PCR methods and applications最新文献_第2页

A simple "universal" DNA extraction procedure using SDS and proteinase K is compatible with direct PCR amplification. 使用SDS和蛋白酶K的简单“通用”DNA提取程序与直接PCR扩增兼容。

PCR methods and applications Pub Date : 1995-06-01 DOI: 10.1101/gr.4.6.368

D Goldenberger, I Perschil, M Ritzler, M Altwegg

引用次数: 192

Generation of DNA-based markers in specific genome regions by two-primer RAPD reactions. 利用双引物RAPD反应在特定基因组区域生成基于dna的标记。

PCR methods and applications Pub Date : 1995-06-01 DOI: 10.1101/gr.4.6.346

J Hu, J van Eysden, C F Quiros

{"title":"Generation of DNA-based markers in specific genome regions by two-primer RAPD reactions.","authors":"J Hu, J van Eysden, C F Quiros","doi":"10.1101/gr.4.6.346","DOIUrl":"https://doi.org/10.1101/gr.4.6.346","url":null,"abstract":"Random amplified polymorphic DNA (RAPD) markers offer quick screening of different regions of the genome for genetic polymorphisms. The standard RAPD procedure uses a single 10-base-long random oligonucleotide as a primer to amplify short stretches of the genome by PCR. We modified the procedure by using two primers in each reaction in a Brassica napus mapping project. We found that the two-primer RAPD tends to amplify more and smaller fragments than the standard RAPD technique. These new bands were always amplified in the two-primer reactions, and Southern analysis revealed that they had no homology to the bands amplified in single-primer reactions involving the same primers. Furthermore, these new markers were not linked to markers amplified with the same primers in the standard RAPD reactions, suggesting that they were amplified from different genomic regions. The advantage of the two-primer RAPDs is that it allows more reactions to be carried out with a limited number of primers to generate more markers. Using a single primer, the number of reactions is equal to the number of primers (n), which in turn limits the total number of markers. When using two primers in all possible combinations, the total number of reactions increases to n x (n-1/2). This method could be useful in conjunction with bulked segregant analysis to develop high density maps of certain chromosomal regions. We used this approach to map a second marker linked to a gene governing low linolenic acid concentration in a B. napus F2 population.","PeriodicalId":77315,"journal":{"name":"PCR methods and applications","volume":"4 6","pages":"346-51"},"PeriodicalIF":0.0,"publicationDate":"1995-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"18585434","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 34

A method for rapid generation of competitive standard molecules for RT-PCR avoiding the problem of competitor/probe cross-reactions. 一种快速生成RT-PCR竞争性标准分子的方法，避免了竞争性/探针交叉反应的问题。

PCR methods and applications Pub Date : 1995-06-01 DOI: 10.1101/gr.4.6.371

R Ross, R Kleiz, A B Reske-Kunz

引用次数: 43

An efficient and optimized PCR method with high fidelity for site-directed mutagenesis. 一种高效、优化的高保真位点诱变PCR方法。

PCR methods and applications Pub Date : 1995-04-01 DOI: 10.1101/gr.4.5.269

Q Liang, L Chen, A J Fulco

{"title":"An efficient and optimized PCR method with high fidelity for site-directed mutagenesis.","authors":"Q Liang, L Chen, A J Fulco","doi":"10.1101/gr.4.5.269","DOIUrl":"https://doi.org/10.1101/gr.4.5.269","url":null,"abstract":"We have developed an efficient method for site-directed mutagenesis using two subsequential rounds of PCR. In this method, PCR conditions are optimized to favor high fidelity of Taq DNA polymerase in the presence of equimolar concentrations of MgCI2 and dNTP in the reaction mixture (pH 5.5-6.2). This method makes use of a pair of universal primers and the multiple cloning site of pUC/M13 vectors. Only one mutagenic primer is required per target site. In the second round of PCR, the 3' extension of the wild-type DNA strand is blocked by the presence of a segment of nonhomologous sequence at its 3' end, and as a consequence, the amplified, full-length DNA fragment is chiefly from the mutant strand. Furthermore, because the mutated DNA fragment has flanking restriction sites different from those of the wild-type DNA fragment, the wild-type DNA fragment is totally excluded in the step involving selective cloning of the mutant DNA fragment. This method was successfully used to introduce four, nonadjacent mutations in the 5' regulatory region of the cytochrome P450BM-3 gene. All 20 analyzed clones from these four cases of mutagenesis carried the desired mutations, and no undesired mutations were observed. We observed that the larger the number of mismatched nucleotide residues in the mutagenic primer, the higher the concentration of MgCI2 was necessary for successful PCR amplification. Our experimental results indicate that this method offers improvements in efficiency, flexibility, and fidelity.","PeriodicalId":77315,"journal":{"name":"PCR methods and applications","volume":"4 5","pages":"269-74"},"PeriodicalIF":0.0,"publicationDate":"1995-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1101/gr.4.5.269","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"18586915","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 18

