(shí)間進(jìn)行臨床測(cè)試，但是最終人們將通過(guò)簡(jiǎn)單的樣本——例如血液,、尿液等分析癌細(xì)胞的DNA標(biāo)記,。此外醫(yī)生還可以簡(jiǎn)便且低成本的監(jiān)測(cè)病人狀態(tài)。一旦治療起效,，變異DNA將不存在,，病人也就被治愈了。
這一被稱為PAMP的技術(shù)基于一種酶反應(yīng),，它發(fā)生在一片DNA被刪除或者異常的和另一DNA結(jié)合時(shí),。而變異的具體位置并不重要，該方法將從正常DNA中探測(cè)出任何變異的片斷,，然后將這些變異分子放大,。
當(dāng)癌細(xì)胞變異時(shí)，通常會(huì)使兩片應(yīng)該分離的DNA結(jié)合起來(lái),。而酶反應(yīng)就在此時(shí)起作用,，這一技術(shù)能放大變異DNA，然后利用微陣列技術(shù)確定具體的變異,。
原文鏈接：http://www.physorg.com/news96302531.html
譯自：physorg.com
原始出處:
A Novel Approach for Determining Cancer Genomic Breakpoints in the Presence of Normal DNA
Yu-Tsueng Liu*, Dennis A. Carson
Moores UCSD Cancer Center, University of California San Diego, La Jolla, California, United States of America
CDKN2A (encodes p16INK4A and p14ARF) deletion, which results in both Rb and p53 inactivation, is the most common chromosomal anomaly in human cancers. To precisely map the deletion breakpoints is important to understanding the molecular mechanism of genomic rearrangement and may also be useful for clinical applications. However, current methods for determining the breakpoint are either of low resolution or require the isolation of relatively pure cancer cells, which can be difficult for clinical samples that are typically contaminated with various amounts of normal host cells. To overcome this hurdle, we have developed a novel approach, designated Primer Approximation Multiplex PCR (PAMP), for enriching breakpoint sequences followed by genomic tiling array hybridization to locate the breakpoints. In a series of proof-of-concept experiments, we were able to identify cancer-derived CDKN2A genomic breakpoints when more than 99.9% of wild type genome was present in a model system. This design can be scaled up with bioinformatics support and can be applied to validate other candidate cancer-associated loci that are revealed by other more systemic but lower throughput assays.
Introduction
Tumors evolve through the continuous accumulation and selection of randomly mutated genes. While sets of advantageous mutations are selected in tumors, neutral or even slightly detrimental mutations may also occur due to genomic instability and genetic drift. Recently, much effort has been expended to identify in primary human cancers point mutations in the exons of cancer-related genes. However, systemic mapping of genomic DNA rearrangements has lagged behind, due to technical difficulties in detecting smaller deletions, tumor heterogeneity, and the necessity to purify malignant from normal cells [1]. Historically, such work was done by time consuming and labor intensive genetics and molecular cloning on established cancer cell lines [2], [3], [4]. One of the most striking examples is the homozygous deletion of the CDKN2A (INK4A/ARF) tumor suppressor locus, which was discovered in this and other laboratories [3], [4], [5], [6], [7], [8]. The CDKN2A deletions occur early during tumor development [9], [10], [11]. The p16INK4a (one of the CDKN2A products [12]) protein constrains cell cycle progression by the Rb pathway and may be responsible for the decline in the replicative potential of stem cells during aging [13]. The p14ARF (the other alternative reading frame of CDKN2A [14]) gene product regulates the expression of MDM2, the turnover of p53, and thereby controls the cellular response to stress (reviewed in [6], [7], [8], [15], [16], [17]). Because the Rb and p53 pathways are central to cancer gate-keeping and caretaking [18], [19], strong selection pressures exist for the disruption of the entire CDKN2A gene segment on both chromosomes. Few other deletions are as well characterized, although it is expected that more will be found when more data from array based comparative genomic hybridization (array-CGH) are reported and also through The Cancer Genome Atlas (TCGA) project [20], [21], [22], [23], [24]. It will be important to validate the relevance of those genomic rearrangements to cancer development since many of the genomic structural changes may be simply due to genome instability in cancer. Large scale studies with clinical samples will be the most reliable confirmation.
While point mutations and very small insertions or deletions in genomic DNA can be detected by exon re-sequencing, it can be more difficult to detect gene dosage changes of larger genomic fragments, especially deletions [1]. Current established techniques for deletion mapping, including Southern blotting [25], fluorescent in situ hybridization (FISH) [26], quantitative PCR [26], [27], [28], [29], [30], and array-CGH [31] rely on the absence of a detectable wild type signal [1]. This is problematic when a significant number of normal cells are present in a tumor sample. Array-CGH has the potential to analyze alterations of DNA copy number on a genome-wide scale with relatively high resolution, depending on whether BACs, PCR products or oligonucleotides are used for the array elements. However, these techniques often fail where there is a heterogeneous cell population or samples of poor quality [31]. FISH is less vulnerable to the presence of heterogeneous cell populations, but has relatively low resolution and is difficult to scale up. Except for FISH, the other techniques mentioned are not practical for mapping genomic translocations and inversions. End-sequencing profiling was developed to address this issue but the approach was costly and hard to scale up [32]. Therefore, there is a need to develop a scalable approach for detecting such genomic structural changes in solid tumors where heterogeneous cell populations are present.
Here we report a novel approach, designated as Primer Approximation Multiplex PCR (PAMP), to enrich small amounts of deleted genomic DNA sequences in the presence of wild type DNA. The genomic locations of the enriched sequences are subsequently decoded by a genomic tiling array and confirmed by sequencing.
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