Tracing B-genome chromatin in Brassica napus × B. juncea interspecific progeny

Genome ◽  
2006 ◽  
Vol 49 (11) ◽  
pp. 1490-1497 ◽  
Author(s):  
C.J. Schelfhout ◽  
R. Snowdon ◽  
W.A. Cowling ◽  
J.M. Wroth
Keyword(s):  
Chromosome Count ◽  
Repeat Sequence ◽  
Chain Reaction ◽  
B Genome ◽  
A Genome ◽  
Pcr Products ◽  
Polymerase Chain ◽  
Pcr Assays ◽  
Selection Of

We used polymerase chain reaction (PCR) and fluorescence in situ hybridization (FISH) techniques to demonstrate the presence of Brassica B-genome chromosomes and putative B-genome introgressions in B. napus × B. juncea interspecific progeny. The B-genome - specific repeat sequence pBNBH35 was used to generate PCR products and FISH probes. The highest frequencies of viable progeny were obtained when B. napus was the maternal parent of the interspecific hybrid and the first backcross. B-genome - positive PCR assays were found in 34/51 fertile F2 progeny (67%), which was more than double the proportion found in fertile BC1 progeny. Four B-genome - positive F2-derived families and 1 BC1-derived family were fixed or segregating for B. juncea morphology in the F4 and BC1S2, respectively, but in only 2 of these families did B. juncea-type plants exhibit B. juncea chromosome count (2n = 36) and typical B-genome FISH signals on 16 chromosomes. The remaining B. juncea-type plants had B. napus chromosome count (2n = 38) and no B-genome FISH signals, except for 1 exceptional F4-derived line that exhibited isolated and weak B-genome FISH signals on 11 chromosomes and typical A-genome FISH signals. B. juncea morphology was associated with B-genome - positive PCR signals but not necessarily with 16 intact B-genome chromosomes as detected by FISH. B-genome chromosomes tend to be eliminated during selfing or backcrossing after crossing B. juncea with B. napus, and selection of lines containing B-genome chromatin during early generations would be promoted by use of this B-genome repetitive marker.

Application of riboprinting for the identification of isolates of cutaneousLeishmaniaspp.

Parasitology ◽  
2003 ◽  
Vol 127 (3) ◽  
pp. 201-205 ◽  
Author(s):  
L. F. NIMRI ◽  
H. D. F. H. SCHALLIG
Keyword(s):  
Fragment Length Polymorphism ◽  
Restriction Pattern ◽  
Chain Reaction ◽  
Restriction Patterns ◽  
Single Genome ◽  
Pcr Rflp ◽  
Pcr Products ◽  
Polymerase Chain ◽  
Selection Of ◽  
Restriction Fragment Length

Riboprinting is one of several molecular methods that can generate comparative data independently of the complexity of the organism's morphology. Restriction fragment length polymorphism (RFLP) profiles derived from digestion of polymerase chain reaction (PCR) products of the ribosomal 18S fromLeishmaniaspp. yields a typical ‘riboprint’ profile that can vary intraspecifically. A selection of 76 stocks ofL. majorandL. tropica, isolated from patients with cutaneous leishmaniasis, was analysed by riboprinting to assess divergence within and between species.L. majorandL. tropicacould be easily differentiated from each other. Analysis of PCR–RFLP profiles indicated that stocks ofLeishmaniaspp. could be broadly partitioned into 2 species corresponding toL. majorandL. tropica. To test if ribosomal 18S sequences were homogeneous within each species, several isolates of each of theLeishmaniaspp. were digested. Interpretation of the riboprint profiles of the 18S independently amplified by PCR, there would appear to be one restriction pattern present within eachLeishmaniaspp. Homogeneity within copies of the ribosomal 18S within a single genome has, therefore, been demonstrated. The species designation established by riboprinting results were in agreement with the zymodeme analysis of the same isolates. The restriction patterns produced were simple, reproducible and easy to interpret.

