,1,2Co-amplification at lower denaturation temperature-PCR: methodology and applications
Hui Liang1,2, Guojie Chen3, Yan Yu4, Likuan Xiong,1,2通讯作者:
第一联系人:
编委: 卢大儒
收稿日期:2017-11-6修回日期:2018-02-2网络出版日期:2018-03-20
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Received:2017-11-6Revised:2018-02-2Online:2018-03-20
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梁卉, 陈国杰, 于燕, 熊礼宽. 低温变性下复合PCR技术及其应用. 遗传[J], 2018, 40(3): 227-236 doi:10.16288/j.yczz.17-369
Hui Liang, Guojie Chen, Yan Yu, Likuan Xiong.
随着分子诊断技术的不断发展和疾病分子标志物的发现,基因突变检测越来越多地被应用于个体化医疗。突变可分为胚系突变和体细胞突变(嵌合突变),前者的细胞遗传缺陷呈均一性,后者的细胞遗传缺陷呈嵌合性,突变比例多变(0%~100%)[1]。当个体存在体细胞突变时,特别是当嵌合突变比例低于分型技术的检测阈值时,常因方法限制而出现假阴性。目前已有多种基于PCR原理的低频突变检测法,根据检测的位点是否已知,主要分为两类：(1)用于检测已知突变位点的方法,包括等位基因特异性扩增PCR(allele-specific amplification PCR, AS-PCR)、野生型阻滞扩增PCR(wild-type blocking PCR, WTB- PCR)和数字PCR等;(2)用于检测未知突变位点的方法,包括变性高效液相色谱法(denaturing high performance liquid chromatography, dHPLC)和基于反向PCR的限制性片段长度多态性(inverse PCR-based amplified restriction fragment length polymorphism, iFLP)等[2]。这些技术的应用范围、灵敏度和优缺点各有不同。
低温变性下复合PCR(co-amplification at lower denaturation temperature polymerase chain reaction, COLD-PCR)是一种在高丰度的野生型序列背景下选择性变性和扩增低丰度突变型序列的方法,主要目的是富集突变序列,使产物中低丰度的突变序列比例增高,高于后续分型技术的检测阈值。通过优化变性温度和反应条件,COLD-PCR可富集目的片段中所有位点的已知或未知的各类型突变,具有敏感、特异、精确、廉价和易操作等优点,可在不增加实验设备的前提下提高其他基因突变分型技术的敏感性[3]。
COLD-PCR在肿瘤、微生物、产前筛查和动植物等方面的研究日益增多,如COLD-PCR可用于体液样本或肿瘤组织混合样本中KRAS等基因热点突变的检测,进行肿瘤的早期诊断、病程和治疗监测、临床预后评估和靶向药物的选择等[4]。本文对COLD-PCR技术的原理、关键技术、衍生方法及其应用展开综述。
1 COLD-PCR原理
COLD-PCR是常规PCR的改进方法,最大特点是变性温度由高温变成一个较低的可选择性扩增突变型序列的精确的温度,定义为Tc(critical denaturation temperature)。基因突变可能导致序列的熔解温度(melting temperature, Tm)发生改变,在低于野生型序列Tm温度下变性,野生型序列的扩增效率低于突变型序列,从而实现富集突变型序列的目的[3]。Tc值的确定是COLD-PCR的关键。Tc值主要取决于两个因素：(1)突变(G:C>A:T)导致的目的片段Tm值降低：≥200 bp大小的序列发生点突变时,Tm值一般改变0.2~1.5℃[3]。(2)异源双链DNA分子的形成：突变型序列和野生型序列杂交形成异源双链DNA分子,由于不完全配对,异源双链DNA分子比同源双链DNA分子稳定性低,因此具有更低的Tm值;Tc温度下,异源双链DNA分子变性为单链,而同源双链DNA分子仍保持双链[3]。Tc值可通过实验确定：荧光定量PCR检测野生型序列Tm,然后进行一系列PCR,初始变性温度为Tm,并依次递减0.5℃,直到无产物出现,此时温度记为Tm°。在Tm′基础上,再依次递增0.1~0.2℃作为变性温度,继续进行一系列PCR反应,直到产物出现,此时的温度被定义为Tc。大量的实验发现,Tc = Tm-(1.0~2.0℃)。
COLD-PCR可分为两种形式,完全COLD- PCR(full COLD-PCR)和快速COLD-PCR(fast COLD- PCR)。Full COLD-PCR的基本过程为：第一步进行常规PCR反应,增加模板数量;第二步进入COLD- PCR阶段,富集突变型序列：95℃变性,70℃杂交形成异源双链DNA分子,Tc温度下变性,58~ 62℃(基于引物Tm)退火,72℃延伸,循环30~45次(图1A)。Full COLD-PCR形成异源双链DNA分子,可富集所有类型的突变,但扩增时间长。Fast COLD- PCR无高温变性阶段和70℃杂交形成异源双链DNA分子阶段,直接在Tc温度下变性和扩增(图1B)。Fast COLD-PCR不生成异源双链DNA分子,扩增时间短。Full COLD-PCR富集倍数低于fast COLD-PCR,但fast COLD-PCR仅能富集Tm值降低的突变[3]。然而,最新的研究发现将反应体系中的dGTP和dATP分别用dITP和dDTP代替,fast COLD-PCR也可富集Tm增加的突变[5]。因此,fast-COLD-PCR也适用于所有类型的突变。
图1
新窗口打开|下载原图ZIP|生成PPT图1Full COLD-PCR和fast COLD-PCR原理图
A：Full COLD-PCR技术原理。DNA95℃高温变性后,降低温度至70℃复性,杂交形成同源双链DNA分子和异源双链DNA分子。由于突变型序列罕见,大部分突变型序列与野生型序列错配形成异源双链DNA分子,具有较低的熔解温度。在Tc温度下变性时,异源双链DNA分子优先解链,随后进行扩增富集。B：Fast COLD-PCR技术原理。基本过程与常规PCR相似,仅将变性温度由高温95℃改为Tc。在Tc温度下,突变型序列由于Tm值低于野生型序列而优先变性和富集。
Fig. 1Schematic view of full COLD-PCR and fast COLD-PCR
结合COLD-PCR,基因分型技术的检测敏感性可提高10~100倍。常规PCR扩增后,Sanger测序、焦磷酸测序、高分辨熔解曲线(high-resolution melting, HRM)、数字PCR和第二代测序技术(next generation sequencing, NGS)的基因分型检测阈值分别为10%~30%,10%,5%~15%,0.1%和2%~5%;改用COLD-PCR富集后,分别提高到0.5%~3%,0.5%~ 1.0%[3],1%~2%[6],0.2%~1.2%[7]和0.02%~1%[8](表1)。为进一步增加检测敏感性,Li等[9]在fast COLD- PCR的基础上增加一轮nested fast COLD-PCR,结合Sanger测序后,可检测到0.01%的突变。但Kristensen等[10]对KRAS基因突变进行两轮相同的COLD-PCR扩增后,并未获得更高的检测敏感性,可能与第二轮COLD-PCR未使用巢式引物有关。Song等[6]在COLD-PCR/HRM中加入7%的DMSO可检测到0.2%~0.3%的TP53突变,与不加DMSO相比(1%~2%),敏感性提高近10倍。Hashida等[11]使用突变富集COLD-PCR/HRM检测到0.01%的EGFR基因T790M突变。该方法加入BstUⅠ酶,将野生型序列酶切成不能扩增富集的小片段,进一步提高了敏感性。
2 COLD-PCR衍生方法
COLD-PCR的最大挑战为Tc值要求非常精确,反应过程中温差不能超过±0.2℃。普通的PCR仪精密度不够,不能满足常规COLD-PCR的实验条件。因此,需要变温更快且精密度更高的变温气流式PCR仪。且Tc值还与COLD-PCR的反应体系和实验室环境有关,因此同一实验室在不同的时期即使进行同一片段富集时,也可能由于实验室环境和操作人员改变而需要重新确定Tc值。因此,研究人员开发了一系列对Tc值要求不高的COLD-PCR衍生方法。2.1 改良型完全富集COLD-PCR (improved and complete enrichment COLD-PCR, ice-COLD-PCR)
Ice-COLD-PCR是COLD-PCR的衍生方法,主要特点是加入非扩增的参考序列(reference sequence, RS),与突变序列杂交形成异源双链DNA分子。RS具有以下特征：(1)与野生型序列中的一条单链完全匹配;(2)不与引物结合;(3)3°端磷酸化确保RS不扩增。Tc温度下变性时,RS-突变型异源二聚体更容易解链,进行突变序列的类线性扩增。与full- COLD-PCR相比,虽然均形成异源双链DNA分子,但ice-COLD-PCR杂交时间仅为30 s,远低于full COLD-PCR的5~8 min,大大缩短了反应时间。Ice- COLD-PCR可富集所有类型的突变,富集程度略高于full COLD-PCR(3~5倍),略低于fast COLD-PCR(~10倍)。结合下游分型技术,ice-COLD-PCR检测阈值分别为1%(Sanger测序)和0.1%(焦磷酸测序)[12],比常规COLD-PCR敏感性提高10倍,该方法已商业化用于KRAS基因突变的检测(Transgenomic, Omaha, NE)。虽然,ice-COLD-PCR部分弥补了COLD-PCR的不足,但仍需要精密的Tc值,临床应用仍受到限制。Table 1
表1
表1 COLD-PCR的方法学比较
Table 1
| 常规PCR | Full COLD-PCR | Fast COLD-PCR | Ice-COLD-PCR | E-ice-COLD-PCR | TT-COLD-PCR | |
|---|---|---|---|---|---|---|
| 突变类型 | - | 所有类型 | Tm降低突变 | 所有类型 | 所有类型 | 所有类型 |
| 富集倍数 | - | 3~10倍 | 10~100倍 | 16倍 | ~16倍 | 6~17倍 |
| 时间 | - | 耗时 | 快速 | 较耗时 | 最快 | 最耗时 |
| RS/浓度 | - | - | - | 有(任意链 20 nmol/L ) | LNA(不能为GC突变链10 nmol/L) | - |
| Tc范围 | - | ±2℃ | ±2℃ | ±2℃ | 5~10℃ | 2.5~3℃ |
| 预扩增 | - | 需要 | 需要 | 需要 | 不需要 | 需要 |
