Genome walking by Klenow polymerase

2012 ◽  
Vol 430 (2) ◽  
pp. 200-202 ◽  
Author(s):  
Mariateresa Volpicella ◽  
Claudia Leoni ◽  
Immacolata Fanizza ◽  
Sebastian Rius ◽  
Raffaele Gallerani ◽  
...  
Keyword(s):  
Genome Walking ◽  
Klenow Polymerase

scholarly journals Genome walking in eukaryotes

FEBS Journal ◽  
2011 ◽  
Vol 278 (21) ◽  
pp. 3953-3977 ◽  
Author(s):  
Claudia Leoni ◽  
Mariateresa Volpicella ◽  
Francesca De Leo ◽  
Raffaele Gallerani ◽  
Luigi R. Ceci
Keyword(s):  
Genome Walking

scholarly journals Directional Genome Walking Using PCR

BioTechniques ◽  
2002 ◽  
Vol 33 (4) ◽  
pp. 830-834 ◽  
Author(s):  
R.N. Mishra ◽  
S.L. Singla-Pareek ◽  
S. Nair ◽  
S.K. Sopory ◽  
M.K. Reddy
Keyword(s):  
Genome Walking

scholarly journals Palindromic sequence-targeted (PST) PCR: a rapid and efficient method for high-throughput gene characterization and genome walking

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Ruslan Kalendar ◽  
Alexandr V. Shustov ◽  
Mervi M. Seppänen ◽  
Alan H. Schulman ◽  
Frederick L. Stoddard
Keyword(s):  
Thermal Cycling ◽  
Annealing Temperature ◽  
Phleum Pratense ◽  
Genome Walking ◽  
Palindromic Sequence ◽  
Linear Amplification ◽  
Gene Characterization ◽  
Intron 1 ◽  
Thermal Cycling Profile ◽  
Palindromic Sequences

AbstractGenome walking (GW) refers to the capture and sequencing of unknown regions in a long DNA molecule that are adjacent to a region with a known sequence. A novel PCR-based method, palindromic sequence-targeted PCR (PST-PCR), was developed. PST-PCR is based on a distinctive design of walking primers and special thermal cycling conditions. The walking primers (PST primers) match palindromic sequences (PST sites) that are randomly distributed in natural DNA. The PST primers have palindromic sequences at their 3′-ends. Upstream of the palindromes there is a degenerate sequence (8–12 nucleotides long); defined adapters are present at the 5′-termini. The thermal cycling profile has a linear amplification phase and an exponential amplification phase differing in annealing temperature. Changing the annealing temperature to switch the amplification phases at a defined cycle controls the balance between sensitivity and specificity. In contrast to traditional genome walking methods, PST-PCR is rapid (two to three hours to produce GW fragments) as it uses only one or two PCR rounds. Using PST-PCR, previously unknown regions (the promoter and intron 1) of the VRN1 gene of Timothy-grass (Phleum pratense L.) were captured for sequencing. In our experience, PST-PCR had higher throughput and greater convenience in comparison to other GW methods.

Isolation and evaluation of three novel native promoters in Brassica napus

Botany ◽  
2013 ◽  
Vol 91 (6) ◽  
pp. 414-419 ◽  
Author(s):  
Limin Wu ◽  
Aliaa El-Mezawy ◽  
Saleh Shah
Keyword(s):  
Brassica Napus ◽  
Reporter Gene ◽  
Genetic Modification ◽  
Gus Activity ◽  
The Other ◽  
Genome Walking ◽  
Constitutive Promoter ◽  
Brassica Napus L ◽  
Transgenic Canola ◽  
Uida Reporter Gene

To provide effective and specific native promoters for canola (Brassica napus L.) genetic modification, three promoters were isolated by genome walking from this species. These three promoters were fused to the uidA reporter gene (GUS) and were independently used to generate populations of transgenic canola plants. Plants transformed with BnPGPro-GUS (B. napus putative germin promoter) exhibited GUS activity in all the tissues tested at a level comparable to those transformed with CaMV35 S promoter. This indicates that BnPGPro may serve as a native constitutive promoter for canola. The other two promoters, BnPro3-GUS and BnPro5-GUS (B. napus, promoter 3 and 5), exhibited GUS activity in various tissues. None of these two promoters expressed in embryo, however. These novel Brassica native promoters can be used to modify canola genes for various purposes.

Unknown Sequence Amplification: Application to in vitro Genome Walking in Chlamydia trachomatis L2

1991 ◽  
Vol 9 (1) ◽  
pp. 74-79 ◽  
Author(s):  
C.G. Copley ◽  
C. Boot ◽  
K. Bundell ◽  
W.L. McPheat
Keyword(s):  
Chlamydia Trachomatis ◽  
Genome Walking ◽  
Unknown Sequence

scholarly journals Hyperglycemia Enhances DNA Fragmentation after Transient Cerebral Ischemia

2001 ◽  
Vol 21 (5) ◽  
pp. 568-576 ◽  
Author(s):  
Ping-An Li ◽  
Ingrid Rasquinha ◽  
Qing Ping He ◽  
Bo K. Siesjö ◽  
Katalin Csiszár ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Cell Death ◽  
Cytochrome C ◽  
Dna Fragmentation ◽  
Caspase 3 ◽  
Cingulate Cortex ◽  
Tunel Staining ◽  
Dna Fragments ◽  
Cytochrome C Release ◽  
Klenow Polymerase

Previous histopathologic results have suggested that one mechanism whereby hyperglycemia (HG) leads to exaggerated ischemic damage involves fragmentation of DNA. DNA fragmentation in normoglycemia (NG) and HG rats subjected to 30 minutes of forebrain ischemia was studied by terminal deoxynucleotidyl transferase mediated DNA nick-labeling (TUNEL) staining, by pulse-field gel electrophoresis (PFGE), and by ligation-mediated polymerase chain reaction (LM-PCR). High molecular weight DNA fragments were detected by PFGE, whereas low molecular weight DNA fragments were detected using LM-PCR techniques. The LM-PCR procedure was performed on DNA from test samples with blunt (without Klenow polymerase) and 3′-recessed ends (with Klenow polymerase). In addition, cytochrome c release and caspase-3 activation were studied by immunocytochemistry. Results show that HG causes cytochrome c release, activates caspase-3, and exacerbates DNA fragments induced by ischemia. Thus, in HG rats, but not in control or NGs, TUNEL-stained cells were found in the cingulate cortex, neocortex, thalamus, and dorsolateral crest of the striatum, where neuronal death was observed by conventional histopathology, and where both cytosolic cytochrome c and active caspase-3 were detected by confocal microscopy. In the neocortex, both blunt-ended and stagger-ended fragments were detected in HG, but not in NG rats. Electron microscopy (EM) analysis was performed in the cingulate cortex, where numerous TUNEL-positive neurons were observed. Although DNA fragmentation was detected by TUNEL staining and electrophoresis techniques, EM analysis failed to indicate apoptotic cell death. It is concluded that HG triggers a cell death pathway and exacerbates DNA fragmentation induced by ischemia.

A specific and versatile genome walking technique

Gene ◽  
2006 ◽  
Vol 381 ◽  
pp. 18-23 ◽  
Author(s):  
Haitao Guo ◽  
Jin Xiong
Keyword(s):  
Genome Walking
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