scholarly journals Cloning Polymerase Chain Reaction (PCR) Products: TOPO TA Cloning

2021 ◽  
Vol 2021 (9) ◽  
pp. pdb.prot101311 ◽  
Author(s):  
Michael R. Green ◽  
Joseph Sambrook
Keyword(s):  
Polymerase Chain Reaction ◽  
Chain Reaction ◽  
Pcr Products ◽  
Polymerase Chain ◽  
Set Up ◽  
Topo Cloning ◽  
First Time

This protocol describes the use of TOPO-activated TA vectors for cloning. Manufacturers of cloning kits provide excellent manuals that explain in detail what to do and why to do it. This makes TOPO cloning easy, but not foolproof. When setting up TOPO cloning for the first time, set up a trial experiment as described here.

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.

Detection of a few DNA copies by real-time electrochemical polymerase chain reaction

The Analyst ◽  
2017 ◽  
Vol 142 (18) ◽  
pp. 3432-3440 ◽  
Author(s):  
M. Moreau ◽  
S. Delile ◽  
A. Sharma ◽  
C. Fave ◽  
A. Perrier ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Current Work ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
First Time ◽  
Accurate Quantification

In the current work, accurate quantification over 10 to 108 DNA copies has been successfully achieved for the first time by real-time electrochemical PCR.

Automated closed-vessel system for in vitro diagnostics based on polymerase chain reaction

1993 ◽  
Vol 39 (9) ◽  
pp. 1927-1933 ◽  
Author(s):  
J B Findlay ◽  
S M Atwood ◽  
L Bergmeyer ◽  
J Chemelli ◽  
K Christy ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Pcr Amplification ◽  
Automated System ◽  
Closed Vessel ◽  
Chain Reaction ◽  
Panel Testing ◽  
Pcr Product ◽  
Pcr Products ◽  
Polymerase Chain

Abstract An automated system for polymerase chain reaction (PCR) amplification and detection combats false-positive results caused by "PCR product carryover." The system uses a single vessel for both PCR amplification and the subsequent detection of PCR products, eliminating the need to handle PCR products in an open environment and risk product carryover. The sample and PCR reagents are introduced into one compartment within the vessel, and amplification occurs as they are thermally cycled. Other compartments contain the reagents for detection of PCR products. Pressure from a roller provides for sequential delivery of the contents of the compartments to a detection area. The PCR products are biotinylated at their 5' ends during amplification through the use of biotinylated primers. After delivery to the detection area, they are specifically captured by hybridization with immobilized oligonucleotide probes. Subsequent reaction with streptavidin-horseradish peroxidase conjugate forms a complex that catalyzes dye formation from dye precursor. Wash steps minimize nonspecific background. This format is amenable to multiplexing, permitting internal controls, speciation of bacteria, typing of viruses, and panel testing. An HIV assay performed with this system demonstrated 100% sensitivity and 95% specificity for 64 patients' samples relative to a conventional PCR assay based on 32P solution hybridization. Similarly, an automated closed-vessel assay of cytomegalovirus exhibited 97.5% sensitivity and 100% specificity.

scholarly journals Detection of the chromosomal translocation t(11;14) by polymerase chain reaction in mantle cell lymphomas

Blood ◽  
1994 ◽  
Vol 83 (7) ◽  
pp. 1871-1875 ◽  
Author(s):  
R Rimokh ◽  
F Berger ◽  
G Delsol ◽  
I Digonnet ◽  
JP Rouault ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Chromosomal Translocation ◽  
Nucleotide Sequencing ◽  
Chromosome 11 ◽  
Chromosome 14 ◽  
Chain Reaction ◽  
Mantle Cell ◽  
Pcr Products ◽  
Polymerase Chain ◽  
Ig Heavy Chain

