降落PCR及克隆测序法检测DNA甲基化的实验研究

目的 探索优化PCR及测序法检测DNA甲基化的实验条件,检测肺癌hsa-mir-34b(mir-34b)基因启动子DNA甲基化谱。方法 肺癌细胞株A549 DNA进行亚硫酸盐处理后,设计引物扩增mir-34b基因启动子序列,使用普通PCR、巢式PCR和降落(Touchdown)PCR方法以及不同DNA聚合酶进行扩增,甲基化谱检测采用PCR直接测序和克隆后测序两种方法。结果 降落 PCR与巢式PCR方法相结合优于普通PCR及单独的巢式PCR或降落 PCR。HotStart酶优于普通Taq酶及Ex Taq酶。直接PCR测序法的测序图普遍出现套峰难以判读,PCR产物克隆后测序可见清晰的测序峰,甲基化位点可明确判读,并可以结合测序克隆数目提高甲基化检测敏感度。结论 对于重亚硫酸盐处理的DNA甲基化状态检测,推荐使用HotStart酶、降落 PCR与巢式PCR相结合、克隆测序法检测基因甲基化谱,序列图明确且敏感度高。     

肺癌线粒体DNA突变的研究

背景与目的：近年来已经在多种肿瘤中发现了线粒体DNA（mitochondrial DNA,mtDNA)突变,但其意义尚不明确。本研究通过检测肺癌组织中的mtDNA突变,探讨mtDNA突变在人肺癌发生中的作用。方法：提取58例肺癌组织及其相应病灶的远端非癌肺组织和外周血淋巴细胞的总DNA（包括核DNA和mtDNA),用巢式PCR（nested PCR)方法扩增相互重叠且涵盖mtDNA全长的58个DNA片段。经PCR产物直接测序法测定mtDNA序列,通过与外周血淋巴细胞比较,确定肺癌组织mtDNA突变。结果：在36例（62．1％）肺癌组织中发现66个突变,其中点突变58个,插入突变4个,缺失突变4个。这些突变分散地分布于mtDNA,以D－loop区突变最多,为18个,在多肽编码区没有明显的热点突变区。在多肽编码区检出的43个点突变中,20个突变不改变氨基酸编码序列。在8例病灶远端非癌肺组织中发现了与相应肿瘤相同的突变。结论：肺癌组织中mtDNA突变可能是随机发生的,且大部分突变对肿瘤的发生可能没有影响,但其有望成为一种肿瘤诊断的分子标记。     

血浆游离DNA的K-ras基因突变丰度与转移性结直肠癌患者疗效和预后的相关性

目的 探讨肽核酸钳制PCR(PNA-PCR)法和传统巢式PCR法检测血浆游离DNA中K-ras基因突变丰度与转移性结直肠癌患者疗效和预后的相关性.方法 收集106例转移性结直肠癌患者外周血并提取血浆游离DNA,分别采用PNA-PCR法和传统巢式PCR法检测血浆游离DNA的K-ras基因状态.根据是否突变及突变丰度,将患者分为野生型组、低丰度突变组和高丰度突变组,分析K-ras基因突变丰度与患者疗效和预后的关系.结果 106例患者肿瘤组织K-ras基因的突变率为40.6％,采用PNA-PCR法检测血浆游离DNA中K-ras基因的突变率为31.1％,显著高于传统巢式PCR法检测的K-ras基因的突变率(15.1％,P=0.006).PNA-PCR法检测的血浆K-ras基因状态与肿瘤组织K-ras基因状态的检测一致率高达83.0％.野生型组(73例)、低丰度突变组(17例)和高丰度突变组(16例)患者的中位生存时间(OS)分别为23.5、17.3和13.9个月(P =0.002),其中各组一线单纯化疗者的中位无进展生存时间(PFS)分别为6.8、6.1和3.2个月(P =0.002),中位OS分别为23.0、15.5和13.9个月(P=0.036).K-ras基因野生型患者一线接受西妥昔单抗联合化疗的有效率为75.0％,好于单纯化疗者(23.4％,P=0.058);二线接受西妥昔单抗联合化疗的疾病控制率为100％,明显好于单纯化疗(35.7％,P＜0.001).Cox多因素回归分析显示,PNA-PCR法检测血浆K-ras基因状态、ECOG评分、手术次数和首发转移部位均为影响转移性结直肠癌患者预后的独立因素.结论 PNA-PCR法检测血浆游离DNA可替代肿瘤组织用于K-ras基因突变检测.血浆游离DNA的K-ras基因突变丰度是影响转移性结直肠癌患者预后的独立因素.     

