scholarly journals PCR Amplification Protocol for GC Rich Protamine Gene from Chicken Testis cDNA

2015 ◽  
Vol 2 (11) ◽  
pp. 599-605 ◽  
Author(s):  
Sanjeev Kumar Sharma
Keyword(s):  
Pcr Amplification ◽  
Amplification Protocol ◽  
Testis Cdna ◽  
Chicken Testis

Investigations of Genetic Variation Between Olive (Olea europaea L.) Cultivars Using Arbitrarily Primed Polymerase Chain Reaction (AP-PCR)

2003 ◽  
Vol 58 (11-12) ◽  
pp. 837-842 ◽  
Author(s):  
Feray Kockar ◽  
Rahsan Ilıkcı
Keyword(s):  
Genetic Relationship ◽  
Genomic Dna ◽  
Pcr Amplification ◽  
Dna Fingerprint ◽  
Factors Affecting ◽  
Genomic Dna Isolation ◽  
Amplification Protocol ◽  
Olive Cultivars ◽  
Arbitrarily Primed Pcr ◽  
Relationship Of

Abstract Characterization and selection of olive clones for the production of olive oil is essential in Turkey because of its profitable exploitation. AP-PCR (Arbitrarily-Primed PCR) is a technique that can distinguish the genetic relationship among plant species and other organisms. In this study, AP-PCR approach was used in order to determine the genetic relationship of different six olive clones. The purity of DNA is one of the most important factors affecting the product of the AP-PCR method. In this respect, modified genomic DNA isolation procedure from Oleae europaea clones was developed so that this procedure can be used to obtain plant genomic DNA from diverse aromatic plants, which produce essential oils and secondary metabolites. By following the optimized AP-PCR amplification protocol, unique DNA fingerprint profiles for each olive clone were produced. AP-PCR-generated unique DNA fingerprint profiles can be used in the identification, distribution and diversity of various olive cultivars.

scholarly journals Rapid, quantitative nonisotopic assay for telomerase activity in human tumors

1998 ◽  
Vol 44 (10) ◽  
pp. 2133-2138 ◽  
Author(s):  
Stefania Gelmini ◽  
Anna Caldini ◽  
Lucia Becherini ◽  
Sergio Capaccioli ◽  
Mario Pazzagli ◽  
...  
Keyword(s):  
Pcr Amplification ◽  
Telomerase Activity ◽  
Quantitative Information ◽  
Cellular Protein ◽  
Large Variability ◽  
Trap Assay ◽  
Mean Values ◽  
Double Stranded Dna ◽  
Amplification Protocol ◽  
Cell Immortalization

Abstract Telomerase is a ribonucleoprotein enzyme that adds TTAGGG repeats onto human telomeres, preventing their shortening. The activation of this enzyme is an important step in cell immortalization and carcinogenesis and seems to represent a new and promising marker in cancer diagnosis and management. Telomerase activity is usually detected in cellular protein extract by the telomeric repeat amplification protocol (TRAP) assay, which can provide only a qualitative (presence/absence) evaluation. Here we present a modification of this method that can provide quantitative information without requiring time-consuming post-PCR procedures such as gel electrophoresis with radioactive materials and autoradiography. The detection and measurement of telomerase activity is performed by evaluating the amount of double-stranded DNA generated in the telomerase reaction and PCR amplification, with the use of the sensitive DNA fluorescent dye PicoGreen®. In a subset of tumors, the presence of telomerase activity was confirmed by the conventional TRAP assay. By this method we evaluated telomerase activity in unselected groups of breast (n = 15), ovarian (n = 12), endometrial (n = 12), gastric (n = 20), and renal (n = 12) carcinomas, in meningiomas (n = 8), and in pheochromocitomas (n = 10). The results indicate substantial differences of telomerase activity among cancer groups; however, a large variability among patients of the same group is observed. Kidney, ovarian, and breast carcinomas showed the highest mean values (31.8 ± 28.9, 29.2 ± 26.7, and 35.3 ± 15.9 ng DNA/μg protein, respectively, mean ± SD), whereas gastric and endometrial cancers had a lower activity (17.2 ± 8.8 and 13.5 ± 7.9 ng DNA/μg protein, respectively). Very low or no detectable telomerase activity was found in meningiomas (with the exception of one malignant atypical variant) and pheochromocitomas (9.7 ± 12.9 and 2.8 ± 2.1 ng DNA/μg protein, respectively). In conclusion, our method seems to be an accurate and reasonable procedure for measuring telomerase activity in human cancers.