Abstract The t(11;14)(q13;q32) and its molecular counterpart, BCL1 rearrangement, are consistent features of mantle cell lymphoma (MCL). Rearrangement is thought to deregulate the nearby CCND1 (BCL1/PRAD1) proto-oncogene, a member of the cyclin G1 gene family, and thereby to contribute to tumorigenesis. We and others have previously shown that the BCL1 locus is rearranged in 55% to 60% of MCL patients and that, on chromosome 11, more than 80% of the breakpoints are localized within a 1-kbp DNA segment known as the major translocation cluster (MTC). We have determined the nucleotide sequence for a portion of the MTC region, and constructed chromosome 11-specific oligonucleotides that were in conjunction with a consensus immunoglobulin (Ig) heavy chain joining region (JH) primer used to perform the polymerase chain reaction (PCR) to amplify t(11;14) chromosomal junctional sequences in DNA from 16 MCL patients with breakpoints in the MTC region. 15 of the 16 breakpoints that occurred at the MTC region were amenable to PCR detection. The sizes of the amplified bands, the existence or not of a Sac I site in the PCR products, and nucleotide sequencing of the amplified DNA from four patients showed that the breakpoints share a remarkable tendency to tightly cluster within 300 bp on chromosome 11, some of them occurring at the same nucleotide. On chromosome 14, the breakpoints were localized within the Ig JH. Our findings indicate that a BCL1 rearrangement can be detected using this approach in roughly one half of the MCL patients. This has implications for both the diagnosis and the clinical management of MCL.

Detection of Subclinical Systemic Disease in Primary CNS Lymphoma by Polymerase Chain Reaction of the Rearranged Immunoglobulin Heavy-Chain Genes

2006 ◽  
Vol 24 (29) ◽  
pp. 4754-4757 ◽  
Author(s):  
Kristoph Jahnke ◽  
Michael Hummel ◽  
Agnieszka Korfel ◽  
Thomas Burmeister ◽  
Philipp Kiewe ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Polymerase Chain Reaction ◽  
Systemic Disease ◽  
Primary Cns Lymphoma ◽  
Cns Lymphoma ◽  
Chain Reaction ◽  
Blood Samples ◽  
Pcr Products ◽  
Polymerase Chain

Purpose To search for subclinical systemic disease in bone marrow and peripheral blood in patients with primary CNS lymphoma (PCNSL) to elucidate whether extracerebral relapse may represent a sequel of initial occult systemic disease rather than true extracerebral spread. Patients and Methods Bone marrow and peripheral-blood specimens of 24 PCNSL patients were examined using polymerase chain reaction (PCR) for analysis of clonally rearranged immunoglobulin heavy-chain (IgH) genes. Results Identical dominant PCR products were found in bone marrow aspirates, blood samples, and tumor biopsy specimens of two patients, indicating that the same tumor cell population is present in the CNS and in extracerebral sites. Follow-up IgH PCR performed in one of these patients in complete remission 24 months after diagnosis yielded a persistent monoclonal product in the blood. An oligoclonal IgH rearrangement pattern was found in the tumor specimen of two other patients, whereas bone marrow and blood samples demonstrated the same dominant PCR products. Follow-up PCR showed a persistent monoclonal amplificate in blood in one of these patients 27 months after diagnosis. Conclusion It could be demonstrated for the first time that subclinical systemic disease can be present in PCNSL patients at initial diagnosis. Our findings may have an impact on the understanding of PCNSL pathogenesis and the extent of staging and treatment.

Biofunctionalization of Polyoxometalates with DNA Primers, Their Use in the Polymerase Chain Reaction (PCR) and Electrochemical Detection of PCR Products

2015 ◽  
Vol 21 (49) ◽  
pp. 17721-17727 ◽  
Author(s):  
Ahmed M. Debela ◽  
Mayreli Ortiz ◽  
Valerio Beni ◽  
Serge Thorimbert ◽  
Denis Lesage ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Electrochemical Detection ◽  
Chain Reaction ◽  
Pcr Products ◽  
Polymerase Chain ◽  
Dna Primers
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