基因组DNA快速提取方法的探讨与应用

目的 探讨以硫氰酸胍作为组织细胞裂解液的可行性,并建立基因组DNA快速提取方法.方法 利用硫氰酸胍等作为裂解消化液,分别从组织、细胞和全血中提取基因组DNA.采用紫外分光光度仪检测基因组DNA含量及纯度,琼脂糖凝胶电泳检测基因组DNA的完整性,以提取的基因组DNA作为模板进行PCR扩增GAPDH基因,并用该方法检测凋亡细胞DNA ladder.结果 基因组DNA提取时间短(耗时30～50 min),基因组DNA含量85 μg/mg(组织),52 μg/1×107细胞,27 μg/ml(血液).A260 nm/A280 nm比值在1.8～1.9之间,电泳显示基因组DNA完整性好,无蛋白质和RNA污染,基因组DNA长度大于48 kb.PCR扩增出306 bp GAPDH目的 基因,肿瘤细胞经抗癌基因诱导凋亡后琼脂糖凝胶电泳可见180～200 bp典型的凋亡带.结论 硫氰酸胍可作为基因组DNA提取裂解消化液,该方法具有简便、快捷、稳定性好等特点.     

改良碘化钠法提取人类微量全血基因组DNA的探索

目的通过改良碘化钠(NaI)法,建立一种简单、快速、经济的从人微量全血中提取基因组DNA的方法。方法采用经典NaI法和改良NaI法分别从人微量全血中提取基因组DNA,并进行常规和荧光定量聚合酶链反应(PCR),比较两种方法DNA提取的效果。结果采用改良NaI法从人外周全血中提取的DNA浓度和纯度与经典NaI法相比较,差异无统计学意义(P0.05),并且可以在较短时间内获得满意的常规和荧光定量PCR结果,且耗时较短。结论改良NaI法是一种简便、快速的提取人微量全血基因组DNA的方法,在临床和基础研究中具有较大的应用价值。     

自口腔粘膜上皮脱落细胞中快速提取人基因且DNA的方法比较

目的 建立简单、快速的人基因组DNA的提取方法。方法 用四种不同取材方法采集人口腔粘膜上皮脱落细胞，利用三种不同提取步骤提取人基因且DNA，并将各种方法所提的DNA进行比较。结果 通过对所提DNA进行OD值测定并计算DNA的质和量后发现，用牙签刮取人口腔粘膜上皮细胞、利用细胞裂解法提取的DNA其纯度和产量最高，以各种方法所提的DNA为模板作PCR后均出现了预定条带。结论 用本文建立的人基因且DNA样品的制备方法取材方便、提取简单、对身体无创伤且适合不同需要，值得推广应用。     

纳米磁珠法与试剂盒法分析载脂蛋白E基因的多态性

背景：相对于血液标本,口腔拭子标本更利于大规模载脂蛋白E基因多态性分析的研究,但目前对口腔拭子标本基因组DNA的提取尚无统一方法。 目的：探索合适的口腔拭子标本基因组提取方法以分析载脂蛋白E基因的多态性。 方法：收集50例散发性阿尔茨海默病患者口腔拭子标本,每份标本分别应用纳米磁珠法与PicoDNA微量核酸提取试剂盒法提取基因组DNA,对比分析两种方法获得的基因组DNA纯度和浓度,后续进行PCR反应,应用DNA电泳确认有无成功扩增出目的条带,通过DNA测序方法分析载脂蛋白E基因的多态性。 结果与结论：两种方法提取的基因组DNA纯度均较好,纳米磁珠法提取的基因组DNA浓度要高于PcioDNA微量核酸提取试剂盒法所得浓度（P〈0.05）。两组获取的基因组DNA均可成功进行PCR扩增,但电泳结果示纳米磁珠法扩增的目的条带更清楚。两组PCR产物DNA测序结果一致,载脂蛋白E基因ε2、ε3、ε4基因型的比例分别是6%,71%,23%。表明纳米磁珠法相对于PcioDNA微量核酸提取试剂盒法提取口腔拭子标本基因组DNA更适合用于大样本载脂蛋白E基因多态性的研究。     