多重PCR-SSCP法检测妇科肿瘤患者hMSH2基因突变

目的：建立经济快捷地筛查基因突变的技术，研究妇科肿瘤患者hMSH2基因突变情况，寻找与妇科肿瘤发生相关的分子标记物。方法：通过PCR优化策略，建立多重PCR—SSCP法；收集42名妇科肿瘤病人的基础资料及血液标本，抽提血液DNA，用多重PCR法扩增hMSH2基因第六、第七外显子，并进行单链构象多态性分析(SSCP)及DNA序列分析。结果：检测出2位妇科肿瘤病人在hMSH2基因第七外显子有杂合性突变，且为Leu→Phe的错义突变。未发现hMSH2基因第六外显子上存在突变。结论：多重PCR—SSCP是筛查基因突变的一种快速有效的方法，hMSH2基因第七外显子上的突变可能是与妇科肿瘤发生相关的一种分子标记物。     

定点诱变法构建p53基因多态质粒

目的：为了研究p53codon72多态的功能,采用定点诱变法构建人p53基因的多态质粒。方法：设计带有突变碱基的引物,通过PCR扩增引入突变碱基,再将突变后的p53序列插入载体中,经测序确定所扩增的DNA序列,并用体外转录翻译系统制备p53蛋白和i ASPP蛋白,免疫沉淀法检测蛋白相互作用。结果：通过PCR获得的含突变碱基的p53序列经连接后插入真核表达载体pReceiver-M01中,构建了p53多态位点为72Arg的表达质粒pReceiver-M01-p53（Arg72）。测序证明序列正确,表达的p53蛋白能与i ASPP相互作用,具有生物学功能。结论：成功构建了p53多态（72Arg）真核表达载体,为进一步深入研究p53多态的生物学功能奠定了基础。     

现代分子生物学技术简介（续）

四、变性-高压液相色谱分析变性-高压液相色谱(denaturing high-performance liquid chromatography,DHPLC)是利用DNA构型改变检测基因突变和遗传多态性的方法之一,其可以检测单核苷酸多态和可遗传的突变。实验主要过程包括PCR扩增待测和正常DNA样品,将扩增产物变性后再复性,若     

鼻咽癌组织线粒体DNA突变的研究

[目的]通过检测鼻咽癌组织中线粒体DNA(mitochodrial DNA,mtDNA)部分区域的突变,探讨mtDNA突变在人鼻咽癌发生中的作用.[方法]提取23例鼻咽癌组织及其同一患者外周血白细胞的总DNA,经PCR扩增mtDNA,产物直接测序测定mtDNA序列.[结果]23例鼻咽癌组织中8例(34.8%)发现33个突变,包括点突变27个,插入突变3个,缺失突变3个;这些突变分布于mtDNA序列中,27个mtDNA突变位于D-loop区,其中21个是属于基因库中的多态变化,6个为新发现的突变;另外6个mtDNA突变位于线粒体编码区.[结论]鼻咽癌组织线粒体DNA的D-loop区是一个高度多态性和突变性的区域,mtDNA的突变可能与鼻咽癌的发生有一定的联系,有望成为肿瘤诊断的分子标记.     

apc基因突变与B细胞性非霍奇金淋巴瘤发病机理相关性的研究

目的 探讨apc基因第15外显子密集突变区(Mutation cluster region, MCR)突变在B细胞性非霍奇金淋巴瘤(B-NHL)发生、发展中的作用.方法 应用石蜡组织DNA抽提、PCR扩增和2%琼脂糖凝胶电泳技术检测40例B-NHL和20例直肠腺癌(阳性对照)组织中DNA的apc基因MCR突变情况.结果 在相同PCR反应体系和条件下,从筛选出的20例直肠腺癌组织DNA中扩增出apc基因MCR的产物,而40例B-NHL组织DNA中未扩增出相应产物.结论 apc抑癌基因第15外显子MCR突变是结、直肠腺癌等恶性肿瘤的常见分子改变,但在B-NHL中可能不是普遍现象并有可能发生纯合性缺失.     