scholarly journals Real-Time Quantitative Telomeric Repeat Amplification Protocol Assay for the Detection of Telomerase Activity2

2001 ◽  
Vol 47 (3) ◽  
pp. 519-524 ◽  
Author(s):  
Mi Hou ◽  
Dawei Xu ◽  
Magnus Björkholm ◽  
Astrid Gruber
Keyword(s):  
Real Time ◽  
Half Life ◽  
Pcr Amplification ◽  
Human Tumor ◽  
Telomerase Activity ◽  
Quantitative Information ◽  
Telomeric Repeat ◽  
Telomeric Repeat Amplification Protocol ◽  
Trap Assay ◽  
Amplification Protocol

Abstract Background: Telomerase is a ribonucleoprotein enzyme associated with immortalization and transformation of human cells. The telomeric repeat amplification protocol (TRAP) is widely used for the detection of telomerase activity. The TRAP method, although highly sensitive and specific because it includes PCR amplification, is laborious and does not provide precise quantitative information. Methods: We developed a real-time quantitative TRAP (RTQ-TRAP) system by combining a real-time PCR technique with the conventional TRAP method. Telomerase activity in human tumor cell lines and in 13 lymphoma samples was measured using the RTQ-TRAP assay, and the results obtained from the samples using the RTQ-TRAP method were compared with the conventional TRAP method. Results: The RTQ-TRAP method was both accurate and reproducible in measuring telomerase activity in a dilution series of protein extracts from HL60 cells. Telomerase activity in 13 lymphoma samples, as determined by the RTQ-TRAP method, was ninefold lower than that measured by the conventional TRAP method. The half-life of telomerase activity in human tumor cells, as determined using RTQ-TRAP, was much shorter than the half-life reported previously. Conclusions: Our results suggest that the conventional TRAP assay frequently overestimates telomerase activity in tumor samples. The RTQ-TRAP method is thus a useful tool to rapidly and precisely quantify telomerase activity.

scholarly journals Efficient PCR Amplification Protocol of Nuclear Microsatellites for Exuviae-Derived DNA of Cicada, Yezoterpnosia nigricosta

2021 ◽  
Vol 1 ◽  
Author(s):  
Keisuke Yumoto ◽  
Takashi Kanbe ◽  
Yoko Saito ◽  
Shingo Kaneko ◽  
Yoshiaki Tsuda
Keyword(s):  
Nuclear Dna ◽  
Molecular Ecology ◽  
Pcr Amplification ◽  
Adult Individual ◽  
Non Invasive ◽  
Amplification Protocol ◽  
Nuclear Microsatellite ◽  
Dna Microsatellite ◽  
Dna Microsatellite Markers ◽  
The Relationship

Although insect exuviae-based genetics is challenging, it can be a valuable method for obtaining reliable DNA resources by non-invasive sampling. This approach is especially effective when the target species is endangered/endemic or when sampling the adult is difficult. One example is cicadas, which during molt leave their exoskeletons on tree trunks, making them easily collectable. While cicada exuviae-derived DNA has previously been employed for mitochondrial DNA sequencing, this study aimed to develop a reliable method for the PCR amplification of nuclear microsatellite loci from cicada exuviae derived DNA for application in molecular ecology, conservation and population genetics. Five different PCR amplification protocols were performed, and the fragment patterns compared with those obtained using DNA extracted from adult individuals. Moreover, the relationship between the freshness of the exuviae and genotyping success was evaluated. TaKaRa LA Taq provided the best performance in the PCR amplification of DNA isolated from cicada exuviae and the electropherogram showed a clear fragment pattern that was equivalent to that obtained from the DNA extracted from the adult individual. This result suggests that cicada exuviae-derived DNA can be amplified by PCR and that multiple independent loci of nuclear DNA microsatellite markers can be easily genotyped. This study demonstrates that fresh cicada exuviae provide high quality DNA, which can be used for microsatellite genotyping. The methods developed in this study are applicable not only for cicada but other insect species for which exuviae are available. Thus, this study can make a significant contribution to insect sciences.