Genetic subtyping of human immunodeficiency virus using a heteroduplex mobility assay. 人类免疫缺陷病毒的遗传分型使用异双工流动性测定。

PCR methods and applications Pub Date : 1995-04-01 DOI: 10.1101/gr.4.5.s202

E L Delwart, B Herring, A G Rodrigo, J I Mullins

引用次数: 150

DNA fingerprinting of crude bacterial lysates using degenerate RAPD primers. 利用退化RAPD引物对细菌裂解物进行DNA指纹图谱分析。

PCR methods and applications Pub Date : 1995-04-01 DOI: 10.1101/gr.4.5.265

S A Sakallah, R W Lanning, D L Cooper

{"title":"DNA fingerprinting of crude bacterial lysates using degenerate RAPD primers.","authors":"S A Sakallah, R W Lanning, D L Cooper","doi":"10.1101/gr.4.5.265","DOIUrl":"https://doi.org/10.1101/gr.4.5.265","url":null,"abstract":"Methods for identifying isolates of various pathogenic bacteria by DNA fingerprinting with random primers (RAPD) have been described recently. In these methods many primers are screened and the primers that generate the most informative DNA pattern are selected. A new strategy that simplifies the primer selection process for RAPD fingerprinting has been developed in our laboratory. In this approach, one or more degenerate nucleotides is introduced into the core RAPD primer sequence at various nucleotide positions. Results show that a single degenerate nucleotide in the primer sequence can significantly change the DNA profile obtained for the same template. The more removed the degenerate nucleotide is from the 3' end of the primer, the less dramatic is its effect on banding pattern. This method utilizing degenerate RAPD (D-RAPD) primers was tested on clinical isolates of Legionella pneumoniae, and results were confirmed with nondegenerate RAPD primers. Results obtained with D-RAPD primers were in total agreement with those obtained with nondegenerate RAPD primers. We propose that the use of a core RAPD primer sequence with one or more degenerate nucleotide(s) at various positions can expedite the generation of unique DNA fingerprints individual organisms. A general method for selecting the most useful fingerprinting RAPD primers is discussed.","PeriodicalId":77315,"journal":{"name":"PCR methods and applications","volume":"4 5","pages":"265-8"},"PeriodicalIF":0.0,"publicationDate":"1995-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"18586914","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 21

Genomic fingerprinting by microsatellite-primed PCR: a critical evaluation. 微卫星引物PCR的基因组指纹鉴定:一个关键的评估。

PCR methods and applications Pub Date : 1995-04-01 DOI: 10.1101/gr.4.5.249

K Weising, R G Atkinson, R C Gardner

引用次数: 167

Multiple fluorescence-based PCR-SSCP analysis with postlabeling. 多重荧光PCR-SSCP分析与后标记。

PCR methods and applications Pub Date : 1995-04-01 DOI: 10.1101/gr.4.5.275

H Iwahana, K Adzuma, Y Takahashi, R Katashima, K Yoshimoto, M Itakura

引用次数: 17

Multiplex PCR of bcr-abl fusion transcripts in Philadelphia positive acute lymphoblastic leukemia. 费城阳性急性淋巴细胞白血病中bcr-abl融合转录物的多重PCR分析。

PCR methods and applications Pub Date : 1995-04-01 DOI: 10.1101/gr.4.5.283

A Lee, J Kirk, S Edmands, J Radich