| 杂交时间 | - | 2~8 min | - | 30 s | 30 s | 2~8 min |
| Sanger测序 | 10%~30% | 3% | 0.5% | 1% | 0.1% | 3% |
| 焦磷酸测序 | ~10% | 0.5%~1% | 0.1% | 0.01% | - | |
| 高分辨熔解曲线 (HRM) | 5%~10% | 1~2% | 2% | - | - | 3% |
| 数字PCR | 0.1 % | - | 0.2%~1.2% | - | - | - |
| 第二代测序技术 (NGS) | 2%~5% | - | 0.02%~0.2% | - | 0.01%~0.1% | |
新窗口打开|下载CSV
How-Kit等[4]在ice-COLD-PCR的基础上,将RS用类寡核苷酸衍生物锁核酸(locked nucleic acid, LNA)代替,建立了E-ice-COLD-PCR(enhanced-ice- COLD-PCR)。E-ice-COLD-PCR的优点是：(1)无需预扩增样本和省时,反应模板仅需25 ng DNA,结合焦磷酸测序可在3 h内完成基因分型;(2)无需精密的Tc值,Tc值跨度为5~10℃,实验操作难度小,可重复性好[4];(3)可进行批量扩增[13];(4)可实现多重COLD-PCR反应[14];(5)较廉价。E-ice-COLD-PCR阻滞剂的加入量(10 nmol/L)远小于ice-COLD-PCR (20 nmol/L)和WTB-PCR(1~100 μmol/L);(6)检测敏感性高,检测敏感性为0.01~0.05%(焦磷酸测序); (7)不重叠LNA对野生型序列的抑制作用更强[4]; (8)对DNA质量要求不高。E-ice-COLD-PCR适用于所有来源和类型的样本,如冰冻组织、福尔马林固定石蜡包埋(formalin-fixed paraffin-embedded, FFPE)样本和体液样本等[15]。
E-ice-COLD-PCR是一种简便、高效且相对廉价的方法,但也具有一定的局限性。当LNA-DNA单碱基错配发生在G-T错配时,Tm值并不发生明显的改变,因此,E-ice-COLD-PCR在LNA设计时需避免G-T突变单链。此外,E-ice-COLD-PCR对野生型序列的抑制作用部分达到100%,当无产物出 现时,不能确定是由扩增失败或抑制作用过强所导 致[4]。后续的研究还应该考虑是否可通过增加内参序列进行改进。
2.2 温度耐受性COLD-PCR(temperature-tolerant COLD-PCR, TT-COLD-PCR)
Castellanos-Rizaldos等[16]建立了TT-COLD- PCR法,在COLD-PCR基础上,Tc值变为横跨2~3℃的温度窗。TT-COLD-PCR是一种变性温度递增的梯度PCR,每个梯度递增0.3~0.5℃,扩增7~15个循环,共5~10个梯度,以保证反应中的序列均有一个合适的变性温度用于富集。该原理应用于COLD- PCR中,已发展为TT-fast-、TT-full-和TT-ice-COLD- PCR,相对于普通PCR,新方法的富集程度分别提高了8~17倍、6~8倍和12倍。进行多重扩增时,可通过加入DMSO降低Tm值,使得所有序列均位于一定的Tc范围内。但TT-COLD-PCR还存在缺陷：多对引物加入,易形成引物二聚体;TT-COLD-PCR循环次数多,可能超过PCR仪允许的最大循环数(115个循环);多次高温变性可能导致聚合酶失活等。此外,富集效率也低于其他COLD-PCR。虽然TT-COLD-PCR的Tc温度窗使得多重COLD-PCR成为可能,但是由于引物二聚体的形成,在进行多重COLD-PCR时,需要将不同的序列在独立的反应管中扩增,增加了反应操作的复杂性。为了改进上述缺陷,Castellanos-Rizaldos等[17]在油包水乳剂中进行TT-fast-eCOLD-PCR (TT-fast- COLD-PCR in emulsion)反应：将每个PCR反应体系(水相)和油相分别混匀涡旋形成油包水乳剂,然后合并到单管中进行多重TT-fast-eCOLD-PCR。该方法解决了引物二聚体导致的扩增失败。但是,需要3次PCR扩增,且每个序列的反应体系均要进行乳剂形成,操作过程极其繁琐且耗时,在临床上使用受限,仅适合特殊样本的处理。随后,该研究小组用修饰核苷酸dITP和dDTP代替dGTP和dATP后,可同时富集包括Tm增高的所有类型的突变。使用商业化试剂盒构建文库后,增加1或2轮的多重fast- TT-COLD-PCR,提高了NGS (0.01%~0.1%)和Sanger测序的检测阈值(0.1%~0.3%)[5]。该方法比其他COLD-PCR/NGS(0.02%~1%)的检测敏感性更高[8],也是实现多重COLD-PCR的方法之一。
3 COLD-PCR的应用
3.1 COLD-PCR与肿瘤
3.1.1 COLD-PCR与KRAS基因COLD-PCR多用于肿瘤特异性突变检测的研究。COLD-PCR可提高肿瘤样本中低频KRAS基因突变的检出率,常规方法检测到30%~40%的结直肠癌(colorectal cancer, CRC)患者携带KRAS基因突变,而COLD-PCR富集后,CRC患者中KRAS基因突变携带率可提高到50%[18,19],部分研究甚至高达80%以上[10]。KRAS基因突变与EGFR抑制剂的个体化治疗有关,因此COLD-PCR在肿瘤化疗药物的合理使用中具有指导意义[3]。
此外,由于COLD-PCR提高了低丰度突变检测的敏感性,该方法从技术层面上增加了样本来源的多样性,组织样本(如新鲜冰冻组织、FFPE)和体液样本(如循环肿瘤细胞、循环肿瘤DNA(circulating tumor DNA, ctDNA)、唾液和腹水等)均适用基因突变检测,可实现实体肿瘤的“液态活检”。Liu等[20]使用COLD-PCR在所有已证实的16例突变携带患者中,ctDNA中检出13例,肿瘤组织样本中检出12例,对同一患者的ctDNA和肿瘤组织分别检测KRAS基因突变,基因分型结果一致率为75%。由于外周血可重复获得,COLD-PCR可通过对ctDNA的多次检测实现病程和疗效的监控[21]。
COLD-PCR用于KRAS基因突变的分型检测阈值为0.01%~5%。Sanger测序法是基因分型的金标准,但常规PCR/Sanger测序法仅能检测高于10~30%的突变,而COLD-PCR/Sanger测序可检测到~5%突变[21]。相对于Sanger测序法,HRM法更为敏感,且省时,更适用于临床样本的常规检测：常规PCR/ HRM可检测到5%~10%的KRAS突变,而COLD- PCR/HRM可将检测敏感性提高到3%[22]。Pritchard等[19]使用fast COLD-PCR/HRM-cy5.5标记探针后,检测敏感性可达到1%。COLD-PCR/焦磷酸测序其检测敏感性提高到0.5%~1%,E-ice-COLD-PCR更是将检测敏感性提高到0.1~0.01%,优于目前推荐的ARMS方法(0.1%~1%)[4]。
3.1.2 COLD-PCR与异柠檬酸脱氢酶1(isocitrate dehydrogenase 1, IDH1)基因
胶质瘤活检组织中常混有大量的正常脑组织、脉管系统和浸润淋巴细胞,大大增加了肿瘤基因的检测难度。COLD-PCR可富集混杂于正常组织中的肿瘤特异性突变,不仅提高了检测敏感性,结合其他技术,还可以实现术中快速诊断。Pang等[23]利用COLD-PCR/荧光熔解曲线法(fluorescence melting curve analysis, FMCA)将IDH1基因的检测敏感性提高到1%。Boisselier等[24]比较了常规PCR/HRM、常规PCR/COLD-PCR/HRM和COLD-PCR/COLD-PCR/ HRM,发现胶质瘤特异性IDH1基因的检测敏感性分别为25%、2%和0.25%。虽然COLD-PCR/COLD- PCR/HRM具有更高的检测敏感性,但在临床应用 中,COLD-PCR/FMCA更为简单、省时,且可避免多次PCR导致的污染。基于COLD-PCR/FMCA的特点,Kanamori等[25]建立了术中快速检测IDH1和IDH2突变的方法,其结合了商业化冰冻快速切片DNA提取试剂盒(15 min)、fast COLD-PCR快速扩增(40~45 min)和FMCA检测技术(5 min),可在60~ 65 min内实现术中组织样本的快速分子病理检测。该术中快速检测方法的建立,将术中快速病理检测技术拓展到分子水平,将会极大推动术中病理学的发展。
先前的研究发现胶质瘤患者的ctDNA存在杂合性丢失和表观遗传学改变,可能对胶质瘤的预后评估具有一定的价值。由于血脑屏障作用,胶质瘤的ctDNA相对于其他部位的肿瘤如乳腺癌、直肠癌等的可检测性较低,因此需要高敏感性检测技术用于胶质瘤ctDNA的检测。Boisselier等[26]使用COLD- PCR结合数字PCR对不同程度的胶质瘤进行ctDNA检测,发现低级别胶质瘤和高级别胶质瘤患者的IDH1突变携带率分别为37.5%和70.6%,提示COLD- PCR对高级别胶质瘤具有较高的敏感性。虽然COLD-PCR不能解决所有胶质瘤的分子检测,但是对于拒绝病理活检的患者,以及需要在治疗前后进行多次检测的患者具有重要意义。尽管E-ice-COLD- PCR还未用于胶质瘤IDH1基因的检测,但其高的野生序列抑制作用和高的突变序列富集能力,可以预测,对于IDH1基因在胶质瘤中的“液体活检”中具有广泛的应用前景。
3.1.3 COLD-PCR与其他肿瘤相关基因
当肿瘤的病理形态难以区分时,COLD-PCR还可用于肿瘤的鉴别诊断。肌内黏液瘤是一种罕见的疾病,病理形态学上与低度粘液纤维肉瘤难以区分。Fast COLD-PCR/突变特异性限制性酶切(mutation- specific restriction enzyme digestion, MARED)发现GNAS1基因突变在肌内黏液瘤的携带率为60.71% (17/28),且在低度粘液纤维肉瘤样本和正常对照样本中并未发现突变,该研究提示,可用COLD-PCR检测GNAS1基因的突变情况来辅助临床医生鉴别诊断这两种疾病[27]。
COLD-PCR的高敏感性还可用于鉴定肿瘤相关基因。以往的研究认为先天性黑素细胞痣(congenital melanocytic nevus, CMN)与NRAS和BRAF基因突变均有关,不同的研究小组发现NRAS突变携带率不同(22%~80%)。Charbel等[28]使用E-ice-COLD-PCR方法确认了这2个基因在不同分型的CMN中的意义。在巨大(>20 cm)CMN和大(10~20 cm)CMN中,NRAS基因突变携带率高达94.7%,BRAF基因突变仅在1例患者中检测到;而在中-小CMN中,NRAS基因突变携带率为70%,BRAF基因突变携带率为30%。因此NRAS可能为CMN的主要致病基因,提示恶性黑色素瘤的风险增加。
此外,COLD-PCR还被用于其他肿瘤相关基因,如EGFR、p53、BRAF(黑色素瘤) [15]、NF2(神经纤维瘤病2型) [29]、H-ras(膀胱癌) [30]、c-KIT(犬肥大细胞肿瘤) [31]基因的分型研究中,均可提高检测敏感性,提示COLD-PCR可广泛应用于肿瘤相关基因的精准检测。