人类性别决定基因（SRY）的检测及其临床应用

目的 探讨SRY基因与两性性别发育的关系. 方法 提取40例健康人外周血总DNA,加入SRY基因的特异性扩增引物和内参引物,运用聚合酶链式反应（PCR）技术进行SRY基因扩增,后经琼脂糖凝胶电泳进行检测. 结果 40例的基因组DNA经 PCR扩增后在500 bp和600 bp之间出现β-actin条带,与预期的大小为517 bp的β-actin片段相吻合,说明本实验的实验条件的可靠性和准确性.其中20例男性在600 bp至700 bp之间出现条带,与预期的SRY的677 bp片段大致相符,而20例女性则无677 bp片段产生.用该方法检测一例外生殖器异常,染色体为46,XY社会性别为女性的患者,其SRY检测结果为阳性. 结论 SRY基因阳性是雄性决定基因,用PCR技术扩增SRY 基因能快速准确检出Y染色体.SRY基因的检测对性连锁遗传性疾病和单基因突变病的无创性产前诊断有重要意义.     

Chelex-100法快速提取石蜡组织中EB病毒基因组DNA

目的:以鼻咽癌石蜡组织为材料,用Chelex-100法提取鼻咽癌组织中EB病毒基因组DNA,并以此为模板扩增出EBNA-3C片段,以建立快速提取石蜡组织中EB病毒基因组DNA的方法。方法:应用原位杂交方法检测30例鼻咽癌石蜡切片组织中EB病毒编码的小RNA(EBER)的情况,应用Chelex-100法快速提取鼻咽癌组织中EB病毒基因组DNA,并以此为模板用PCR扩增出EBNA-3C片段。结果:(1)30例鼻咽癌石蜡组织中原位杂交检测EBER全为阳性。(2)用Chelex-100法从30例鼻咽癌石蜡组织中提取了EB病毒基因组DNA并成功扩增出EBNA-3C片段。结论:Chelex-100法是快速提取鼻咽癌石蜡组织中EB病毒基因组DNA的高效方法。     

碳离子辐射引起Hela细胞线粒体DNA 4 977 bp的缺失

背景:线粒体DNA 4 977 bp缺失的累积对衰老的多细胞动物来说是一个显著的特征,同时也与各种肿瘤细胞的转移和凋亡有关.目前的研究己证明辐射可特异性地诱导此缺失的产生,有望将其作为新的DNA水平的辐射损伤生物剂量标记.目的:用聚合酶链反应方法检测碳离子辐射引起的Hela细胞线粒体DNA 4 977 bp突变缺失.并观察碳离子辐射剂量及辐射时间与线粒体DNA 4 977 bp缺失的相关性.设计、时间及地点:细胞DNA水甲的对照实验,于2008-10/11在甘肃省兰州市近代物理研究所完成.材料:人宫颈癌细胞Hela细胞株.方法:Hela细胞经2-8 Gy的碳离子辐射后,于2～24 h各个时间点分别提取包含线粒体DNA的全基因组DNA,并用聚合酶链反应法扩增线粒体DNA 4 977 bp缺失的特异片段,通过限制反应模板浓度和聚合酶链反应循环数的方法进行定性分析.主要观察指标:碳离子辐射剂量及辐射时间对线粒体DNA 4 97H7 bp缺失的影响.结果:经2 Gy辐照处理的Hela细胞在2,4,8,24 h 4个时间点的检测中均无聚合酶链反应阳性产物,8 Gy辐照处理的Hela细胞在12,16,24 h 3个时间点可检测到线粒体DNA 4 977 bp缺失,且随时间延长而累积.辐照后12 h Hela细胞在8 Gy辐照处理后可检测到线粒体DNA 4 977 bp缺失,辐照后16和24 h Hela细胞在6,8 Gy辐照处理后可检测到线粒体DNA4 977 bp缺失.结论:碳离子辐射引起的Hela细胞线粒体DNA 4 977 bp缺失存在剂量相关性,且其累计程度可能与辐射时间有关.     

