Amplification of Salmonella Chromosomal DNA Using the Polymerase Chain Reaction

1994 ◽  
Vol 38 (1) ◽  
pp. 119 ◽  
Author(s):  
Anh V. Nguyen ◽  
Mazhar I. Khan ◽  
Zhiqiang Lu
Keyword(s):  
Polymerase Chain Reaction ◽  
Chromosomal Dna ◽  
Chain Reaction ◽  
Polymerase Chain

An initiation zone of chromosomal DNA replication located upstream of the c-myc gene in proliferating HeLa cells

1990 ◽  
Vol 10 (9) ◽  
pp. 4899-4904
Author(s):  
L Vassilev ◽  
E M Johnson
Keyword(s):  
Polymerase Chain Reaction ◽  
Dna Replication ◽  
Hela Cells ◽  
Polymerase Chain Reaction Amplification ◽  
Chromosomal Dna ◽  
Chain Reaction ◽  
Initiation Zone ◽  
Myc Gene ◽  
Reaction Amplification ◽  
Polymerase Chain

Studies on origins of DNA replication in mammalian cells have long been hampered by a lack of methods sensitive enough for the localization of such origins in chromosomal DNA. We have employed a new method for mapping origins, based on polymerase chain reaction amplification of nascent strand segments, to examine replication initiated in vivo near the c-myc gene in human cells. Nascent DNA, pulse-labeled in unsynchronized HeLa cells, was size fractionated and purified by immunoprecipitation with anti-bromodeoxyuridine antibodies. Lengths of the nascent strands that allow polymerase chain reaction amplification were determined by hybridization to probes homologous to amplified segments and used to calculate the position of the origin. We found that DNA replication through the c-myc gene initiates in a zone centered approximately 1.5 kilobases upstream of exon I. Replication proceeds bidirectionally from the origin, as indicated by comparison of hybridization patterns for three amplified segments. The initiation zone includes segments of the c-myc locus previously reported to drive autonomous replication of plasmids in human cells.

cDNA genes formed after infection with retroviral vector particles lack the hallmarks of natural processed pseudogenes

1990 ◽  
Vol 10 (1) ◽  
pp. 68-74 ◽  
Author(s):  
R Dornburg ◽  
H M Temin
Keyword(s):  
Polymerase Chain Reaction ◽  
Full Length ◽  
Target Cells ◽  
Hygromycin B ◽  
Direct Repeats ◽  
Chromosomal Dna ◽  
Chain Reaction ◽  
Cis Acting ◽  
Processed Pseudogenes ◽  
Polymerase Chain

Retroviral proteins can encapsidate RNAs without retroviral cis-acting sequences. Such RNAs are reverse transcribed and inserted into the genomes of infected target cells to form cDNA genes. Previous investigations by Southern blot analysis of such cDNA genes suggested that they were truncated at the 3' and the 5' ends (R. Dornburg and H. M. Temin, Mol. Cell. Biol. 8:2328-2334, 1988). To analyze such cDNA genes further, we cloned three cDNA genes (derived from a hygromycin B phosphotransferase gene) in lambda vectors and analyzed them by DNA sequencing. We found that they did not correspond to the full-length mRNA: they were truncated at both the 3' and the 5' ends, did not contain a poly(A) tract, and were not flanked by direct repeats. The 3'-end junctions to chromosomal DNA of five more cDNA genes were amplified by polymerase chain reaction, cloned in pUC vectors, and sequenced. All of these cDNA genes had 3'-end truncations, and no poly(A) tracts were found. Further polymerase chain reaction experiments were performed to detect hygromycin B phosphotransferase cDNA genes with a poly(A) tract in DNA extracted from a pool of about 500 colonies of cells containing cDNA genes. No hygromycin B phosphotransferase cDNA gene with a poly(A) tract was found. Investigation of two preintegration sites by Southern analysis revealed that deletions were present in chromosomal DNA at the site of the integration of the cDNA genes. Naturally occurring processed pseudogenes correspond to the full-length mRNA, contain a poly(A) sequence, and are flanked by direct repeats. Our data indicate that cDNA genes formed by infection with retrovirus particles lack the hallmarks or natural processed pseudogenes. Thus, it appears that natural processed pseudogenes were not generated by retrovirus proteins.

scholarly journals Optimization of Polymerase Chain Reaction for the Detection of Borrelia Burgdorferi in Biologic Specimens

1993 ◽  
Vol 5 (4) ◽  
pp. 548-554 ◽  
Author(s):  
Abigail C. Kaufman ◽  
Craig E. Greene ◽  
Royal A. McGraw
Keyword(s):  
Polymerase Chain Reaction ◽  
Borrelia Burgdorferi ◽  
Base Pair ◽  
Ethylenediaminetetraacetic Acid ◽  
Chromosomal Dna ◽  
Chain Reaction ◽  
Clinical Specimens ◽  
Pcr Diagnosis ◽  
Blood And Urine Samples ◽  
Polymerase Chain

This study describes the use of a newly constructed set of primers that amplifies an U-base pair (bp) segment of Borrelia burgdorferi chromosomal DNA. This 85-bp product is not produced when other Borrelia species, Leptospira, or other bacteria are subjected to polymerase chain reaction (PCR). We also describe a rapid method of optimizing the amplification of B. burgdorferi DNA from canine ethylenediaminetetraacetic acid-treated blood and urine samples that circumvents some of the problems encountered due to low number of spirochetes in clinical specimens and that removes inhibiting substances, which improves the PCR diagnosis of canine Lyme borreliosis.