3.2 COLD-PCR与微生物感染
在感染性疾病中,耐药基因的检测对抗感染治疗有重要作用。由于携带耐药突变的菌种混杂于微生物群中,特别是在用药前比例较低,耐药株不易被检出。COLD-PCR在结核杆菌和乙肝病毒耐药突变的检测研究中,可将检测敏感性提高5~20倍。在进行COLD-PCR时,微生物在血浆中的浓度不能过低,如乙肝病毒耐药突变的检测,需要病毒数达到50 IU/mL(fast COLD-PCR)~100 IU/mL(full COLD-PCR),否则可能由于模板量太低导致检测失败[32]。单链探针反向杂交(line probe assay, LiPA)仅可检测已知的突变[33],而COLD-PCR还可检测未知突变,用于微生物耐药突变应用范围更广,但可能存在漏检(COLD-PCR法：148/215,LiPA法：155/215)。在微生物的混合感染中,当一种微生物处于绝对优势时,其他微生物很难被检测到。Takahashi等[34]使用改进的COLD-PCR(modified COLD-PCR, mCOLD- PCR)特异性抑制优势微生物片段的扩增,间接提高产物中劣势微生物的比例。结合梯度变性凝胶电泳(denaturing gradient gel electrophoresis, DGGE)可检测出1/10 000的劣势微生物。随后在发酵饮品中,使用mCOLD-PCR/DGGE可检测出浓度远低于优势酿酒酵母菌的劣势微生物(如假丝酵母菌、枝孢霉菌和汉森氏酵母菌),因此,COLD-PCR可帮助工作人员在一些致病菌浓度较低时发现食品污染问题,采取预防措施,避免致病菌的爆发,提高食品安全。
mCOLD-PCR结合NGS用于微生物群的研究,产物中优势微生物的丰度从80.8%降低到8.8%,有效避免了优势细菌的干扰,获得更为广泛的微生物群数据。鉴于该方法的优势,也可用于混合感染中低丰度基因型片段的富集和检测,提高多重感染时的检出率,相关研究还有待开发。
COLD-PCR应用于微生物的检测还存在一定的缺陷,由于微生物的基因组不稳定,扩增序列中的新发突变和多个突变混杂使得Tc值的确定存在巨大的挑战。此外,经过临床干预后,感染灶中的微生物的浓度降低,当低于COLD-PCR扩增的最低浓度时,也可能导致检测失败。但是由于该方法廉价、易操作和高敏感性,COLD-PCR仍可作为微生物基因型检测的有效方法[35]。
3.3 COLD-PCR与产前筛查
传统的绒毛膜取样和羊膜腔穿刺等侵入性产前诊断对母体和胎儿有一定的风险。胎儿游离DNA (cell-free fetal DNA, cfDNA)的发现为无创产前筛查与诊断提供了新思路,已用于染色体非整倍体和部分单基因病的检测[36]。cfDNA在母外周血中含量较低,仅占总游离DNA的3%~20%,COLD-PCR对低丰度突变的富集效能可提高母外周血中胎儿父源突变的检出率。COLD-PCR用于评估胎儿患病风险是一种廉价且有效的方法[36,37]。Full COLD-PCR/Sanger用于检测孕早期母血中胎儿β地中海贫血致病突变,与绒毛基因型结果一致[38]。Galbiati等[39]使用COLD-PCR在母血中检测到胎儿携带的TWIST 18 bp缺失突变,提示COLD-PCR对中等大小的缺失突变也有富集效力,拓展了应用范围。COLD-PCR/限制性酶切法用于母血中胎儿CYP21 p.Q318X突变的检测,获得明显的分型结果,提示其在基层医院具有推广前景[40]。Galbiati等[41]比较了Full-和fast- COLD-PCR对β地中海贫血和囊胞性纤维症的常见突变富集效率和检测敏感性,发现fast COLD-PCR虽然具有较高的富集效率,但存在一定的漏检(16%(5/31))。与肿瘤常见的突变(Tm降低)不同,引起遗传性疾病的致病突变多样,因此,full COLD-PCR可能更适用于无创产前筛查[38,41]。
随着二胎政策的开放和对优生优育的认识,产前筛查的需求量日益增加。COLD-PCR较高的敏感性、廉价、易操作、不需要复杂仪器等优点对于产前筛查项目的开展和基层医院的推广有重要的意义。但是,COLD-PCR应用于产前筛查的研究主要集中于小样本量和单个病例,研究采用横跨1℃或5℃的Tc窗,缺少必要的突变梯度丰度检测和常规方法学的比较,使用COLD-PCR进行无创产前筛查的检测阈值还不清楚,敏感度更高的E-ice-COLD- PCR尚待研究[40]。
3.4 COLD-PCR在其他方面的应用
重亚硫酸盐处理后,未甲基化的胞嘧啶(C)全部转化为尿嘧啶(U),使序列Tm值下降约5℃。因此,COLD-PCR可用于未甲基化DNA的富集扩增。Castellanos-Tizaldos等[42]用fast COLD-PCR结合甲基化特异性PCR(methylation-specific PCR, MS- PCR)检测O6-甲基鸟嘌呤-DNA甲基转移酶基因,发现fast COLD-MS-PCR对未甲基化DNA的检测敏感性为0.05%,而常规MS-PCR的检测敏感性为5%。COLD-PCR还被用于植物育种研究,Chen等[43]利用COLD-PCR/HRM提高了PpAG和PpTFL1基因突变的检测敏感性,对缩短植物诱变育种周期具有重要意义。
4 结语与展望
COLD-PCR是一种敏感、特异、精确、廉价和易操作的PCR扩增方法,可在“非均一性”基因型样本中富集低丰度的突变,提高突变的检出率。含有“非均一性”基因型的样本均可考虑使用COLD- PCR进行富集,如肿瘤的嵌合突变样本、感染灶样本和包含游离DNA的血浆样本等已用于COLD- PCR的研究。此外,COLD-PCR还被用于法医学[44],但目前还未有 COLD-PCR 用于线粒体 DNA(mitochondrial DNA, mtDNA)突变检测的研究。对于遗传与环境共同作用导致的线粒体疾病,低丰度mtDNA突变的检测对于早期避免环境暴露具有重要意义,可通过临床干预降低线粒体疾病的发病率[45]。虽然COLD-PCR在多个领域均有研究,但其临床应用目前仅限于KRAS基因热点突变检测。在我国COLD-PCR还未有相关试剂盒的开发,遗传性疾病父源致病突变的无创产前筛查和微生物耐药突变研究有待进一步深入。此外鉴于E-ice-COLD-PCR的优势,在肿瘤、微生物感染、无创产前筛查以及线粒体疾病检测具有更为广泛的应用前景。
参考文献 原文顺序
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URL [本文引用: 1]
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Abstract BACKGROUND: The ability to identify low-level somatic DNA mutations and minority alleles within an excess wild-type sample is becoming essential for characterizing early and posttreatment tumor status in cancer patients. Over the past 2 decades, much research has focused on improving the selectivity of PCR-based technologies for enhancing the detection of minority (mutant) alleles in clinical samples. Routine application in clinical and diagnostic settings requires that these techniques be accurate and cost-effective and require little effort to optimize, perform, and analyze. CONTENT: Enrichment methods typically segregate by their ability to enrich for, and detect, either known or unknown mutations. Although there are several robust approaches for detecting known mutations within a high background of wild-type DNA, there are few techniques capable of enriching and detecting low-level unknown mutations. One promising development is COLD-PCR (coamplification at lower denaturation temperature), which enables enrichment of PCR amplicons containing unknown mutations at any position, such that they can be subsequently sequenced to identify the exact nucleotide change. SUMMARY: This review summarizes technologies available for detecting minority DNA mutations, placing an emphasis on newer methods that facilitate the enrichment of unknown low-level DNA variants such that the mutation can subsequently be sequenced. The enrichment of minority alleles is imperative in clinical and diagnostic applications, especially in those related to cancer detection, and continued technology development is warranted.
URLPMID:18408729 [本文引用: 7]
Nature Medicine is the premier journal for biomedical research. Respected internationally for the quality of its papers on areas ranging from infectious disease to cancer and neurodegeneration, Nature Medicine aims to bridge the gap between basic research and medical advances and is consistently ranked the number one journal by the Institute of Scientific Investigation in the Medicine, Research and Experimental category.
