共查询到20条相似文献,搜索用时 15 毫秒
1.
Jonathan Shum 《Analytical biochemistry》2009,388(2):266-3618
Multiplex polymerase chain reaction (PCR), the amplification of multiple targets in a single reaction, presents a new set of challenges that further complicate more traditional PCR setups. These complications include a greater probability for nonspecific amplicon formation and for imbalanced amplification of different targets, each of which can compromise quantification and detection of multiple targets. Despite these difficulties, multiplex PCR is frequently used in applications such as pathogen detection, RNA quantification, mutation analysis, and (recently) next generation DNA sequencing. Here we investigated the utility of primers with one or two thermolabile 4-oxo-1-pentyl phosphotriester modifications in improving multiplex PCR performance. Initial endpoint and real-time analyses revealed a decrease in off-target amplification and a subsequent increase in amplicon yield. Furthermore, the use of modified primers in multiplex setups revealed a greater limit of detection and more uniform amplification of each target as compared with unmodified primers. Overall, the thermolabile modified primers present a novel and exciting avenue for improving multiplex PCR performance. 相似文献
2.
一种快速提取肠道微生物总DNA的方法 总被引:3,自引:2,他引:3
采集的兔肠道内容物及其粪便样品,通过分散浸泡、震荡洗涤、分级离心、滤器过滤、DNA提取试剂盒提取纯化,可以获得纯度很高的DNA样品。经0.8%琼脂糖凝胶电泳检测和紫外分光光度计测定,样品A260/A280的比值为1.72±0.02。分别以提取的DNA样品为模板,通过设计的细菌特异引物,对其16S rDNA基因进行PCR扩增,获得了1.6 kb大小特异性很好的预期条带。这为肠道微生物群落的分子生态学研究提供了一种简便、可靠的DNA提取方法。 相似文献
3.
Sachiko Shiokai Hiroyasu Kitashiba Kenta Shirasawa Kuniaki Nagano Takeshi Nishio 《Molecular breeding : new strategies in plant improvement》2009,23(2):329-336
DNA preparation is indispensable for genotyping by DNA polymorphism analysis, and that for a large number of plants is laborious.
In the present study, a small leaf disk of rice, 1–2 mm in diameter, punched by a mini cork borer was found to be directly
usable as a PCR template. DNA fragments <300 bp were amplified efficiently. Leaf disks of 1–1.5 mm in diameter were better
than those of 2 mm for a small volume of reaction mixture. Multiplex PCR was possible with four or eight primer pairs using
the small leaf disk as a template. Leaf disks of Arabidopsis, Lotus, wheat, soybean, tomato, Chinese cabbage, and melon were also good PCR templates. This method for preparation of PCR templates,
named the leaf-punch method, was applicable to SNP analysis of a large number of plants by dot-blot-SNP analysis.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
4.
To simulate single gene retrieval from ancient DNA, several related factors have been investigated. By monitoring a 889 bp
polymerase chain reaction (PCR) product and genomic DNA degradation, we find that heat and oxygen (especially heat) are both
crucial factors influencing DNA degradation. The heat influence, mainly represented by temperature and heating time, affects
the DNA degradation via DNA depurination followed by cleavage of nearby phosphodiesters. The heating time influence is temperature-dependent.
By reactive oxygen species (ROS) scavenging and 1,3-diphenyl-isobenzofuran (DPBF) bleaching experiments the influence of oxygen
on DNA thermal degradation was shown to occur via a singlet oxygen pathway. A comparative study of the thermal degradation
of cellular DNA and isolated DNA showed that cellular lipids can aggravate DNA thermal degradation. These results confirm
the possibility of gene amplification from thermally degraded DNA. They can be used to evaluate the feasibility of the retrieval
of single gene from ancient remains. 相似文献
5.
6.
An efficient method for DNA isolation from red algae 总被引:4,自引:0,他引:4
Zimin Hu Xiaoqi Zeng Aihua Wang Cuijuan Shi Delin Duan 《Journal of applied phycology》2004,16(3):161-166
A simple, inexpensive and efficient method was developed for rapid isolation of totalgenomic DNA from 15 red algal species. It resulted in 0.1 g high quality DNAfrom 1 mg fresh algal material, with an A260/A280ratio of 1.68–1.90.Using this rapidly isolated DNA, the 18S ribosomal RNA genes (rDNA) and the nuclearribosomal DNA of the internal transcribed spacer (ITS) regions were amplified. Thetested DNA was suitable for restriction endonuclease digestion, genetic markeranalysis and polymerase chain reaction (PCR) amplification, and may be valid forother genetic manipulation. 相似文献
7.
