首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 0 毫秒
1.
This study introduces a DNA microarray-based genotyping system for accessing single nucleotide polymorphisms (SNPs) directly from a genomic DNA sample. The described one-step approach combines multiplex amplification and allele-specific solid-phase PCR into an on-chip reaction platform. The multiplex amplification of genomic DNA and the genotyping reaction are both performed directly on the microarray in a single reaction. Oligonucleotides that interrogate single nucleotide positions within multiple genomic regions of interest are covalently tethered to a glass chip, allowing quick analysis of reaction products by fluorescence scanning. Due to a fourfold SNP detection approach employing simultaneous probing of sense and antisense strand information, genotypes can be automatically assigned and validated using a simple computer algorithm. We used the described procedure for parallel genotyping of 10 different polymorphisms in a single reaction and successfully analyzed more than 100 human DNA samples. More than 99% of genotype data were in agreement with data obtained in control experiments with allele-specific oligonucleotide hybridization and capillary sequencing. Our results suggest that this approach might constitute a powerful tool for the analysis of genetic variation.  相似文献   

2.
Synthetic DNA oligonucleotides can serve as efficient primers for DNA synthesis even when there is a single base mismatch between the primers and the corresponding DNA template. However, when the primer-template annealing is carried out with a mixture of primers and at low stringency the binding of a perfectly matched primer is strongly favored relative to a primer differing by a single base. This primer competition is observed over a range of oligonucleotide sizes from twelve to sixteen bases and with a variety of base mismatches. When coupled with the polymerase chain reaction, for the amplification of specific DNA sequences, competitive oligonucleotide priming provides a simple general strategy for the detection of single DNA base differences.  相似文献   

3.
4.
Summary A method is described for identifying variants at the bovine κ-casein locus by combining polymerase chain reaction (PCR) amplification and restriction enzyme digests.  相似文献   

5.
We have developed a polymerase chain reaction-based procedure for reproducible detection of the E6-E7 gene in human papilloma virus DNA sequences using formalin-fixed, paraffin-embedded tissue sections. This procedure is a simple one-step procedure which does not require any elaborate hybridization following polymerase chain reaction amplification. The protocol combines modified tissue treatment and proper primer selection for efficient amplification of target DNA in a highly specific manner allowing identification in ethidium bromide-stained gels. The procedure described here is useful for a variety of tissue preparations, particularly formalin-fixed, paraffin-embedded archival tissues.  相似文献   

6.
Detection of Nitrosomonas spp. by polymerase chain reaction   总被引:2,自引:0,他引:2  
Abstract A unique genomic DNA fragment was isolated from Nitrosomonas europaea ATCC 19718. Based on the sequence of this fragment, oligonucleotide primers for polymerase chain reaction amplification were prepared which amplify sequences of 775 and 658 bp. The predicted DNA fragments were both amplified from the genome of N. europaea and a Nitrosomonas spp. isolated from a local oxidation pond. The primers failed to amplify DNA from the genomes of the ammonia oxidiser Nitrosolobous multiformis , the nitrite oxidiser Nitrococcus mobilis as well as from the genomes of other unrelated heterotrophic bacteria. These DNA sequences could be amplified from 0.01 ng of N. europaea genomic DNA or from 100 intact cells, and it was possible to detect Nitrosomonas DNA in a DNA mixture extracted from water samples drawn from a local oxidation pond.  相似文献   

7.
8.
The use of DNA primers with 3'-ends complementary to specific genetic point mutations allowed for the rapid detection of such mutations in genomic DNA by polymerase chain reaction. The sensitivity of this approach was such that mutations could be detected in DNA samples mixed with a 10(7)-fold excess of normal non-mutated DNA. To increase the practicality of this approach for the detection of point mutations affecting all 3 of the known ras oncogenes we synthesized mutation-specific primers complementary to all 3 genes by substituting inosine residues at positions corresponding to ambiguous bases on the genes.  相似文献   

9.
, , and 1992. Detection of pathogenic Entamoeba histolytica DNA in liver abscess fluid by polymerase chain reaction. International Journal for Parasitology 22: 1193–1196. A sensitive method for detection of pathogenic Entamoeba histolytica DNA in drained fluids from liver abscess patients, using the polymerase chain reaction (PCR), has been developed. The PCR employs oligonucleotide primers specific for the gene encoding the 30 kDa molecule of pathogenic E. histolytica. Liver abscess fluids (19 samples), from 14 patients with a presumptive amebic liver abscess, were examined microscopically and by the PCR method. Only two of the 19 samples were positive microscopically, whereas all 19 samples tested positive by PCR. This technique can be used to confirm the diagnosis of amebic liver abscess.  相似文献   