探讨从人血凝块中提取基因组DNA

目的从人血凝块中提取基因组DNA。方法室温自然解冻,人工磨碎,用Relax Gene Blood DNA System血液基因组DNA提取试剂盒提取DNA。结果用此试剂盒从人血凝块中成功提取基因组DNA,提取DNA浓度为16.1±4.7mg/L。结论用此试剂盒提取的基因组DNA的总量较高,提取效率高,PCR扩增效果好,可很好的用于血凝块基因组DNA的提取。     

从单个细胞扩增靶基因片段的技术

目的 建立从人的单个细胞扩增特定靶基因片段进行基因诊断的技术。方法 利用显微操作技术挑取单个纤维母细胞，各置于0.2ml簿壁反应管的裂解液中，合成针对抑癌基因P53第5－9外显子（e）的引物，运用15mer高度随机引物或一个特异引物进行单个细胞的整修基因组DNA或特异靶片段的预扩增，再以其为模板进行巢式聚合酶链反应（PCR）扩增P53基因第e5-9的1900bp片段及含第5，6外显子的580bp片段，并用ABI－377测序仪测定其序列。结果 运用稀释至0.01ng的基因组DNA扩增1900bp片段，优化PCR的条件，分别从300个单个纤维母细胞扩增上述1900bp片段，10个获得1900bp片段，成功率为33％。拉坟580bp片段，则阳性率可达605。序列分析证明确为P53基因第5，6外显子序列。结论 运用本实验室建立的技术可从人的单个细胞扩增特定靶基因片段，并测定其序列，作出基因诊断。     

荧光定量PCR检测鼻咽部脱落细胞内EBV-DNA

目的 建立荧光定量PCR（FQ－PCR）检测鼻咽部脱落细胞中EBV－DNA的方法。方法 以患者鼻咽部脱落细胞内DNA作为样本，以EB病毒基因序列中EBNA1（核抗原1）基因片段作为扩增的靶DNA，同时从人β－珠蛋白DNA片段作为内参照，合成了两对引物，并设计了两个特异的荧光探针，优化了FQ－PCR反应体系，同时对这两个片段进行扩增，每一标本得到两个Ct值；CtE和Ctβ，得到单位细胞EB病毒的相对量。结果 临床标本29例中，阳性22例，阴性7例，临床资料证实阳性者全部为鼻咽癌（NPC），阴性者全部为非鼻咽癌。结论 建立的FQ－PCR检测鼻咽部脱落细胞中EBV－DNA的方法，能反映单位细胞病毒的复制情况，可用于NPC的筛查，并可能应用于该病的风险评估和疗效观察。     

在HeLa细胞中表达人βIVS—Ⅱ—nt—654C→突变基因

OBJECTIVE: To establish a cell model expressing human betaIVS- II -nt 654C --> T allele (beta654 mutant). METHODS: DNA fragment of entire beta-globin gene encompassing the codon region and poly(A) signal of the allele was amplified by long PCR from the genomic DNA of a homozygote with beta65.4 mutation. The amplified fragments were cloned into the Hind III and Xba I sites of the pcDNA3.1 vector. After reidentification of the clone with beta654 mutant by DNA sequencing, the recombinant plasmid was transfected into HeLa cell using liposome method. The expression of the beta654 mutant gene in the transfected cells was detected by RT-PCR. RESULTS: Both the normally processed beta globin mRNA (183bp) and aberrant processed beta globin mRNA (256bp) were identified in the transfected HeLa cells, while no RT-PCR product was detected in the controls. CONCLUSION: The transfected HeLa cells can express human beta654 allele.     

Identification of Aeromonas trota (hybridization group 13) by amplification of the aerolysin gene using polymerase chain reaction

Aeromonas trota is recognized as an important enteropathogen, and its haemolysin (aerolysin) is purported to be one of the virulence factors. Rapid detection and identification of A. trota is important for early and specific diagnosis of the infectious diseases that it causes. Synthetic oligonucleotide primers were used in a polymerase chain reaction (PCR) technique to amplify a species-specific sequence of the aerA gene, which encodes the aerolysin of A. trota. A DNA fragment of 622 bp was amplified from both lysed cells and isolated DNA from A. trota. The identity of the amplified 622 bp fragment was confirmed by digestion with BamH I restriction endonuclease, which produced the predicted 557 and 65 bp fragments. The lower limit for detection of the aerA gene by PCR amplification was 10 pg of total DNA or 10-15 cells ml-1. Primer specificity for A. trota was determined by the PCR assay with cells of 55 strains of Aeromonas sppincluding all of the 14 currently recognized DNA hybridization groups. A strain of Aeromonas enteropelogenes that had been reclassified as A. trota was also PCR positive. The method described here can be used to detect aerolysin-producing A. trota (hybridization group 13) strains from environmental and clinical samples without the use of selective media or additional biochemical tests.     

Expression of DeltaF508 CFTR in normal mouse lung after site-specific modification of CFTR sequences by SFHR.