利用微滴数字PCR进行CpG岛甲基化表型结直肠癌分子分型

目的：利用微滴数字PCR(droplet digital PCR, DD-PCR)技术进行CpG岛甲基化表型(CpG island methylation phenotype, CIMP)结直肠癌分型, 并探讨以血浆游离DNA进行CIMP分型的可行性。方法：收集2008到2012年在长海医院行结直肠癌手术的患者216例,抽提肿瘤组织DNA和血浆游离DNA, 重亚硫酸盐处理后,利用DD-PCR进行CIMP分型。CIMP分型选用CACNA1G、IGF2、NEUROG1、RUNX3、SOCS1五个基因的甲基化位点,利用免疫组织化学技术（IHC）检测P53突变情况, 以巢式PCR测序方法检测K-RAS 和BRAF突变。结果：216例肿瘤中17例(7.9%)存在BRAF突变, 96例(444%) 存在K-RAS 突变和112例 (51.9%) P53-IHC(+)。31.5%(68/216)的结直肠癌为CIMP（+）(5个基因位点中高甲基化位点数目≥3),823%(14/17)的BRAF突变属于CIMP(+), 而只有9.4% (9/96)的K-RAS 突变和7.1% (8/112) 的P53突变属于CIMP(+)。除了K-RAS ,BRAF和P53突变外,同CIMP（-）结直肠癌相比,CIMP（+）肿瘤还具有独特的临床病理特征：肿瘤更易发生在结直肠的近端位置,黏液性癌比例偏高,分化程度较高,CIMP(+) 患者生存时间显著短于CIMP (-)患者。利用血浆游离DNA进行分型和利用肿瘤组织进行分型的一致性为93.4%、灵敏度为872%、特异性为100%。结论：CIMP（+）结直肠癌具有独特的临床病理特征, 且预后较差;在缺乏肿瘤组织的情况下, 利用DD-PCR对血浆游离DNA进行CIMP分型是可行的。     

甲状腺癌p53基因甲基化的研究

目的 探讨甲状腺癌 p53基因甲基化状态.方法 用限制性内切酶HpaⅠ和MspⅠ酶切甲状腺癌及癌旁组织DNA,通过聚合酶链反应(PCR)扩增p53基因第5外显子,经琼脂糖凝胶电泳后,分析其电泳图谱.结果12例甲状腺癌中9例p53基因第5外显子出现高甲基化状态,而5例癌旁组织为低甲基化状态.结论p53基因高甲基化状态与甲状腺癌发生以及p53基因点突变有关.     

Application of Multiplex Nested Methylated Specific PCR in Early Diagnosis of Epithelial Ovarian Cancer

Objective: To explore the application of multiplex nested methylated specific polymerase chain reaction (PCR)in the early diagnosis of epithelial ovarian carcinoma (EOC). Materials and Methods: Serum and fresh tissuesamples were collected from 114 EOC patients. RUNX3, TFPI2 and OPCML served as target genes. Methylationlevels of tissues were assessed by multiplex nested methylated specific PCR, the results being compared withthose for carcinoma antigen 125 (CA125). Results: The serum free deoxyribose nucleic acid (DNA) methylationspectrum of EOC patients was completely contained in the DNA spectrum of cancer tissues, providing an accuratereflection of tumor DNA methylation conditions. Serum levels of CA125 and free DNA methylation in the EOCgroup were evidently higher than those in benign lesion and control groups (p<0.05). Patients with early EOC hadmarkedly lower serum CA125 than those with advanced EOC (p<0.05), but there was no significant differencein free DNA methylation (p>0.05). The sensitivity, specificity and positive predicative value (PPV) of multiplexnested methylated specific PCR were significantly higher for detection of all patients and those with early EOCthan those for CA125 (p<0.05). In the detection of patients with advanced EOC, the PPV of CA125 detectionwas obviously lower than that of multiplex nested methylated specific PCR (p>0.05), but there was no significantdifference in sensitivity (p>0.05). Conclusions: Serum free DNA methylation can be used as a biological markerfor EOC and multiplex nested methylated specific PCR should be considered for early diagnosis since it canaccurately determine tumor methylation conditions.     

