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 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 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

A specific and versatile genome walking technique

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

A tailing genome walking method suitable for genomes with high local GC content

2013 ◽  
Vol 441 (2) ◽  
pp. 101-103 ◽  
Author(s):  
Taian Liu ◽  
Yongxiang Fang ◽  
Wenjuan Yao ◽  
Qisai Guan ◽  
Gang Bai ◽  
...  
Keyword(s):  
Gc Content ◽  
Genome Walking

Cloning and Recombinant Expression of Chitosanase Gene from Bacillus Sp. S-1

2011 ◽  
Vol 343-344 ◽  
pp. 994-999
Author(s):  
Yu Ying Sun ◽  
Shu Jun Wang ◽  
Ji Quan Zhang
Keyword(s):  
Recombinant Expression ◽  
Full Length ◽  
Genome Walking ◽  
Predicted Amino Acid Sequence ◽  
Accession Number ◽  
Physiological Properties ◽  
A Genome ◽  
Full Length Sequence ◽  
Flanking Sequences

In this work, we report the characterization of a chitosanase-producing bacterium isolated from soil. This strain was grouped under the genus Bacillus by virtue of its morphological, physiological properties and 16S rDNA gene sequence and named it Bacillus sp. S-1. According to the information of chitosanase full-length sequences deposited in NCBI, a pair of degenerated primes was designed and a partial sequence of chitosanase gene was obtained by polymerase chain reaction (PCR) using Bacillus sp. S-1 genome DNA as the template. A genome walking library was constructed followed as the protocol provided by CLONTECH Company. The flanking sequences of the 5’ and 3’ terminal was obtained by genome walking method and two-step PCR technique. After overlapped and confirmed, the full-length sequence of chitosanase from Bacillus sp. S-1 was achieved and it contained 1362 bp coding 453 amino acids (accession number is EU924147). The predicted amino acid sequence was 96% similar to that of Bacillus cereus ATCC 14579 (accession number is NC_004722). The fusion protein containing BSCHITO was produced in Escherichia coli and purified using Ni-NTA affinity chromatography. The purified rBSCHITO degraded the chitosan (the degree of deacetylation of 99%) to produce mixture of chitooligosaccharides. The BSCHITO is thus an endo-chitosanase.

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