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URLPMID:4281501 [本文引用: 2]
Abstract BACKGROUND: Multiplexed detection of low-level mutations presents a technical challenge for many technologies, including cancer gene panels used for targeted-resequencing. Analysis of mutations below approximately 2%-5% abundance in tumors with heterogeneity, samples with stromal contamination, or biofluids is problematic owing to increased noise from sequencing errors. Technologies that reduce noise via deep sequencing unavoidably reduce throughput and increase cost. Here we provide proof of principle that coamplification at lower denaturation temperature (COLD)-PCR technology enables multiplex low-level mutation detection in cancer gene panels while retaining throughput. METHODS: We have developed a multiplex temperature-tolerant COLD-PCR (fast-TT-COLD-PCR) approach that uses cancer gene panels developed for massively parallel sequencing. After multiplex preamplification from genomic DNA, we attach tails to all amplicons and perform fast-TT-COLD-PCR. This approach gradually increases denaturation temperatures in a step-wise fashion, such that all possible denaturation temperatures are encompassed. By introducing modified nucleotides, fast-COLD-PCR is adapted to enrich for melting temperature (Tm)-increasing mutations over all amplicons, in a single tube. Therefore, in separate reactions, both Tm-decreasing and Tm-increasing mutations are enriched. RESULTS: Using custom-made and commercial gene panels containing 8, 50, 190, or 16 000 amplicons, we demonstrate that fast-TT-COLD-PCR enriches mutations on all examined targets simultaneously. Incorporation of deoxyinosine triphosphate (dITP)/2,6-diaminopurine triphosphate (dDTP) in place of deoxyguanosine triphosphate (dGTP)/deoxyadenosine triphosphate (dATP) enables enrichment of Tm-increasing mutations. Serial dilution experiments demonstrate a limit of detection of approximately 0.01%-0.1% mutation abundance by use of Ion-Torrent and 0.1%-0.3% by use of Sanger sequencing. CONCLUSIONS: Fast-TT-COLD-PCR improves the limit of detection of cancer gene panels by enabling mutation enrichment in multiplex, single-tube reactions. This novel adaptation of COLD-PCR converts subclonal mutations to clonal, thereby facilitating detection and subsequent mutation sequencing. 2014 American Association for Clinical Chemistry.
Magsci [本文引用: 2]
Melting Temperature shift(Tm-shift)是一种新的基因分型方法, 主要通过在两条特异性引物5′端加入不同长度的GC序列, PCR扩增后根据熔解曲线中产物<em>Tm</em>值的差异来完成分型。文章建立了Tm-shift法对2 048份样品的29个SNP进行分型, 通过分型成功率、重复检测一致率、测序验证准确度综合评价分型效果。结果显示, 29个SNP中有27个可以采用本方法分型, 分型成功率为93.1%。测序验证准确性达到100%。3种基因型阳性标准对照重复检测一致率为100%; 100个随机样品重复检测, 重复性为97%。因此, Tm-shift基因分型法是一种成本低廉、准确灵敏、稳定可靠、通量灵活、操作简便的基因分型方法, 可在遗传学研究中推广应用。
Magsci [本文引用: 2]
Melting Temperature shift(Tm-shift)是一种新的基因分型方法, 主要通过在两条特异性引物5′端加入不同长度的GC序列, PCR扩增后根据熔解曲线中产物<em>Tm</em>值的差异来完成分型。文章建立了Tm-shift法对2 048份样品的29个SNP进行分型, 通过分型成功率、重复检测一致率、测序验证准确度综合评价分型效果。结果显示, 29个SNP中有27个可以采用本方法分型, 分型成功率为93.1%。测序验证准确性达到100%。3种基因型阳性标准对照重复检测一致率为100%; 100个随机样品重复检测, 重复性为97%。因此, Tm-shift基因分型法是一种成本低廉、准确灵敏、稳定可靠、通量灵活、操作简便的基因分型方法, 可在遗传学研究中推广应用。
URLPMID:25772705 [本文引用: 1]
The use of droplet digital PCR (ddPCR) for low-level DNA mutation detection in cancer, prenatal diagnosis, and infectious diseases is growing rapidly. However, although ddPCR has been implemented successfully for detection of rare mutations at pre-determined positions, no ddPCR adaptation for mutation scanning exists. Yet, frequently, clinically relevant mutations reside on multiple sequence positions in tumor suppressor genes or complex hotspot mutations in oncogenes. Here, we describe a combination of coamplification at lower denaturation temperature PCR (COLD-PCR) with ddPCR that enables digital mutation scanning within approximately 50-bp sections of a target amplicon. Two FAM/HEX-labeled hydrolysis probes matching the wild-type sequence are used during ddPCR. The ratio of FAM/HEX-positive droplets is constant when wild-type amplicons are amplified but deviates when mutations anywhere under the FAM or HEX probes are present. To enhance the change in FAM/HEX ratio, we employed COLD-PCR cycling conditions that enrich mutation-containing amplicons anywhere on the sequence. We validated COLD-ddPCR on multiple mutations in TP53 and in EGFR using serial mutation dilutions and cell-free circulating DNA samples, and demonstrate detection down to approximately 0.2% to 1.2% mutation abundance. COLD-ddPCR enables a simple, rapid, and robust two-fluorophore detection method for the identification of multiple mutations during ddPCR and potentially can identify unknown DNA variants present in the target sequence.