非小细胞肺癌组织化疗前后表皮生长因子受体基因的突变

目的:探讨非小细胞肺癌(non-small cell lung cancer,NSCLC)患者化疗前、后肿瘤组织中表皮生长因子受体(epidermal growth factor receptor,EGFR)基因外显子19和21的突变状况。方法:提取31例NSCLC患者化疗前、后肿瘤组织标本中的基因组DNA,采用巢式PCR技术扩增EGFR基因外显子19和21,并进行测序分析。结果:6例患者化疗前、后EGFR基因发生突变,其中4例为19号外显子发生缺失突变,2例为2l号外显子发生替代突变,且化疗前、后的突变状况一致。女性患者突变率(2/3)高于男性(4/28)(P=0.029),非吸烟者的突变率(4/9)高于吸烟者(2/22)(P=0.043)。结论:NSCLC组织EGFR基因外显子19和21突变在化疗前、后无明显改变。     

Comparison of Two Methods to Extract DNA from Formalin- Fixed,Paraffin-Embedded Tissues and their Impact on EGFR Mutation Detection in Non-small Cell Lung Carcinoma

Objective: Molecular pathology tests are often carried for clinicopathological diagnosis and pathologists haveestablished large collections of formalin-fixed, paraffin-embedded tissue (FFPE) banks. However, extraction ofDNA from FFPE is a laborious and challenging for researchers in clinical laboratories. The aim of this studywas to compare two widely used DNA extraction methods: using a QIAamp DNA FFPE kit from Qiagen and aCobas Sample Preparation Kit from Roche, and evaluated the effect of the DNA quality on molecular diagnostics.Methods: DNA from FFPE non-small cell lung carcinoma tissues including biopsy and surgical specimens wasextracted with both QIAamp DNA FFPE and Cobas Sample Preparation Kits and EGFR mutations of nonsmallcell lung carcinomas were detected by real-time quantitative PCR using the extracted DNA. Results andConclusion: Our results showed that DNA extracted by QIAamp and Cobas methods were both suitable to detectdownstream EGFR mutation in surgical specimens. Howover, Cobas method could yield more DNA from biopsyspecimens, and gain much better EGFR mutation results.     

外周血DNA和hTERT在肺癌诊断中的应用

目的检测肺癌患者血清DNA含量及hTERT的表达水平,寻求微创、高敏感性及特异性的检测方法,为肺癌早期诊断提供新指标。方法41例病理证实肺癌患者,22例正常人设为对照组。磁珠悬浮法提取测定血清DNA含量;巢式PCR法扩增血清hTERT片段,测定灰度比。结果肺癌患者、正常人血清DNA含量均数分别为33.464和18.420,肺癌组血清DNA浓度显著高于正常对照组(P<0.01)。巢式PCR法扩增血清hTERT片段,测定灰度比作为诊断分界点,绘制ROC曲线,AZ=0.852,AZ的95%置信区间为CI(0.750,0.953,P=0.000),因此可以认为血清hTERT对于肺癌的诊断具有中等准确性。诊断分界点=0.4时,其Kappa=0.477,(P<0.01),有统计学意义,其与病理诊断的一致性较好。结论外周血DNA和hTERT在肺癌诊断中具有较好的敏感性和特异性,可能成为肺癌早期诊断的新检测手段。     

Comparison of Polymerase Chain Reaction–Restriction Fragment Length Polymorphism,Immunohistochemistry, and DNA Sequencing for the Detection of IDH1 Mutations in Gliomas