The development of gene targeting strategies for specific modification of genomic DNA in human somatic cells has provided a potential gene therapy for the treatment of inherited diseases. One approach, small fragment homologous replacement (SFHR), directly targets and modifies specific genomic sequences with small fragments of exogenous DNA (400-800 bp) that are homologous to genomic sequences except for the desired modification. This approach has been effective for the in vitro modification of exon 10 in the cystic fibrosis transmembrane conductance regulator (CFTR) gene in human airway epithelial cells. As another step in the development of SFHR for gene therapy, studies were carried out to target and modify specific genomic sequences in exon 10 of the mouse CFTR (mCFTR) in vivo. Small DNA fragments (783 bp), homologous to mCFTR except for a 3-bp deletion (DeltaF508) and a silent mutation which introduces a unique restriction site (KpnI), were instilled into the lungs of normal mice using four different DNA vehicles (AVE, LipofectAMINE, DDAB, SuperFect). Successful modification was determined by PCR amplification of DNA or mRNA-derived cDNA followed by KpnI digestion. The results of these studies showed that SFHR can be used as a gene therapy to introduce specific modifications into the cells of clinically affected organs and that the cells will express the new sequence.     

The facile detection of the nt 1226 mutation of glucocerebrosidase by 'mismatched' PCR

The most common Gaucher disease-producing mutation among Ashkenazi Jews is an A----G substitution at cDNA nt 1226 (genomic nt 5841). We describe a simple method for detecting this mutation both in genomic DNA and in cDNA by performing polymerase chain reaction (PCR) using a 5'-primer mismatched at one nucleotide so as to create an Xho I restriction site. When the mutation is present. the 105 bp fragment formed is cleaved to 89 and 16 nt fragments. The 89 bp fragment is easily visualized on a gel making it possible to distinguish individuals who do not have the mutation from heterozygotes and homozygotes.     

Construction and use of PCR primers from a 65 kDa mannoprotein gene for identification of C. albicans

A method for detection of Candida albicans in biological samples (blood, serum, urine) was developed by the use of polymerase chain reaction (PCR) amplification of a DNA fragment of a gene coding for a 65 kDa mannoprotein of C. albicans (CaMP65). The PCR amplifies a 220 bp fragments whose specificity for C. albicans was demonstrated by Southern blot with a non-radioactive probe, leading to the differentiation from all other yeast species or human and bacterial DNA. The sensitivity of this assay was 5-10 C. albicans cells per milliliter of biological sample.     

Extraction, detection and persistence of extracellular DNA in forest litter microcosms

A DNA extraction method was developed that preferentially extracted extracellular DNA rather than intracellular DNA from forest litter. The method purposely avoided the use of harsh chemicals and physical disruption steps used in total DNA extraction to release DNA from cells. The detection limit of PCR, determined by spiking forest litter samples with a dilution series of Choristoneura fumiferana MNPVegt(-)/lacZ(+) genomic DNA, was about 1 ng DNA or 6.85 x 10(6) target copies 0.5 g(-1) moist forest litter or 0.14 g(-1) dry forest litter. In this study, outdoor terrestrial microcosms, each spiked with 49.2 microg of genomic DNA (from the baculovirus CfMNPVegt(-)/lacZ(+)), were exposed to summer conditions. A 530 bp DNA fragment from the genome of the baculovirus CfMNPVegt(-)/lacZ(+) was detected in these microcosms for about 3 months. The DNA may have persisted for a longer period but was below the detection limit of the PCR analysis.     

连接酶反应技术检测冠心病三个相关基因的单核苷酸多态性

目的 利用聚合酶链反应-连接酶反应（polymorphism chain reaction-ligase detection reaction,PCR-LDR）技术检测瘦素受体基因Lys109Arg （A/G）、Gln223Arg （A/G）,脂联素基因G276T、T45G,护骨素基因T950C、G1181C六个多态性位点的单核苷酸多态性（single nucleotide polymorphism,SNP）.方法 参照待测多态性位点所在DNA序列设计并合成一对引物及3条探针,先通过PCR反应获得含有待检测突变位点的基因片段,再进行LDR,根据测序电泳结果所显示的含荧光标记的LDR产物的片段长度来判断基因型别.结果 采用PCR技术成功扩增出包含瘦素受体基因（110 bp,130 bp）、脂联素基因（400 bp）、护骨素基因（118 bp,163 bp）多态性位点的基因片段.根据LDR产物片段长度不同进行基因分型,纯合子只有一种片段长度,杂合子包含两种纯合子的片段长度,如瘦素受体基因SNP Lys109Arg （A/G）的PCR产物长度为110 bp,LDR产物片段的长度为110 bp时则判断为AA基因型,112 bp则判断为GG基因型,AG基因型则具有110 bp、112 bp两种片段长度.PCR-LDR基因分型结果与DNA测序技术的检测结果一致（Kappa=1,P=0.00）.结论 PCR-LDR技术简单、快速、准确、成本低,适用于大批量SNP检测.     