URLPMID:22194627 [本文引用: 2]
Despite widespread interest in next-generation sequencing (NGS), the adoption of personalized clinical genomics and mutation profiling of cancer specimens is lagging, in part because of technical limitations. Tumors are genetically heterogeneous and often contain normal/stromal cells, features that lead to low-abundance somatic mutations that generate ambiguous results or reside below NGS detection limits, thus hindering the clinical sensitivity/specificity standards of mutation calling. We applied COLD-PCR (coamplification at lower denaturation temperature PCR), a PCR methodology that selectively enriches variants, to improve the detection of unknown mutations before NGS-based amplicon resequencing. We used both COLD-PCR and conventional PCR (for comparison) to amplify serially diluted mutation-containing cell-line DNA diluted into wild-type DNA, as well as DNA from lung adenocarcinoma and colorectal cancer samples. After amplification of TP53 (tumor protein p53), KRAS (v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog), IDH1 [isocitrate dehydrogenase 1 (NADP(+)), soluble], and EGFR (epidermal growth factor receptor) gene regions, PCR products were pooled for library preparation, bar-coded, and sequenced on the Illumina HiSeq 2000. In agreement with recent findings, sequencing errors by conventional targeted-amplicon approaches dictated a mutation-detection limit of approximately 1%-2%. Conversely, COLD-PCR amplicons enriched mutations above the error-related noise, enabling reliable identification of mutation abundances of approximately 0.04%. Sequencing depth was not a large factor in the identification of COLD-PCR-enriched mutations. For the clinical samples, several missense mutations were not called with conventional amplicons, yet they were clearly detectable with COLD-PCR amplicons. Tumor heterogeneity for the TP53 gene was apparent. As cancer care shifts toward personalized intervention based on each patient's unique genetic abnormalities and tumor genome, we anticipate that COLD-PCR combined with NGS will elucidate the role of mutations in tumor progression, enabling NGS-based analysis of diverse clinical specimens within clinical practice.
URLPMID:19760750 [本文引用: 1]
Abstract Reliable identification of cancer-related mutations in TP53 is often problematic, as these mutations can be randomly distributed throughout numerous codons and their relative abundance in clinical samples can fall below the sensitivity limits of conventional sequencing. To ensure the highest sensitivity in mutation detection, we adapted the recently described coamplification at lower denaturation temperature-PCR (COLD-PCR) method to employ two consecutive rounds of COLD-PCR followed by Sanger sequencing. Using this highly sensitive approach we screened 48 nonmicrodissected lung adenocarcinoma samples for TP53 mutations. Twenty-four missense/frameshift TP53 mutations throughout exons 5 to 8 were identified in 23 out of 48 (48%) lung adenocarcinoma samples examined, including eight low-level mutations at an abundance of approximately 1 to 17%, most of which would have been missed using conventional methodologies. The identified alterations include two rare lung adenocarcinoma mutations, one of which is a "disruptive" mutation currently undocumented in the lung cancer mutation databases. A sample harboring a low-level mutation ( approximately 2% abundance) concurrently with a clonal mutation (80% abundance) revealed intratumoral TP53 mutation heterogeneity. The ability to identify and sequence low-level mutations in the absence of elaborate microdissection, via COLD-PCR-based Sanger sequencing, provides a platform for accurate mutation profiling in clinical specimens and the use of TP53 as a prognostic/predictive biomarker, evaluation of cancer risk, recurrence, and further understanding of cancer biology.
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URLPMID:24842519 [本文引用: 1]
The T790M mutation in the epidermal growth factor receptor (EGFR) gene is known to be associated with the acquired resistance of lung adenocarcinoma patients to EGFR-tyrosine kinase inhibitors (EGFR-TKIs). The minor T790M mutant allele is occasionally detected in EGFR-TKI-naive tumor samples, yet findings concerning the clinical impact of the minor T790M mutation vary among previous studies. In the present study, we assessed the clinical impact of the minor T790M mutation using a novel, highly sensitive assay combining high-resolution melting (HRM), mutant-enriched PCR and co-amplification at a lower denaturation temperature (COLD)-PCR. We determined the T790M mutational status in 146 surgically resected lung adenocarcinomas without a history of EGFR-TKI treatment using mutant-enriched COLD-HRM (MEC-HRM) and standard HRM assays. The sensitivities of the MEC-HRM and standard HRM assays for the detection of T790M-mutant alleles among wild-type alleles were 0.01 and 10%, respectively. Although the T790M mutation was not detected using a standard HRM assay, we identified 19 (13%) T790M mutations using the MEC-HRM assay and defined these 19 mutations as minor T790M mutations. The proportion of T790M alleles was <0.1% in 17 (84%) of the 19 samples. Multivariate analyses revealed that a minor T790M mutation was significantly associated with the presence of EGFR exon 19 deletions or the L858R mutation (both of which are drug-sensitive EGFR mutations) (P=0.04). In conclusion, the minor EGFR T790M mutations were present in 13% of EGFR-TKI-naive surgically resected lung adenocarcinomas and were associated with drug-sensitive EGFR mutations.
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[本文引用: 2]
URLPMID:22587896 [本文引用: 1]
Low-level mutations in clinical tumor samples often reside below mutation detection limits, thus leading to false negatives that may impact clinical diagnosis and patient management. COLD-PCR (coamplification at lower denaturation temperature PCR) is a technology that magnifies unknown mutations during PCR, thus enabling downstream mutation detection. However, a practical difficulty in applying COLD-PCR has been the requirement for strict control of the denaturation temperature for a given sequence, to within 卤0.3 C. This requirement precludes simultaneous mutation enrichment in sequences of substantially different melting temperature (T(m)) and limits the technique to a single sequence at a time. We present a temperature-tolerant (TT) approach (TT-COLD-PCR) that reduces this obstacle. We describe thermocycling programs featuring a gradual increase of the denaturation temperature during COLD-PCR. This approach enabled enrichment of mutations when the cycling achieves the appropriate critical denaturation temperature of each DNA amplicon that is being amplified. Validation was provided for KRAS (v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog) and TP53 (tumor protein p53) exons 6-9 by use of dilutions of mutated DNA, clinical cancer samples, and plasma-circulating DNA. A single thermocycling program with a denaturation-temperature window of 2.5-3.0 C enriches mutations in all DNA amplicons simultaneously, despite their different T(m)s. Mutation enrichments of 6-9-fold were obtained with TT-full-COLD-PCR. Higher mutation enrichments were obtained for the other 2 forms of COLD-PCR, fast-COLD-PCR, and ice-COLD-PCR. Low-level mutations in diverse amplicons with different T(m)s can be mutation enriched via TT-COLD-PCR provided that their T(m)s fall within the denaturation-temperature window applied during amplification. This approach enables simultaneous enrichment of mutations in several amplicons and increases significantly the versatility of COLD-PCR.
URLPMID:3516544 [本文引用: 1]
Multiplex detection of low-level mutant alleles in the presence of wild-type DNA would be useful for several fields of medicine including cancer, pre-natal diagnosis and infectious diseases. COLD-PCR is a recently developed method that enriches low-level mutations during PCR cycling, thus enhancing downstream detection without the need for special reagents or equipment. The approach relies on the differential denaturation of DNA strands which contain Tm-lowering mutations or mismatches, versus 'homo-duplex' wild-type DNA. Enabling multiplex-COLD-PCR that can enrich mutations in several amplicons simultaneously is desirable but technically difficult to accomplish. Here we describe the proof of principle of an emulsion-PCR based approach that demonstrates the feasibility of multiplexed-COLD-PCR within a single tube, using commercially available mutated cell lines. This method works best with short amplicons; therefore, it could potentially be used on highly fragmented samples obtained from biological material or FFPE specimens.Following a multiplex pre-amplification of TP53 exons from genomic DNA, emulsions which incorporate the multiplex product, PCR reagents and primers specific for a given TP53 exon are prepared. Emulsions with different TP53 targets are then combined in a single tube and a fast-COLD-PCR program that gradually ramps up the denaturation temperature over several PCR cycles is applied (temperature-tolerant, TT-fast-eCOLD-PCR). The range of denaturation temperatures applied encompasses the critical denaturation temperature (T(c)) corresponding to all the amplicons included in the reaction, resulting to a gradual enrichment of mutations within all amplicons encompassed by emulsion.Validation for TT-fast-eCOLD-PCR is provided for TP53 exons 6-9. Using dilutions of mutated cell-line into wild-type DNA, we demonstrate simultaneous mutation enrichment between 7 to 15-fold in all amplicons examined.TT-fast-eCOLD-PCR expands the versatility of COLD-PCR and enables high-throughput enrichment of low-level mutant alleles over multiple sequences in a single tube.
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[本文引用: 2]
URLPMID:23060932 [本文引用: 1]
Abstract Despite the improved ability to detect mutations in recent years, tissue specimens cannot always be procured in a clinical setting, particularly from patients with recurrence of tumors or metastasis. Therefore, the aim of this study was to investigate whether plasma is able to be used for mutation analysis instead of tissue specimens. We collected plasma from 62 patients with colorectal cancer (CRC) prior to treatment. DNA extracted from plasma and matched tumor tissues were obtained. Mutations in KRAS were amplified from the tissue specimens and sequenced by regular polymerase chain reaction (PCR) and co-amplification at lower denaturation temperature (COLD)-PCR. Plasma KRAS gene mutation on codon 12 (GGT>GAT) was detected using a nested COLD-PCR/TaqMan() -MGB probe. Mutations in plasma and matched tumors were compared. KRAS mutation on codon 12 (GGT>GAT) was found in 13 (21.0%) plasma specimens and 12 (19.4%) matched tumor tissues. The consistency of KRAS mutations between plasma and tumors was 75% (9/12), which indicated a high correlation between the mutations detected in plasma DNA and the mutations detected in the corresponding tumor DNA (P<0.001; correlation index, k=0.649). Notably, four (6.5%) patients with plasma DNA mutations had no detectable KRAS mutations in the corresponding primary tumors, and three (4.8%) patients with tumor DNA mutations had no detectable KRAS mutations in the corresponding plasma DNA samples. Thus, KRAS mutations in plasma DNA correlate with the mutation status in matched tumor tissues of patients with CRC. Our study provides evidence to suggest that plasma DNA may be used as a potential medium for KRAS mutation analysis in CRC using the COLD-PCR/TaqMan-MGB probe method.