Background: IDH1 mutation shows diagnostic, prognostic, and predictive value in gliomas. Direct Sanger sequencing is considered the gold standard to detect IDH1 mutation. However, this technology is not available in most neuropathological centers in developing countries such as Indonesia. Immunohistochemistry (IHC) and polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) have also been used to detect IDH1 mutation. This study aimed to compare DNA sequencing, IHC, and PCR–RFLP in detecting IDH1 mutations in gliomas. Methods: Research subjects were recruited from Dr. Sardjito Hospital. Genomic DNA was extracted from fresh or formalin-fixed paraffin-embedded samples of tumor tissue. DNA sequencing, PCR–RFLP and IHC were performed to detect IDH1 mutation. Sensitivity, specificity, and accuracy of PCR–RFLP and IHC were calculated by comparing them to DNA sequencing as the gold standard. Results: Among 61 recruited patients, 13 (21.3%) of them carried a mutation in codon 132 of the IDH1 gene, as shown by DNA sequencing. PCR–RFLP and DNA sequencing have a concordance value of 100%. Meanwhile, the concordance value between IDH1 R132H IHC and DNA sequencing was 96.7%. The sensitivity, specificity, positive predictive values, negative predictive values, and accuracy for PCR–RFLP were all 100%. On the other hand, the sensitivity, specificity, and accuracy of IHC were 92.3%, 97.9%, and 96.7%, respectively. Conclusion: This study showed that both PCR–RFLP and IHC have high accuracy in detecting IDH1 mutation. We recommend a combination of PCR–RFLP and IHC to detect IDH1 mutation in resource-limited settings.     

Evaluation of Primers and PCR Performance on HPV DNA Screening in Normal and Low Grade Abnormal Cervical Cells

High risk human papillomaviruses (HR-HPVs) are associated with increased risk of normal cervical cellsdeveloping to dysplasia and cervical carcinoma. Therefore, HR-HPV DNA testing can predict an endpoint ofcervical carcinogenesis that is earlier than the development of cervical abnormalities. Not only the sensitivity ofmethods but also the amount of HPV DNA are very important and might be parameters to distinguish HPVdetection. In this study, we evaluated the effects of primer sets and the polymerase chain reaction (PCR)performance with low viral load samples with normal cervical cytology (140 samples) and mild dysplasia (140samples) using two consensus primers MY09/MY11 and GP5+/6+. The PCR was performed with single andnested PCR. Positive samples with both primer sets were then HPV genotyped by dot blot hybridization. Resultsshowed higher sensitivity of single PCR using primer GP5+/GP6+ than primer MY09/MY11. HPV DNA wasdetected in 15% (21 of 140)and 20.7% (29 of 140) of normal cervical samples, respectively. For mild dysplasiasamples, HPV DNA was detected in 37.1% (52 of 140) with MY09/MY11 and 50% (70 of 140) using GP5+/GP6+. In normal cervical samples, the positivity rate was increased to 38.5% (54 of 140) by nested PCR usingprimer GP5+/6+, but only 2 mild dysplasia samples that were negative by single GP5+/6+ were positive by autonestedPCR. These results suggested that, in low viral load samples, the sensitivity of HPV DNA detectiondepends not only on primer sets but also PCR performance. HPV 16 was the most common in mild dysplasiasamples (20.8%), whereas HPV type 58 was found in 11.1%. This study suggested that nested PCR might benecessary for HPV DNA detection in cervical samples of women participating in cervical cancer screening.     

KRAS and BRAF mutation status in circulating colorectal tumor cells and their correlation with primary and metastatic tumor tissue