Xin Xu Shinji Kawasaki Tatsuhito Fujimura Chuntai Wang 《Plant Molecular Biology Reporter》2005,23(3):291-295
Current DNA isolation methods have limitations between speed and purity in high-throughput molecular genetic analysis such
as gene mapping and marker-assisted selection programs. We have optimized a simple and rapid method for isolating high-quality
genomic DNA from rice that significantly minimizes time and the use of laboratory materials. One person can process as many
as 384 samples in 2 h. The isolated DNA is suitable for polymerase chain reaction-based techniques and is stable for no less
than 6 mo of storage at 4°C. 相似文献
8.
K. B. Ignatov A. I. Miroshnikov V. M. Kramarov 《Russian Journal of Bioorganic Chemistry》2003,29(4):368-371
A new approach to enhanced specificity and product yield of polymerase chain reaction is proposed. It is based on control of DNA polymerase activity during PCR by changing the magnesium ion concentration, which depends on the temperature of the reaction mixture. A slightly soluble magnesium salt, magnesium oxalate, whose solubility depends on temperature, was used as a source of magnesium ions. During PCR, magnesium oxalate was maintained at saturating concentration by the presence of an insoluble excess of this salt, and the concentration of magnesium ions depended on the salt solubility: binding of magnesium ions at lower temperatures and their release at higher temperatures was shown to affect the DNA polymerase activity and to favor the specific PCR amplification of the target DNA fragment. 相似文献
9.
A noninvasive method for distinguishing among canid species: amplification and enzyme restriction of DNA from dung 总被引:12,自引:0,他引:12
Endangered San Joaquin kit foxes Vulpes macrotis mutica can be sympatrically distributed with as many as four other canids: red fox, gray fox, coyote and domestic dog. Canid scats are often found during routine fieldwork, but cannot be reliably identified to species. To detect and study the endangered kit fox, we developed mitochondrial DNA markers that can be amplified from small amounts of DNA extracted from scats. We amplified a 412-bp fragment of the mitochondrial cytochrome- b gene from scat samples and digested it with three restriction enzymes. The resulting restriction profiles discriminated among all five canid species and correctly identified 10 'unknown' fox scats to species in blind tests. We have applied our technique to identify canids species for an environmental management study and a conservation study. We envision that our protocol, and similar ones developed for other endangered species will be greatly used for conservation management in the future. 相似文献
10.
狗獾粪便DNA提取方法的初步探讨 总被引:1,自引:0,他引:1
在京杭大运河扬州段堤坝上采集狗獾的新鲜粪便,采用预处理-酚/氯仿抽提法、NaCl改良法、异硫氰酸胍裂解法、淀粉吸附法及QIAamp试剂盒法5种方法对粪便样品中狗獾DNA进行提取,探讨狗獾粪便样品中DNA提取方法和优化条件。结果表明,在5种提取方法中,淀粉吸附法的效果明显优于其它4种方法。采用粪便裂解液快速裂解细胞后,加入淀粉去除其中的大量PCR抑制物,然后用蛋白酶K裂解、酚/氯仿/异戊醇抽提,最后使用UNIQ-10柱纯化粪便DNA,对线粒体控制区和微卫星位点的PCR扩增反应及测序结果证实该方法的可行性。以上结果表明,通过该方法获得的粪便DNA能够用于更深入的分子遗传学等学科的研究。 相似文献
11.
一种简便、快捷的胰蛋白酶抑制剂基因的分离与克隆方法 总被引:2,自引:0,他引:2
从 3个豇豆品种幼嫩叶片中分离出核基因组 DNA,参照已知的几种 Bowman-Birk型胰蛋白酶抑制剂基因序列 ,设计合成了 2 7bp,且含有 Bam H I位点的寡核苷酸引物 ,分别以 3种豇豆核基因组 DNA为模板 ,PCR扩增 ,均得到长度约为 3 40 bp的 DNA片段。产物 DNA片段经 DNA序列分析 ,结果表明三者的碱基序列相同 ,与报道的胰蛋白酶抑制剂基因相比 ,同源性为 1 0 0 %和 99.7%。 相似文献
12.
A method for isolation and purification of peanut genomic DNA suitable for analytical applications 总被引:2,自引:0,他引:2
Numerous methods are available for isolation of plant genomic DNA, but in practice these procedures are empirical due to variability
in plant tissue composition. Consistent isolation of quality DNA from peanut (Arachis hypogaea L.) is particularly problematic due to the presence of phenolic compounds and polysaccharides. Inconsistencies in extraction
results can be attributed to the age and growth stage of the plant material analyzed. Mature leaves have higher quantities
of polyphenols, tannins, and polysaccharides that can contaminate DNA during isolation. We show that four published protocols
could not be used to isolate peanut DNA of sufficient quality for PCR amplification or Southern hybridization. We have devised
a new protocol that uses DEAE-cellulose purification to isolate peanut DNA useful for downstream applications. 相似文献
13.