Efficient selection of preimplantation transgenic embryos by an improved procedure using Dpn I - Bal 31 digestion and the polymerase chain reaction

1997 ◽  
Vol 9 (2) ◽  
pp. 263 ◽  
Author(s):  
B. B. Seo ◽  
C.H Kim ◽  
H. Tojo ◽  
S. Tanaka ◽  
K. Yamanouchi ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Dna Sequence ◽  
Mouse Embryos ◽  
Chain Reaction ◽  
Exogenous Dna ◽  
Pcr Products ◽  
Polymerase Chain ◽  
Transgenic Embryos ◽  
Efficient Selection ◽  
Selection Of

Efficient selection of preimplantation transgenic embryos by an improved method after pronuclear injection of exogenous DNA is described. The method is based on subjecting DNA extracted from the embryos to restriction enzymes as well as the polymerase chain reaction (PCR). The incorporated procedure included recovery of the digested DNA with glassmilk before PCR, which markedly enhanced the rate of accurate detection of transgenic embyos. When exogenous DNA sequences in the mouse embryos were not integrated into the genome they were digested with both Dpn I and Bal31, and subsequent PCR analysis generated DNA fragments of the injected DNA sequence in only 1 ·5% of cases examined. However, DNA extracted from mouse embryos containing the transgene sequences integrated into the genome evaded digestion by both enzymes and yielded transgene-specific PCR products in 68· 6% of the embryos tested. When bovine embryos were used, sequences of the endogenous haemoglobin gene used as a control genomic DNA sequence were protected from enzyme digestion (PCR products in 70· 5% of the embryos examined); by contrast, the non-integrated injected sequences were almost completly eliminated by the same treatment (PCR products in 1· 4% of the embryos examined). It is suggested that this method might be useful for the selection of transgenic embryos before embryo transfer, thereby reducing the number of recipient females required.

High performance Nanogold™-in situ hybridzation and its use in the detection of hybridized and PCR-amplified microscopical preparations

1996 ◽  
Vol 54 ◽  
pp. 896-897
Author(s):  
G. W. Hacker ◽  
I. Zehbe ◽  
J. Hainfeld ◽  
A.-H. Graf ◽  
C. Hauser-Kronberger ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Messenger Rna ◽  
High Performance ◽  
Detection System ◽  
Direct Methods ◽  
Genetic Alterations ◽  
Chain Reaction ◽  
Protein Encoding ◽  
Polymerase Chain

In situ hybridization (ISH) with biotin-labeled probes is increasingly used in histology, histopathology and molecular biology, to detect genetic nucleic acid sequences of interest, such as viruses, genetic alterations and peptide-/protein-encoding messenger RNA (mRNA). In situ polymerase chain reaction (PCR) (PCR in situ hybridization = PISH) and the new in situ self-sustained sequence replication-based amplification (3SR) method even allow the detection of single copies of DNA or RNA in cytological and histological material. However, there is a number of considerable problems with the in situ PCR methods available today: False positives due to mis-priming of DNA breakdown products contained in several types of cells causing non-specific incorporation of label in direct methods, and re-diffusion artefacts of amplicons into previously negative cells have been observed. To avoid these problems, super-sensitive ISH procedures can be used, and it is well known that the sensitivity and outcome of these methods partially depend on the detection system used.

scholarly journals Evaluation of Sandwich Enzyme-Linked Immunosorbent Assay and Reverse Transcription Polymerase Chain Reaction for the Diagnosis of Foot-and-Mouth Disease

Intervirology ◽  
2021 ◽  
pp. 1-6
Author(s):  
Salman Khan ◽  
Syed Asad Ali Shah ◽  
Syed Muhammad Jamal
Keyword(s):  
Polymerase Chain Reaction ◽  
Viral Genome ◽  
Foot And Mouth Disease ◽  
Enzyme Linked Immunosorbent Assay ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Kappa Value ◽  
Polymerase Chain ◽  
Pcr Assays ◽  
Level Of Agreement