Asian Soybean Rust Caused by Phakopsora pachyrhizi on Soybean and Kudzu in Florida

2005 ◽  
Vol 6 (1) ◽  
pp. 9 ◽  
Author(s):  
Philip F. Harmon ◽  
M. Timur Momol ◽  
J. J. Marois ◽  
Hank Dankers ◽  
Carrie L. Harmon
Keyword(s):  
Light Microscopy ◽  
Dna Extraction ◽  
Pcr Amplification ◽  
Initial Diagnosis ◽  
Soybean Rust ◽  
Phakopsora Pachyrhizi ◽  
First Report ◽  
Asian Soybean Rust ◽  
Rust Diseases ◽  
Amplification Protocol

Asian soybean rust caused by Phakopsora pachyrhizi was found on soybean and kudzu in Florida in November of 2004. The initial diagnosis of soybean rust was based on observations of symptoms and urediniospores. The two species of Phakopsora that cause rust diseases on soybean, P. pachyrhizi and P. meibomiae, cannot be differentiated with light microscopy. A rapid DNA extraction and PCR amplification protocol discriminated between the two species. The sequence of the amplified DNA product confirmed this first report of P. pachyrhizi in Florida. Accepted for publication 9 May 2005. Published 13 June 2005.

Characterization of a cDNA for Rhesus Monkey Protein C Inhibitor – Evidence for N-Terminal Involvement in Heparin Stimulation

1995 ◽  
Vol 74 (04) ◽  
pp. 1079-1087 ◽  
Author(s):  
Klaus-P Radtke ◽  
José A Fernández ◽  
Bruno O Villoutreix ◽  
Judith S Greengard ◽  
John H Griffin
Keyword(s):  
Rhesus Monkey ◽  
Protein C ◽  
Activated Protein C ◽  
Pcr Amplification ◽  
Amino Acid Sequences ◽  
Heparin Binding ◽  
Reactive Center ◽  
Protein C Inhibitor ◽  
Polymerase Chain

SummarycDNAs for protein C inhibitor (PCI) were cloned from human and rhesus monkey 1 liver RNAs by reverse transcription and polymerase chain reaction (PCR) amplification. Sequencing showed that rhesus monkey and human PCI cDNAs were 93% identical. Predicted amino acid sequences differed at 26 of 387 residues. Pour of these differences (T352M, N359S, R362K, L3631) were in the reactive center loop that is important for inhibitory specificity, and two were in the N-terminal helix (M8T, E13K) that is implicated in glycosaminoglycan binding. PCI in human or rhesus monkey plasma showed comparable inhibitory activity towards human activated protein C in the presence of 10 U/ml heparin. However, maximal acceleration of the inhibition of activated protein C required 5-fold lower heparin concentration for rhesus monkey than for human plasma, consistent with the interpretation that the additional positive charge (E13K) in a putative-heparin binding region increased the affinity for heparin.

Simple and Rapid Detection of Factor V Leiden by Allele-specific PCR Amplification

1996 ◽  
Vol 75 (05) ◽  
pp. 757-759 ◽  
Author(s):  
Rainer Blasczyk ◽  
Markus Ritter ◽  
Christian Thiede ◽  
Jenny Wehling ◽  
Günter Hintz ◽  
...  
Keyword(s):  
Direct Sequencing ◽  
Activated Protein C ◽  
Factor V Leiden ◽  
Pcr Amplification ◽  
Factor V ◽  
Specific Pcr ◽  
Pcr Rflp ◽  
Allele Specific ◽  
Specific Amplification ◽  
Allele Specific Pcr

SummaryResistance to activated protein C is the most common hereditary cause for thrombosis and significantly linked to factor V Leiden. In this study, primers were designed to identify the factor V mutation by allele-specific PCR amplification. 126 patients with thromboembolic events were analysed using this technique, PCR-RFLP and direct sequencing. The concordance between these techniques was 100%. In 27 patients a heterozygous factor VGln506 mutation was detected, whereas one patient with recurrent thromboembolism was homozygous for the point mutation. Due to its time- and cost-saving features allele-specific amplification should be considered for screening of factor VGln506.

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