《Saudi Journal of Biological Sciences》2017,24(7):1465-1469
In case of studies associated with human genetics, genomics, and pharmacogenetics the genomic DNA is extracted from the buccal cells, whole blood etc. Several methods are exploited by the researchers to extract DNA from the whole blood. One of these methods, which utilizes cell lysis and proteolytic properties of sodium dodecyl sulfate (SDS) and proteinase K respectively, might also be called SDS-PK method. It does not include any hazardous chemicals such as phenol or chloroform and is inexpensive. However, several researchers report the same method with different formulas and conditions. During our experiments with whole blood DNA extraction we experienced problems such as protein contamination, DNA purity and yield when followed some SDS-PK protocols reported elsewhere. A260/A280 and A260/A230 ratios along with PCR amplification give a clear idea about the procedure that was followed to extract the DNA. In an effort to increase the DNA purity from human whole blood, we pointed out some steps of the protocol that play a crucial role in determining the extraction of high quality DNA. 相似文献
14.
15.
目的:为了从分子水平上了解厌氧颗粒污泥中微生物的种类和数量,研究一种高效提取环境微生物DNA的方法。方法:厌氧颗粒污泥样品经液氮速冻、沸水浴融化、溶菌酶处理和SDS裂解后,琼脂糖凝胶电泳检测所提取的DNA,以提取的总DNA为模板,进行细菌核糖体小亚基16S rDNA基因V8、V9区的PCR扩增。结果:经检测,其DNA片段约为20 kb,样品D260nm/D280nm值为1.88,扩增结果理想,与OMEGA公司提供的试剂盒提取效果基本一致。结论:为薯类酒糟厌氧发酵污泥中微生物群落的分子生态学研究提供了一种简便、可靠的DNA提取方法。 相似文献
16.
Palakolanu Sudhakar Reddy 《Analytical biochemistry》2008,381(2):248-253
We developed a PCR-based high-throughput genome-walking protocol. The novelty of this protocol is in the random introduction of unique walker primer binding sites into different regions of the genome efficiently by taking advantage of the rolling circle mode of DNA synthesis by Phi29 DNA polymerase after annealing the partially degenerate primers to the denatured genomic DNA. The inherent strand-displacement activity of the Phi29 DNA polymerase displaces the 5′ ends of downstream strands and DNA synthesis continues, resulting in a large number of overlapping fragments that cover the whole genome with the unique walker adapter attached to the 5′ end of all the genomic DNA fragments. The directional genome walking can be performed using a locus-specific primer and the walker primer and Phi29 DNA polymerase-amplified genomic DNA fragments as template. The locus-specific primer will determine the position and direction of the genome walk. Two rounds of successive PCR amplifications by locus-specific and walker primers and their corresponding nested primers effectively amplify the flanking DNA fragments. The desired PCR fragment can be either cloned or sequenced directly using another nested, locus-specific primer. We successfully used this protocol to isolate and sequence 5′ flanking regions/promoters of selected plant genes. 相似文献
17.
可用于微生物群落分子生态学研究的活性污泥总DNA提取方法研究 总被引:47,自引:5,他引:47
活性污泥样品经液氮速冻、沸水浴融化、溶菌酶处理和 SDS裂解后 ,99%以上细胞裂解。所提取的 DNA经琼脂糖凝胶电泳检测和荧光法浓度测定 ,其片断大小在 2 0 kb左右 ,产量可达 1 .75 6± 0 .1 mg/g MLSS。样品 ABS2 6 0 nm/ABS2 80 nm的比值为 1 .96± 0 .2。以提取的总 DNA为模板 ,进行细菌核糖体小亚基 1 6Sr DNA基因 V3区和多组分苯酚羟化酶大亚基基因 (Lm PHs)的 PCR扩增 ,均获得成功 ,为活性污泥中微生物群落的分子生态学研究提供了一种简便、可靠的 DNA提取方法。 相似文献
18.
A method is described for the rapid analysis of DNA ligation products in the assembly of synthetic genes and gene fragments. The method is based on the simultaneous analysis of multiple ligation reactions where a single but different DNA oligomer is radiolabelled per ligation reaction. After each ligation the reaction mixture is electrophoresed on a denaturing, as well as a non-denaturing, polyacrylamide gel allowing one to monitor the ligation reaction products. In addition, a unique method for generating single stranded DNA sizing standards up to approximately 300 nucleotides in length is described. 相似文献
19.
利用T7DNA聚合酶在低温下仍具较高活性的特点,在热变性后低温下进行测序反应,使用该方法对多种PCR产物进行序列分析均取得较好的结果. 相似文献