ALIS-FLP: amplified ligation selected fragment-length polymorphism method for microbial genotyping

A DNA fingerprinting method known as ALIS-FLP (amplified ligation selected fragment-length polymorphism) has been developed for selective and specific amplification of restriction fragments from TspRI restriction endonuclease digested genomic DNA. The method is similar to AFLP, but differs in that only one specific restriction enzyme (TspRI) is used. The cohesive ends of the DNA fragments are ligated with two types of oligonucleotide. A long oligonucleotide containing the primer site and the specific 9 nt 3 prime end, which is complementary to specific 9 nt, cohesive 3 prime end of the TspRI genomic DNA fragment, and a short, degenerated, oligonucleotide covering the remaining TspRI cohesive ends. Other cohesive ends are covered by a short degenerated oligonucleotide lacking the primer site. The ligation mixture is used as a template for amplification using a single primer corresponding to the 5 prime end of the long, specific oligonucleotide. The selection of TspRI digested genomic DNA fragments for amplification is achieved by sequence selective ligation of the specific long oligonucleotide carrying the primer site to both ends of the specific target fragment. This technique allows for differentiation of the organisms without previous knowledge of their DNA sequence. The usefulness of the method is confirmed by genotyping of 70 previously characterized clinical E. coli isolates. The grouping obtained was identical to the results of REA-PFGE. Versatility of the method is highlighted, i.e. its combining the advantages of the AFLP technique with a simple, rapid and cheap polymerase chain reaction product detection method.     

Mycobacterium tuberculosis detection by polymerase chain reaction and identification of M. tuberculosis strain

A rapid multiprimer PCR method for detection of Mycobacterium tuberculosis complex (MTC) and simultaneous identification of M. tuberculosis in clinical samples has been developed. The method is based on simultaneous amplification of two targets: a 401 bp region from the mtp40 species-specific gene sequence of M. tuberculosis and a 544 bp fragment from the RD1 genome region which is specific for MTC but absent in BCG strains. Polymerase inhibitors in this study were detected by internal control in each test. Detection sensitivity was 25 copies of M. tuberculosis genomic DNA. Seven methods for isolation of mycobacterial DNA were compared and the technique with chloroform extraction was selected as the most efficient. The proposed method was used for analysis of 37 clinical samples and the results were compared with the results of culturing, acid-fast bacilli staining, and clinical diagnosis. The method proved to be sufficiently sensitive and specific for detection of mycobacterial DNA. Moreover, in countries with only two main pathogenic species of MTC circulating (M. tuberculosis and M. bovis) this method can be used for differentiation of these two species.     

hVPS4A 基因克隆及真核表达载体的构建

目的：克隆人细胞空泡蛋白分选因子4A（human vacuolar protein sorting 4A,hVPS4A）基因,构建其真核表达质粒。方法以 Huh7细胞 cDNA 为模板,设计引物扩增全长 hVPS4A 基因,再将目的基因插入到真核载体 pRK5中。重组质粒经PCR、酶切和 DNA 测序确认。结果从 Huh7细胞 cDNA 中扩增得到约1350 bp 大小的目的片段,经回收、纯化、酶切后与载体pRK5连接,转化 DH5α大肠杆菌。选择 PCR 鉴定阳性的重组质粒,经 EcoRⅠ单酶切可见约一条5900 bp 片段;经 EcoRⅠ和HindⅢ双酶切可得到4600 bp 和1350 bp 两个片段,分别与载体 pRK5和目的片段 hVPS4A 预期大小一致;DNA 测序结果显示插入片段与 hVPS4A 参考序列一致。结论成功构建了含 hVPS4A 基因的真核表达载体,为进一步研究 hVPS4A 的生物学功能提供了条件。