10.
Despite toxoplasmosis being a common infection among human and other warm-blooded animals worldwide, there are no findings about Toxoplasma gondii evolutionary forms in ancient populations. The molecular techniques used for amplification of genetic material have allowed recovery of ancient DNA (aDNA) from parasites contained in mummified tissues. The application of polymerase chain reaction (PCR) to paleoparasitological toxoplasmosis research becomes a promising option, since it might allow diagnosis, acquisition of paleoepidemiological data, access to toxoplasmosis information related origin, evolution, and distribution among the ancient populations. Furthermore, it makes possible the analysis of parasite aDNA aiming at phylogenetic studies. To standardize and evaluate PCR applicability to toxoplasmosis paleodiagnostic, an experimental mummification protocol was tested using desiccated tissues from mice infected with the ME49 strain cysts, the chronic infection group (CIG), or infected with tachyzoites (RH strain), the acute infection group (AIG). Tissues were subjected to DNA extraction followed by PCR amplification of T. gondii B1 gene. PCR recovered T. gondii DNA in thigh muscle, encephalon, heart, and lung samples. AIG presented PCR positivity in encephalon, lungs, hearts, and livers. Based on this results, we propose this molecular approach for toxoplasmosis research in past populations.  相似文献   

11.
The polymerase chain reaction and oligonucleotide probe hybridization technique were applied to the detection of two common mutations of the beta-globin gene found in Chinese, namely the 4-base pair deletion at the 41-42 codons and the C to T substitution at nucleotide 654 of IVS-2. The accuracy of the method was established using beta-thalassemia cases with known mutations or haplotypes of the restriction fragment length polymorphism (RFLP). A further 11 cases of thalassemia intermediate and thalassemia minor were then analysed with the same approach. Our results showed that 5 of the 11 cases carried the TCTT-deletion at codons 41-42. Our method is economical both in terms of materials and time needed and in an alternative to the use of the molecular RFLP approach in the prenatal diagnosis of beta-thalassemia.  相似文献   

12.
We have developed a strategy to rapidly construct DNA hybridization probes for the isolation of genes disrupted by transposon Tn5 insertions. A single oligonucleotide complementary to and extending outward from the ends of the inverted repeat of Tn5 was used to prime DNA synthesis in the polymerase chain reaction. The amplified product consisted of DNA sequences adjacent to both ends of the transposon insertion. The general feasibility of the approach was tested by amplifying pBR322 sequences from a derivative of pBR322 containing a Tn5 insertion. To amplify genomic DNA sequences flanking a Tn5 insertion in the chromosome of a Pseudomonas syringae strain, circular substrates were generated by ligating EcoRI-digested genomic DNA. Tn5 was contained intact within one such circular molecule, as the transposon does not contain sites for cleavage by EcoRI. The amplified product (approximately 2.5 kb) was used as a DNA hybridization probe to isolate the homologous fragment from a cosmid library of wild-type Pseudomonas syringae genomic DNA. This approach may be applied to the efficient isolation of sequences flanking any Tn5 insertion.  相似文献   

13.
Detection of Salmonella typhi by polymerase chain reaction   总被引:1,自引:0,他引:1  
A rapid and sensitive method for detection of Salmonella typhi would help in preventing the spread of outbreaks and in clinical diagnosis. In order to develop unique PCR primers to detect Salm. typhi , ribosomal RNA genes from Salm. typhi (Rawlings) were cloned in pUC18. The resulting clone was confirmed by sequencing. The cloned DNA fragment contained the 5S, part of the 23S rRNA genes and the 5S-23S spacer region (EMBL/GenBank accession No. U04734).
It was expected that the 5S-23S spacer region is divergent unlike the highly conserved 23S+5S genes. This was confirmed by comparison with the rRNA gene sequences in the EMBL/GenBank database. A pair of PCR primers specific for Salm. typhi was obtained, based on this spacer region sequence. The specificity of this pair of primers was tested with 54 Salm. typhi strains (of 27 different phage types). All these Salm. typhi strains showed the positive 300 bp PCR product with this pair of primers. Six other Salmonella species as well as six other non- Salmonella bacteria were tested and none showed the 300 bp PCR product. The sensitivity of the detection level was 0·1 pg of pure Salm. typhi genomic DNA, or approximately 40 Salm. typhi cells in a spiked food sample. This pair of primers therefore has the potential for development into a diagnostic tool for the rapid diagnosis of typhoid fever.  相似文献   