URLPMID:22042676 [本文引用: 2]
Patients with metastatic colorectal carcinoma (mCRC) carrying activating mutations of the KRAS gene do not benefit from treatment with anti-epidermal growth factor receptor (EGFR) monoclonal antibodies. Therefore, KRAS mutation testing of mCRC patients is mandatory in the clinical setting for the choice of the most appropriate therapy. Co-amplification-at-lower denaturation-temperature PCR (COLD-PCR) is a novel modification of the conventional PCR method that selectively amplifies minority alleles from a mixture of wild-type and mutant sequences irrespective of the mutation type or position within the sequence. In this study, we compared the sensitivity of a COLD-PCR method with conventional PCR/sequencing and the real-time PCR-based Therascreen kit to detect KRAS mutations. By using dilutions of KRAS mutant DNA in wild-type DNA from colon cancer cell lines with known KRAS status, we found that Fast COLD-PCR is more sensitive than the conventional PCR method, showing a sensitivity of 2.5% in detecting G>A and G>T mutations. The detection of G>C transversions was not improved by either Fast COLD-PCR or Full COLD-PCR. We next analyzed by COLD-PCR, conventional PCR and Therascreen 52 formalin-fixed paraffin-embedded samples from mCRC patients. Among 36 samples with >30% tumor cells, 8 samples were negative by conventional PCR, Therascreen and Fast COLD-PCR; 20 mutations identified by conventional PCR were confirmed by Therascreen and Fast COLD-PCR; 8 cases undetermined by conventional PCR were all confirmed to carry KRAS G>A or G>T mutations by using either Therascreen or Fast COLD-PCR. Conventional PCR was able to detect only 2 KRAS mutations among 16 samples with C to A>T changes in the KRAS gene, which represent >90% of the mutations of this oncogene in CRC.
[本文引用: 1]
URLPMID:23370430 [本文引用: 1]
Point mutations in isocitrate dehydrogenase 1 (IDH1) have been identified in many gliomas. The detection of IDH1 mutations becomes challenging on suboptimal glioma biopsies when a limited number of tumor cells is available for analysis. Coamplification at lower denaturing-polymerase chain reaction (COLD-PCR) is a PCR technique that deliberately lowers the denaturing cycle temperature to selectively favor amplification of mutant alleles, allowing for the sensitive detection of low-abundance mutations. We developed a novel COLD-PCR assay on the LightCycler platform (Roche, Applied Science, Indianapolis, IN), using post-PCR fluorescent melting curve analysis (FMCA) for the detection of mutant IDH1 with a detection limit of 1%. Thirty-five WHO grade I to IV gliomas and 9 nonneoplastic brain and spinal cord biopsies were analyzed with this technique and the results were compared with the conventional real-time PCR and the Sanger sequencing analysis. COLD-PCR/FMCA was able to detect the most common IDH1 R132H mutation and rare mutation types including R132H, R132C, R132L, R132S, and R132G mutations. Twenty-five glioma cases were positive for IDH1 mutations by COLD-PCR/FMCA, and 23 gliomas were positive by the conventional real-time PCR and Sanger sequencing. A pilocytic astrocytoma (PA I) and a glioblastoma multiforme (GBM IV) showed low-abundance IDH1 mutations detected by COLD-PCR/FMCA. The remaining 10 glioma and 9 non-neoplastic samples were negative by all the 3 methods. In summary, we report a novel COLD-PCR/FMCA method that provides rapid and sensitive detection of IDH1 mutations in formalin-fixed paraffin-embedded tissue and can be used in the clinical setting to assess the small brain biopsies.
URLPMID:20886613 [本文引用: 1]
The p.Arg132His mutation of isocitrate dehydrogenase 1 (IDH1R132H) is a frequent alteration and a major prognostic marker in gliomas. However, direct sequencing of highly contaminated tumor samples may fail to detect this mutation. Our objective was to evaluated the sensitivity of a newly described amplification method, coamplification at lower temperature-PCR (COLD PCR), combined with high-resolution melting (HRM) for the detection of the IDH1R132H mutation. To this end, we used serial dilutions of mutant DNA with wild-type DNA. PCR-HRM assay detects IDH1R132H at an abundance of 25%, similar to the detection limit of direct Sanger sequencing. Introducing a run of COLD PCR allows the detection of 2% mutant DNA. Using two consecutive runs of COLD PCR, we detected 0.25% mutant DNA in a background of wild-type DNA, that mimics a tumor sample highly contaminated by normal DNA. We then analyzed 10 biopsies of tumor edges, considered free of tumor cells by histological analysis, and showed that immunohistochemistry of IDH1R132H was positive in three cases (30%), whereas double COLD PCR HRM was positive in the 10 cases studied (100%). In summary, COLD PCR HRM analysis is 100-fold more sensitive than Sanger sequencing, rendering this rapid and powerful strategy particularly useful for samples highly contaminated with normal tissue.Hum Mutat 31:1-6, 2010. 2010 Wiley-Liss, Inc.
[本文引用: 1]
[本文引用: 1]
URLPMID:19287459 [本文引用: 1]
Mutation detection plays an important role in diagnostic pathology, not only in providing a tissue diagnosis, but also in predicting response to antitumourigenic agents. However, mutation detection strategies are often hampered by masking of mutant alleles by wild-type sequences. Coamplification at lower denaturation temperature PCR (COLD-PCR) reportedly increases the proportion of rare variant sequences in a wild-type background by using PCR cycles in which the denaturation temperature is reduced to favour product with lower melt temperatures and heteroduplexes arising from minor variants. is a rare that occurs sporadically and less commonly in association with (Mazabraud's syndrome). results from activating mutations, and the same mutations have been identified in small numbers of . The aim of the study was primarily to establish whether COLD-PCR is more sensitive than conventional PCR; this was achieved by testing for mutations in using the two methodologies. Mutations were detected in 8 of 28 (29%) cases of using conventional PCR followed by mutation-specific restriction enzyme (PCR-MSRED) whereas 17 of 28 (61%) mutations were detected using COLD-PCR/MSRED. Mutations were detected in two cases where a diagnosis of low-grade had been favoured over . No mutations were detected in an additional 9 low-grade and 19 high-grade , and another 40 control samples. This study shows the power of COLD-PCR compared with conventional PCR in mutation detection, and shows that mutation detection increases diagnostic accuracy when distinguishing between and low-grade .
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URLPMID:24899029 [本文引用: 1]
Abstract Mutations in the reverse transcriptase (rt) region of the DNA polymerase gene are the primary cause of hepatitis B virus (HBV) drug resistance. In this study, we established a novel method that couples coamplification at lower denaturation temperature (COLD)-PCR and Sanger sequencing, and we applied it to the detection of known and unknown HBV mutations. Primers were designed based on the common mutations in the HBV rt sequence at positions 180 to 215. The critical denaturation temperature (Tc) was established as a denaturing temperature for both fast and full COLD-PCR procedures. For single mutations, when a melting temperature (Tm)-reducing mutation occurred (e.g., C-G → T-A), the sensitivities of fast and full COLD-PCR for mutant detection were 1% and 2%, respectively; when the mutation caused no change in Tm (e.g., C-G → G-C) or raised Tm (e.g., T-A → C-G), only full COLD-PCR improved the sensitivity for mutant detection (2%). For combination mutations, the sensitivities of both full and fast COLD-PCR were increased to 0.5%. The limits of detection for fast and full COLD-PCR were 50 IU/ml and 100 IU/ml, respectively. In 30 chronic hepatitis B (CHB) cases, no mutations were detected by conventional PCR, whereas 18 mutations were successfully detected by COLD-PCR, including low-prevalence mutations (<10%), as confirmed by ultradeep pyrosequencing. In conclusion, COLD-PCR provides a highly sensitive, simple, inexpensive, and practical tool for significantly improving amplification efficacy and detecting low-level mutations in clinical CHB cases. Copyright 08 2014, American Society for Microbiology. All Rights Reserved.