Although anti‐EGFR therapy has established efficacy in metastatic colorectal cancer, only 10‐20% of unselected patients respond. This is partly due to KRAS and BRAF mutations, which are currently assessed in the primary tumor. To improve patient selection, assessing mutation status in circulating tumor cells (CTCs), which possibly better represent metastases than the primary tumor, could be advantageous. We investigated the feasibility of KRAS and BRAF mutation detection in colorectal CTCs by comparing three sensitive methods and compared mutation status in matching primary tumor, liver metastasis and CTCs. CTCs were isolated from blood drawn from 49 patients before liver resection using CellSearch?. DNA and RNA was isolated from primary tumors, metastases and CTCs. Mutations were assessed by co‐amplification at lower denaturation temperature‐PCR (Transgenomic?), real‐time PCR (EntroGen?) and nested Allele‐Specific Blocker (ASB‐)PCR and confirmed by Sanger sequencing. In 43 of the 49 patients, tissue RNA and DNA was of sufficient quantity and quality. In these 43 patients, discordance between primary and metastatic tumor was 23% for KRAS and 7% for BRAF mutations. RNA and DNA from CTCs was available from 42 of the 43 patients, in which ASB‐PCR was able to detect the most mutations. Inconclusive results in patients with low CTC counts limited the interpretation of discrepancies between tissue and CTCs. Determination of KRAS and BRAF mutations in CTCs is challenging but feasible. Of the tested methods, nested ASB‐PCR, enabling detection of KRAS and BRAF mutations in patients with as little as two CTCs, seems to be superior.     

Quantitative Extra Long PCR to Detect DNA Lesions in Patients Exposed to Low Doses of Diagnostic Radiation

Background: Radiation causes oxidative lesions and strand breaks in DNA of exposed cells. Extended lengthPCR is a reliable method for assessing DNA damage. Longer DNA strands with DNA damage are difficult to amplifycompared to smaller DNA strands by PCR. The present study was aimed to evaluate DNA damage caused by ionisingradiation exposure in therapeutic and diagnostic medicine. Materials and Methods: The study group comprised 50cases with low dose single exposure (LDS), low dose multiple exposure (LDM) and low dose angiography (LDA)which were compared with 25 high dose controls (HDC) and 25 controls with no exposure (NEC). Blood samples werecollected within 1 hour of radiation exposure. DNA was isolated using a kit based protocol, 50 ng aliquots of DNAwere used to amplify a long 13kbp DNA fragment of the β-actin gene by conventional PCR and band intensity wasthen quantified. Relative amplification was calculated and damage was expressed in terms of lesions per kilobase (kbp)by assuming a Poisson distribution. Result: Relative amplification was found to be 1.0, 0.87, 0.86, 0.72 and 0.69 withNEC, LDS, LDM, LDA and HDC groups, respectively. Cases undergoing angiography as well as high dose controlshad high values, compared to NEC. The lesions/kbp calculated for LDS was 0.13, for LDM 0.15, for LDA 0.32 andfor HDC 0.37 suggesting a linear increase in quantity with increasing radiation dose. Conclusion: DNA damage, evenat low doses of radiation can be assessed by quantitative extra long PCR.     

Detection of (NAD(P)H:Quinone oxidoreductase-1, EC 1.6.99.2) 609C-->T and 465C-->T polymorphisms in formalin-fixed, paraffin-embedded human tumour tissue using PCR-RFLP

NQO1 is a cytosolic flavoprotein that plays a dual role in the detoxification of potentially carcinogenic compounds and the bioreductive activation of quinone based anticancer drugs. Two polymorphic variants of NQO1 exist (NQO1*2 and NQO1*3) which cause significant phenotypic reductions in NQO1 protein content and activity. Current methods for detecting NQO1 polymorphisms commonly use PCR-RFLP techniques and have exclusively used DNA isolated from fresh tissues. This study describes a method that is suitable for analysing NQO1 polymorphisms in genomic DNA isolated from formalin-fixed paraffin-embedded tissue. The method utilises two rounds of PCR amplification using a nested primer strategy that generates specific PCR products followed by RFLP analysis using either Hinf1 (for NQO1*2) or Msp1 (for NQO1*3). Whilst existing methods proved unsatisfactory (low product yield and poor specificity), the nested primer strategy produced good quality PCR products suitable for RFLP analysis and genotyping of NQO1*2 and NQO1*3 in archival tissue samples. The ability to utilise the vast archives of human tissue held by pathology laboratories would be of considerable benefit as retrospective studies comparing NQO1 genotype status, patient history and treatment outcomes could be conducted.