14.
In order to develop a rapid and specific detection test for bacteria in soil, we improved a method based on the polymerase chain reaction (PCR). Each step of the protocol, including direct lysis of cells, DNA purification, and PCR amplification, was optimized. To increase the efficiency of lysis, a step particularly critical for some microorganisms which resist classical techniques, we used small soil samples (100 mg) and various lytic treatments, including sonication, microwave heating, and thermal shocks. Purification of nucleic acids was achieved by passage through up to three Elutip d columns. Finally, PCR amplifications were optimized via biphasic protocols using booster conditions, lower denaturation temperatures, and addition of formamide. Two microorganisms were used as models: Agrobacterium tumefaciens, which is naturally absent from the soil used and was inoculated to calibrate the validity of the protocol, and Frankia spp., an actinomycete indigenous to the soil used. Specific primers were characterized either in the plasmid-borne vir genes for A. tumefaciens or in the variable regions of the 16S ribosomal gene for Frankia spp. Specific detection of the inoculated A. tumefaciens strain was routinely obtained when inocula ranged from 10(7) to 10(3) cells. Moreover, the strong correlation we observed between the size of the inocula and the results of the PCR reactions permitted assessment of the validity of the protocol in enumerating the number of microbial cells present in a soil sample. This allowed us to estimate the indigenous population of Frankia spp. at 0.2 x 10(5) genomes (i.e., amplifiable target sequences) per g of soil.  相似文献   

15.
Neospora caninum is an intracellular protozoan that infects many domestic and wild animals. Domestic dogs and other canids function as definitive hosts, while other mammals serve as natural intermediate hosts. In the present study, the brain tissues of bats collected in Yunnan Province, Southern China were tested by N. caninum specific-nested PCR, targeting the Nc-5 gene and the internal transcribed spacer 1 (ITS1) region of the ribosomal DNA to determine whether bats could be infected with N. caninum. N. caninum DNA was detected in 1.8% (4/227) of bats, i.e., 1.7% (1/60) in Rousettus leschenaultia, 1.7% (1/58) in Hipposideros pomona, 2.9% (2/69) in Rhinolophus pusillus, and none (0/40) in Myotis daubentoniid. The findings of the present study are only the first indication that bats could serve as an intermediate host, and further studies are necessary to confirm whether bats are involved in the transmission of N. caninum infections.  相似文献   

16.
Lee D  Lee J  Yoon JK  Kim NY  Kim GW  Park C  Oh YK  Kim YB 《Animal biotechnology》2011,22(4):175-180
Here, we report the quantification of porcine endogenous retrovirus (PERV) copy numbers using real time PCR. After generating standard curves using plasmid DNA, copy numbers were determined for PERV pol and for a housekeeping gene, porcine estrogen receptor2 (ER2) with the same amount of genomic DNA. Using this method, we examined 6 pig breeds in Korea including two breeds of miniature pig, one domestic pig from Jeju, and imported pig breeds, Duroc, Landrace, and Yorkshire. All breeds showed PERV copy numbers ranging from 9 to 50. This method will be useful for monitoring of PERVs in a porcine xenograft.  相似文献   

17.
We combined three modern technologies of single base polymorphism detection in human genome: ligase detection reaction, rolling circle amplification and IMAGE hydro-gel microarrays. Polymorphism in target DNA was tested by selective ligation on microarray. Product of the ligase reaction was determined in microarray gel pads by rolling circle amplification. Two different methods were compared. In first, selective ligation of short oligonucleotides immobilized on microarray was used with subsequent amplification on preformed circle probe ("common circle"). The circle probe was designed especially for human genome research. In second variant, allele-specific padlock probes that may be circularized by selective ligation were immobilized on microarray. Polymorphism of codon 72 in human p53 gene was used as a biological model. It was shown that LDR/RCA on microarray is a quantitative reaction and gives high discrimination of alleles. Principles and perspectives of selective ligation and rolling circle amplification are being discussed.  相似文献   

18.
A method was developed for detection of Listeria monocytogenes by polymerase chain reaction amplification followed by agarose gel electrophoresis or dot blot analysis with a 32P-labeled internal probe. The technique identified 95 of 95 L. monocytogenes strains, 0 of 12 Listeria strains of other species, and 0 of 12 non-Listeria strains.  相似文献   

19.
20.
The polymerase chain reaction (PCR) using published primers and probes has been compared to conventional stains and immunofluorescence for diagnosis of Pneumocystis carinii. We have screened 71 bronchoalveolar lavage (BAL) fluids from HIV-immunosuppressed patients. Of 34 samples negative by conventional stains and immunofluorescence, only one was positive by PCR. Thirty of 35 samples positive by conventional stains and immunofluorescence were also positive by PCR. One BAL sample, negative by conventional stains but positive by immunofluorescence, was negative by PCR. These data are discussed in relation to clinical and therapeutic conditions of the patients.  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号