DNA sequence heterogeneity in the gene encoding a 60-kilodalton extracellular protein of Listeria monocytogenes and other Listeria species

1992 ◽  
Vol 38 (8) ◽  
pp. 865-870 ◽  
Author(s):  
Karen A. Gutekunst ◽  
Brian P. Holloway ◽  
George M. Carlone
Keyword(s):  
Polymerase Chain Reaction ◽  
Listeria Monocytogenes ◽  
Fragment Length Polymorphism ◽  
Restriction Pattern ◽  
Extracellular Protein ◽  
Chromosomal Dna ◽  
Chain Reaction ◽  
Gene Encoding ◽  
Listeria Species ◽  
Polymerase Chain

Chromosomal DNA sequences from the 60 kilodalton protein gene of Listeria monocytogenes, amplified by the polymerase chain reaction, were used for restriction fragment length polymorphism differentiation of L. monocytogenes serotypes and other Listeria species. All 24 strains of L. monocytogenes examined produced an extracellular protein of molecular weight 60 000 (p60) as determined by Western blot analysis. Four of six other Listeria species had a protein that cross-reacted to antibodies to p60, but all differed in molecular weight, ranging from approximately 50 000 to 65 000. The gene encoding p60 was amplified from chromosomal DNA in all strains using polymerase chain reaction with a single primer pair. Restriction enzyme digestion with HindIII of the amplified product revealed a restriction pattern that was distinct between serotypes 1/2a and either 4b or 1/2b of L. monocytogenes. Of the other Listeria species, four strains that produced a cross-reacting protein likewise produced a polymerase chain reaction amplification product with the primer pair. Listeria innocua alone had a restriction pattern similar to that of Listeria monocytogenes serotype 4b and 1/2b. Genotypic heterogeneity, as revealed by DNA amplification and restriction endonuclease digestion of the p60 open reading frame, correlates with "electrophoretic type" grouping and may be related to differences in virulence mechanisms of Listeria monocytogenes and other Listeria species. Key words: Listeria monocytogenes, 60-kilodalton extracellular protein, polymerase chain reaction, restriction fragment length polymorphism.

scholarly journals cDNA genes formed after infection with retroviral vector particles lack the hallmarks of natural processed pseudogenes.

1990 ◽  
Vol 10 (1) ◽  
pp. 68-74 ◽  
Author(s):  
R Dornburg ◽  
H M Temin
Keyword(s):  
Polymerase Chain Reaction ◽  
Full Length ◽  
Target Cells ◽  
Hygromycin B ◽  
Direct Repeats ◽  
Chromosomal Dna ◽  
Chain Reaction ◽  
Cis Acting ◽  
Processed Pseudogenes ◽  
Polymerase Chain

Retroviral proteins can encapsidate RNAs without retroviral cis-acting sequences. Such RNAs are reverse transcribed and inserted into the genomes of infected target cells to form cDNA genes. Previous investigations by Southern blot analysis of such cDNA genes suggested that they were truncated at the 3' and the 5' ends (R. Dornburg and H. M. Temin, Mol. Cell. Biol. 8:2328-2334, 1988). To analyze such cDNA genes further, we cloned three cDNA genes (derived from a hygromycin B phosphotransferase gene) in lambda vectors and analyzed them by DNA sequencing. We found that they did not correspond to the full-length mRNA: they were truncated at both the 3' and the 5' ends, did not contain a poly(A) tract, and were not flanked by direct repeats. The 3'-end junctions to chromosomal DNA of five more cDNA genes were amplified by polymerase chain reaction, cloned in pUC vectors, and sequenced. All of these cDNA genes had 3'-end truncations, and no poly(A) tracts were found. Further polymerase chain reaction experiments were performed to detect hygromycin B phosphotransferase cDNA genes with a poly(A) tract in DNA extracted from a pool of about 500 colonies of cells containing cDNA genes. No hygromycin B phosphotransferase cDNA gene with a poly(A) tract was found. Investigation of two preintegration sites by Southern analysis revealed that deletions were present in chromosomal DNA at the site of the integration of the cDNA genes. Naturally occurring processed pseudogenes correspond to the full-length mRNA, contain a poly(A) sequence, and are flanked by direct repeats. Our data indicate that cDNA genes formed by infection with retrovirus particles lack the hallmarks or natural processed pseudogenes. Thus, it appears that natural processed pseudogenes were not generated by retrovirus proteins.

scholarly journals Detection of Rhodococcus Equi by Polymerase Chain Reaction Using Species-Specific Nonproprietary Primers

2002 ◽  
Vol 14 (4) ◽  
pp. 347-353 ◽  
Author(s):  
José Miguel Arriaga ◽  
Noah D. Cohen ◽  
James N. Derr ◽  
M. Keith Chaffin ◽  
Ronald J. Martens
Keyword(s):  
Polymerase Chain Reaction ◽  
Rhodococcus Equi ◽  
Chromosomal Dna ◽  
Chain Reaction ◽  
Specific Primers ◽  
Pcr Product ◽  
Random Amplification ◽  
Rhodococcus Species ◽  
Polymerase Chain ◽  
Species Specific

Species-specific primers for the polymerase chain reaction (PCR) for the detection of Rhodococcus equi were developed. These primers were based on unique DNA fragments produced from R. equi reference strains and field isolates. Following random amplification of polymorphic DNA from R. equi and R. rhodochrous with a set of 40 arbitrary 10–base pair (bp) primers, a pair of species-specific primers was designed to detect a unique 700-bp fragment of R. equi chromosomal DNA. This PCR product was limited to R. equi and was not detectable in other Rhodococcus species or in a panel of additional gram-positive and gram-negative bacteria.

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