<b><i>Background:</i></b> Foot-and-mouth disease (FMD) is an infectious and highly contagious disease of cloven-hoofed domestic and wild animals, causing heavy economic losses to the livestock industry. Rapid and reliable diagnosis of the disease is essential for the implementation of effective control measures. This study compared sandwich enzyme-linked immunosorbent assay (S-ELISA) and conventional reverse transcription polymerase chain reaction (RT-PCR) for the diagnosis of FMD. <b><i>Methods:</i></b> A total of 60 epithelial samples from suspected cases of FMD were tested using both S-ELISA and RT-PCR assays. The level of agreement between the assays was assessed by calculating the Kappa value. <b><i>Results:</i></b> S-ELISA detected 38 (63%) samples positive for FMD virus (FMDV). Being predominant, serotype O was detected in 22 (57.9%) of the total samples tested positive, whereas 9 (23.7%) and 7 (18.4%) samples were found positive for serotypes A and Asia-1, respectively. RT-PCR detected viral genome in 51 (85%) of the samples using pan-FMDV primers set, 1F/1R. Thirty-six samples were found positive and 7 negative by both the tests. The level of agreement between the tests was assessed by calculating the Kappa value, which was found to be fair (Kappa value = 0.303 and 95% CI = 0.089; 0.517) and significant (<i>p</i> = 0.009). However, 2 samples, which were found positive on S-ELISA tested negative on RT-PCR. This may be attributed to the presence of nucleotide mismatch(es) in the primer-binding sites that may have resulted in failure of amplification of the viral genome. The serotype-specific RT-PCR assays not only confirmed serotyping results of S-ELISA but were also able to establish serotype in 9 S-ELISA-negative but pan-FMDV RT-PCR-positive samples. <b><i>Conclusions:</i></b> The RT-PCR assay contributes significantly to establishing a quick, sensitive, and definitive diagnosis of FMD in resource-constrained countries. Samples giving negative results in S-ELISA should be tested in RT-PCR for the disease detection and virus typing.

scholarly journals A Comparative Study of 5S rDNA Non-Transcribed Spacers in Elaeagnaceae Species

Plants ◽  
2020 ◽  
Vol 10 (1) ◽  
pp. 4
Author(s):  
Oleg S. Alexandrov ◽  
Olga V. Razumova ◽  
Gennady I. Karlov
Keyword(s):  
Polymerase Chain Reaction ◽  
Molecular Markers ◽  
Dna Markers ◽  
5S Rdna ◽  
Chain Reaction ◽  
Closely Related Species ◽  
Pcr Products ◽  
Polymerase Chain ◽  
Species Specific ◽  
Shepherdia Canadensis

5S rDNA is organized as a cluster of tandemly repeated monomers that consist of the conservative 120 bp coding part and non-transcribed spacers (NTSs) with different lengths and sequences among different species. The polymorphism in the 5S rDNA NTSs of closely related species is interesting for phylogenetic and evolutional investigations, as well as for the development of molecular markers. In this study, the 5S rDNA NTSs were amplified with universal 5S1/5S2 primers in some species of the Elaeagnaceae Adans. family. The polymerase chain reaction (PCR) products of five Elaeagnus species had similar lengths near 310 bp and were different from Shepherdia canadensis (L.) Nutt. and Sh. argentea (Pusch.) Nutt. samples (260 bp and 215 bp, respectively). The PCR products were cloned and sequenced. An analysis of the sequences revealed that intraspecific levels of NTS identity are high (approximately 95–96%) and similar in the Elaeagnus L. species. In Sh. argentea, this level was slightly lower due to the differences in the poly-T region. Moreover, the intergeneric and intervarietal NTS identity levels were studied and compared. Significant differences between species (except E. multiflora Thunb. and E. umbellata Thunb.) and genera were found. Herein, a range of the NTS features is discussed. This study is another step in the investigation of the molecular evolution of Elaeagnaceae and may be useful for the development of species-specific DNA markers in this family.

scholarly journals Easy and Efficient DNA Extraction from Woody Plants for the Detection of Phytoplasmas by Polymerase Chain Reaction

Plant Disease ◽  
1999 ◽  
Vol 83 (5) ◽  
pp. 482-485 ◽  
Author(s):  
Margaret J. Green ◽  
Dan A. Thompson ◽  
Donald J. MacKenzie
Keyword(s):  
Polymerase Chain Reaction ◽  
Plant Material ◽  
Woody Plant ◽  
Leaf Roll ◽  
Chain Reaction ◽  
Efficient Procedure ◽  
Identical Manner ◽  
Total Dna ◽  
Polymerase Chain ◽  
Selection Of

A simple and efficient procedure for the extraction of high-quality DNA from phytoplasma-infected woody and herbaceous plants for polymerase chain reaction (PCR) detection is described. This procedure does not require phenol, chloroform, or alcohol for the precipitation of nucleic acids. Herbaceous and woody plant material are extracted in an identical manner with no additional purification or enrichment steps required. The method utilizes commercially available microspin-column matrices, and the extraction of total DNA can be achieved in less than 1 h. The method has been used to successfully purify phytoplasma DNA from whole leaves, leaf petioles and midribs, roots, and dormant wood from a diverse selection of plant material. The phytoplasmas detected by PCR include pear decline, western X-disease, peach yellow leaf roll, peach rosette, apple proliferation, Australian grapevine yellows, and Vaccinium witches'-broom.

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