URLPMID:24951803 [本文引用: 1]
Nucleoside/nucleotide analogue for the treatment of chronic hepatitis B virus (HBV) infection is hampered by the emergence of drug resistance mutations. Conventional PCR-sequencing cannot detect minor variants of <20%. We developed a modified CO-amplification at Lower Denaturation temperature-PCR (COLD-PCR) method for the detection of HBV minority drug resistance mutations. The critical denaturation temperature for COLD-PCR was determined to be 78 C. Sensitivity of COLD-PCR sequencing was determined using serially-diluted plasmids containing mixed proportions of HBV reverse transcriptase (rt) wild-type and mutant sequences. Conventional PCR-sequencing detected mutations only if they existed in 25%, whereas COLD-PCR sequencing detected mutations when they existed in 5-10% of the viral population. The performance of COLD-PCR was compared to conventional PCR-sequencing and a line probe (LiPA) assay, using 215 samples obtained from 136 lamivudine- or telbivudine-treated patients with virological breakthrough. Among these 215 samples, drug resistance mutations were detected in 155 (72 %), 148 (69 %) and 113 samples (53 %) by LiPA, COLD-PCR, and conventional PCR-sequencing, respectively. Nineteen (9 %) samples had mutations detectable by COLD-PCR but not LiPA, while 26 (12 %) samples had mutations detectable by LiPA but not COLD-PCR, indicating both methods were comparable (P = 0.371). COLD-PCR was more sensitive than conventional PCR-sequencing: 35 (16 %) samples had mutations detectable by COLD-PCR but not conventional PCR-sequencing, while none had mutations detected by conventional PCR-sequencing but not COLD-PCR (P < 0.0001). COLD-PCR sequencing is a simple method which is comparable to LiPA and superior to conventional PCR-sequencing in detecting minor lamivudine/telbivudine resistance mutations.
URLPMID:24387855 [本文引用: 1]
The detection of low-abundant microorganism is difficult when in a sample in which a specific microorganism represents an overwhelming majority using polymerase chain reaction (PCR)-based methods. A modified CO -amplification at L ower D enaturation temperature PCR (mCOLD-PCR) method was developed to detect low-abundant microorganisms using a double-strand RNA probe to inhibit the amplification of the sequence of a major microorganism. Combining the mCOLD-PCR and downstream application (e.g., denaturing gradient gel electrophoresis (DGGE) and next-generation sequencing (NGS)), low-abundant microorganisms were detected more efficiently, even when a specific microorganism represents an overwhelming majority of the sample. We demonstrated that mCOLD-PCR-DGGE enabled us to detect Schizosaccharomyces pombe in a model sample coexisting with 10,000 times as many Saccharomyces cerevisiae . When mCOLD-PCR-DGGE was applied in the microbiota analysis of a fermenting white wine, Candida sp. and Cladosporium sp., which were not detected by conventional PCR, were detected. According to the NGS analysis after mCOLD-PCR of a fermenting red wine, the detection ratio of Saccharomyces was decreased dramatically, and the detection ratios of other microorganisms and the numbers of genera detected were increased compared with the conventional PCR. Thus, the application of mCOLD-PCR will reveal comprehensive microbiota of fermented foods, beverages, and so on.
URLPMID:26908265 [本文引用: 1]
react-text: 89 In the era of highly effective direct acting antiviral (DAA) drugs for the
URL [本文引用: 2]
URLPMID:3517603 [本文引用: 1]
The multiple endocrine neoplasia type 2A (MEN2A) is a monogenic disorder characterized by an autosomal dominant pattern of inheritance which is characterized by high risk of medullary thyroid carcinoma in all mutation carriers. Although this disorder is classified as a rare disease, the patients affected have a low life quality and a very expensive and continuous treatment. At present, MEN2A is diagnosed by gene sequencing after birth, thus trying to start an early treatment and by reduction of morbidity and mortality. We first evaluated the presence of MEN2A mutation (C634Y) in serum of 25 patients, previously diagnosed by sequencing in peripheral blood leucocytes, using HRM genotyping analysis. In a second step, we used a COLD-PCR approach followed by HRM genotyping analysis for non-invasive prenatal diagnosis of a pregnant woman carrying a fetus with a C634Y mutation. HRM analysis revealed differences in melting curve shapes that correlated with patients diagnosed for MEN2A by gene sequencing analysis with 100% accuracy. Moreover, the pregnant woman carrying the fetus with the C634Y mutation revealed a melting curve shape in agreement with the positive controls in the COLD-PCR study. The mutation was confirmed by sequencing of the COLD-PCR amplification product. In conclusion, we have established a HRM analysis in serum samples as a new primary diagnosis method suitable for the detection of C634Y mutations in MEN2A patients. Simultaneously, we have applied the increase of sensitivity of COLD-PCR assay approach combined with HRM analysis for the non-invasive prenatal diagnosis of C634Y fetal mutations using pregnant women serum.
URLPMID:20974797 [本文引用: 2]
Galbiati S, Brisci A, Lalatta F, Seia M, Makrigiorgos GM, Ferrari M, Cremonesi L.
URLPMID:24706433 [本文引用: 1]
Fish habitat in lakes is strongly constrained by water temperature and available dissolved oxygen (DO). Suitable fish habitat for three fish assemblages (cold-, cool-, and warm-water) in Minnesota (US) lakes was therefore determined from simulated daily water temperature and dissolved oxygen profiles. A total of 27 types of lakes were simulated under past (1961-1979) and a projected 2 x CO2 climate scenarios. The projected climate scenario was derived from the output of the Canadian Climate Center General Circulation Model for a doubling of atmospheric CO2. A verified, process-oriented, unsteady and one-dimensional (vertical) year-round lake water quality model (MINLAKE96) was used for the temperature and DO simulations, which were run in a continuous mode over 19 years. Water temperature and DO criteria for survival and good-growth of each fish guild were provided by the United States Environmental Protection Agency. Simulated suitable fish habitats were compared with fish observations in 3002 Minnesota lakes. Winterkill was simulated to occur in shallow eutrophic and mesotrophic lakes under past climate conditions, and predicted to disappear under the projected 2 x CO2 climate scenario due to a shortening of the ice cover period. Sensitivity of the simulated winterkill to three DO survival limits was analyzed. A lower DO (less than 0.5 mg/l) limit for winterkill produced better agreement with a fish observation database than higher limits. Dependence of the simulated good-growth habitat areas (GGHA) and volumes (GGHV) on the geometry (surface area and maximum depth) and trophic state of a lake was also examined. Fish habitat parameters were found to depend more strongly on geometry and less on trophic state. Climate change is projected to increase GGHA in seasonally stratified (medium-depth and deep) lakes on the average by 50 and 115% for cool-water and warm-water fish guilds, respectively. It is also projected that cold-water fish species will have a small percentage loss in weakly stratified (medium-depth) lakes, but a small percentage gain of GGHA in deep, strongly stratified lakes. (C) 1999 Elsevier Science B.V. All rights reserved.
URLPMID:21163141 [本文引用: 2]
URLPMID:26912453 [本文引用: 2]
Abstract BACKGROUND: Until now, non-invasive prenatal diagnosis of genetic diseases found only limited routine applications. In autosomal recessive diseases, it can be used to determine the carrier status of the fetus through the detection of a paternally inherited disease allele in cases where maternal and paternal mutated alleles differ. METHODS: Conditions for non-invasive identification of fetal paternally inherited mutations in maternal plasma were developed by two independent approaches: coamplification at lower denaturation temperature-PCR (COLD-PCR) and highly sensitive microarrays. Assays were designed for identifying 14 mutations, 7 causing thalassaemia and 7 cystic fibrosis. RESULTS: In total, 87 non-invasive prenatal diagnoses were performed by COLD-PCR in 75 couples at risk for thalassaemia and 12 for cystic fibrosis. First, to identify the more appropriate methodology for the analysis of minority mutated fetal alleles in maternal plasma, both fast and full COLD-PCR protocols were developed for the most common Italian -thalassaemia Cd39 and IVSI.110 mutations. In 5 out of 31 samples, no enrichment was obtained with the fast protocol, while full COLD-PCR provided the correct fetal genotypes. Thus, full COLD-PCR protocols were developed for all the remaining mutations and all analyses confirmed the fetal genotypes obtained by invasive prenatal diagnosis. Microarray analysis was performed on 40 samples from 28 couples at risk for thalassaemia and 12 for cystic fibrosis. Results were in complete concordance with those obtained by both COLD-PCR and invasive procedures. CONCLUSIONS: COLD-PCR and microarray approaches are not expensive, simple to handle, fast and can be easily set up in specialised clinical laboratories where prenatal diagnosis is routinely performed. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/
URLPMID:3984089 [本文引用: 1]
Aberrant hypo-methylation of DNA is evident in a range of human diseases including cancer and diabetes. Development of sensitive assays capable of detecting traces of un-methylated DNA within methylated samples can be useful in several situations. Here we describe a new approach, fast-COLD-MS-PCR, which amplifies preferentially un-methylated DNA sequences. By employing an appropriate denaturation temperature during PCR of bi-sulfite converted DNA, fast-COLD-MS-PCR enriches un-methylated DNA and enables differential melting analysis or bisulfite sequencing. Using methylation on the MGMT gene promoter as a model, it is shown that serial dilutions of controlled methylation samples lead to the reliable sequencing of un-methylated sequences down to 0.05% un-methylated-to-methylated DNA. Screening of clinical glioma tumor and infant blood samples demonstrated that the degree of enrichment of un-methylated over methylated DNA can be modulated by the choice of denaturation temperature, providing a convenient method for analysis of partially methylated DNA or for revealing and sequencing traces of un-methylated DNA. Fast-COLD-MS-PCR can be useful for the detection of loss of methylation/imprinting in cancer, diabetes or diet-related methylation changes.
URLPMID:3120741 [本文引用: 1]
Background Mutation scanning technology has been used to develop crop species with improved traits. Modifications that improve screening throughput and sensitivity would facilitate the targeted mutation breeding of crops. Technical innovations for high-resolution melting (HRM) analysis are enabling the clinic-based screening for human disease gene polymorphism. We examined the application of two HRM modifications, COLD-PCR and QMC-PCR, to the mutation scanning of genes in peach, Prunus persica . The targeted genes were the putative floral regulators PpAGAMOUS and PpTERMINAL FLOWER I . Results HRM analysis of PpAG and PpTFL1 coding regions in 36 peach cultivars found one polymorphic site in each gene. PpTFL1 and PpAG SNPs were used to examine approaches to increase HRM throughput. Cultivars with SNPs could be reliably detected in pools of twelve genotypes. COLD-PCR was found to increase the sensitivity of HRM analysis of pooled samples, but worked best with small amplicons. Examination of QMC-PCR demonstrated that primary PCR products for further analysis could be produced from variable levels of genomic DNA. Conclusions Natural SNPs in exons of target peach genes were discovered by HRM analysis of cultivars from a southeastern US breeding program. For detecting natural or induced SNPs in larger populations, HRM efficiency can be improved by increasing sample pooling and template production through approaches such as COLD-PCR and QMC-PCR. Technical advances developed to improve clinical diagnostics can play a role in the targeted mutation breeding of crops.
[D].
URL [本文引用: 1]
背景来自两个或两个以上个体的混合检材是法医物证检验中经常遇到的疑难检材类型之一。随着高灵敏检测技术、高多态性和性连锁等特殊遗传标记的普及应用,从混合检材中获取证据信息的能力不断提高。但即使对于经验丰富的专家来说,实际案件中一些混合检材的检测和结果解释仍然是一个巨大的挑战。如何对组份含量差异较大的混合检材中的少量成份进行分型,是当前法医DNA分析的难题之一。 目的建立人类Y染色体M175插入/缺失基因座的COLD-PCR(co-ampLification at lower denaturation temperature PCR)技术,比较COLD-PCR与常规PCR对不同比例混合样品的扩增分型效果,初步探讨COLD-PCR用于法医混合检材中低水平等位基因富集扩增分型的可行性。 方法首先建立人Y-M175基因座的常规PCR-聚丙烯酰胺凝胶电泳(PCR-PAGE)银染法分型技术,调查103名河南汉族男性无关志愿者的基因型。用蛋白酶K消化-酚/氯仿法提取插入和缺失型个体的基因组DNA,按不同比例混合制备成混合样品。用梯度保温异源双链消减法测定M175大扩增子(171/166bp)的临界变性温度(critical denaturation temperature,Tc),高分辨率熔解率曲线法(high resolution melting analysis,HRM)测定M175短扩增子(91/86bp)的Tc值。以Tc值为基础,通过在常规PCR的变性和退火之间增加扩增产物杂交和异源双链变性两个步骤,分别建立和优化基于PAGE-银染法(大扩增子)和实时定量法(小扩增子)的COLD-PCR技术,比较常规PCR和COLD-PCR对不同比例混合样品的扩增分型效果。 结果河南汉族Y-M175基因座插入和缺失型的频率分别为0.1650、0.8350。M175大扩增子的Tc值为76.4℃,小扩增子的Tc值为78.4℃,优化后采用的COLD-PCR异源双链变性温度分别为76.4和78.2℃。大扩增子PAGE-银染法,常规PCR可以有效分型的混合样品的组分比例为5∶1,而COLD-PCR至少可以达到20∶1。小扩增子实时定量法,常规PCR可以有效分型的混合样品的组分比例为16∶1,而COLD-PCR至少可以达到64∶1。 结论使用与常规PCR相同的设备和试剂, COLD-PCR能实现混合样品中微量等位基因的富集扩增,扩大可分型混合检材的范围,改进分型效果,在混合检材的法医DNA分型中有潜在应用价值。
URL [本文引用: 1]
背景来自两个或两个以上个体的混合检材是法医物证检验中经常遇到的疑难检材类型之一。随着高灵敏检测技术、高多态性和性连锁等特殊遗传标记的普及应用,从混合检材中获取证据信息的能力不断提高。但即使对于经验丰富的专家来说,实际案件中一些混合检材的检测和结果解释仍然是一个巨大的挑战。如何对组份含量差异较大的混合检材中的少量成份进行分型,是当前法医DNA分析的难题之一。 目的建立人类Y染色体M175插入/缺失基因座的COLD-PCR(co-ampLification at lower denaturation temperature PCR)技术,比较COLD-PCR与常规PCR对不同比例混合样品的扩增分型效果,初步探讨COLD-PCR用于法医混合检材中低水平等位基因富集扩增分型的可行性。 方法首先建立人Y-M175基因座的常规PCR-聚丙烯酰胺凝胶电泳(PCR-PAGE)银染法分型技术,调查103名河南汉族男性无关志愿者的基因型。用蛋白酶K消化-酚/氯仿法提取插入和缺失型个体的基因组DNA,按不同比例混合制备成混合样品。用梯度保温异源双链消减法测定M175大扩增子(171/166bp)的临界变性温度(critical denaturation temperature,Tc),高分辨率熔解率曲线法(high resolution melting analysis,HRM)测定M175短扩增子(91/86bp)的Tc值。以Tc值为基础,通过在常规PCR的变性和退火之间增加扩增产物杂交和异源双链变性两个步骤,分别建立和优化基于PAGE-银染法(大扩增子)和实时定量法(小扩增子)的COLD-PCR技术,比较常规PCR和COLD-PCR对不同比例混合样品的扩增分型效果。 结果河南汉族Y-M175基因座插入和缺失型的频率分别为0.1650、0.8350。M175大扩增子的Tc值为76.4℃,小扩增子的Tc值为78.4℃,优化后采用的COLD-PCR异源双链变性温度分别为76.4和78.2℃。大扩增子PAGE-银染法,常规PCR可以有效分型的混合样品的组分比例为5∶1,而COLD-PCR至少可以达到20∶1。小扩增子实时定量法,常规PCR可以有效分型的混合样品的组分比例为16∶1,而COLD-PCR至少可以达到64∶1。 结论使用与常规PCR相同的设备和试剂, COLD-PCR能实现混合样品中微量等位基因的富集扩增,扩大可分型混合检材的范围,改进分型效果,在混合检材的法医DNA分型中有潜在应用价值。
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线粒体ND6基因(<em>MT-ND6</em>)上的m.14484T>C突变是Leber’s遗传性视神经病变(Leber’s hereditary optic neuropathy, LHON)的一个原发性突变, 但该突变自身不足以产生视力损伤。为研究线粒体单体型对携带该突变人群LHON发病的影响, 文章对1 177例中国汉族LHON患者MT-ND6基因进行了全面系统的筛查, 共筛查到67例患者携带m.14484T>C同质性突变, 在该研究群体中所占比例为5.7%。携带m.14484T>C突变的51例家系LHON的外显率从5.6%~100.0%不等, 平均外显率为21.5%。对家系中51例先证者线粒体全基因组进行分析, 各表现为不同的多态性, 分别属于18个东亚线粒体单体型。其中单体型A和单体型F在病例组频率均明显低于106例对照组。另外, 单体型M10a在病例组中占9.8%, 在对照组中未被发现, 进一步发现该单体型家系LHON的平均外显率(46.13%)显著高于其他单体型家系的平均外显率, 提示线粒体单体型M10a可能增加视力损伤的风险。
URLMagsci [本文引用: 1]
线粒体ND6基因(<em>MT-ND6</em>)上的m.14484T>C突变是Leber’s遗传性视神经病变(Leber’s hereditary optic neuropathy, LHON)的一个原发性突变, 但该突变自身不足以产生视力损伤。为研究线粒体单体型对携带该突变人群LHON发病的影响, 文章对1 177例中国汉族LHON患者MT-ND6基因进行了全面系统的筛查, 共筛查到67例患者携带m.14484T>C同质性突变, 在该研究群体中所占比例为5.7%。携带m.14484T>C突变的51例家系LHON的外显率从5.6%~100.0%不等, 平均外显率为21.5%。对家系中51例先证者线粒体全基因组进行分析, 各表现为不同的多态性, 分别属于18个东亚线粒体单体型。其中单体型A和单体型F在病例组频率均明显低于106例对照组。另外, 单体型M10a在病例组中占9.8%, 在对照组中未被发现, 进一步发现该单体型家系LHON的平均外显率(46.13%)显著高于其他单体型家系的平均外显率, 提示线粒体单体型M10a可能增加视力损伤的风险。
