Genome walking of large fragments: An improved method

A. S. Rishi; Neil D Nelson; Arun Goyal

doi:10.1016/j.jbiotec.2004.03.008

Genome walking of large fragments: An improved method

A. S. Rishi, Neil D Nelson, Arun Goyal

Natural Resources Research Institute

Research output: Contribution to journal › Article › peer-review

21 Scopus citations

Abstract

The PCR-based genome walking method has been commonly used to isolate upstream regions from known cDNA sequences. The limitation of this technique is based on the location of the restriction site upstream to the gene-specific primer in the genome; hence, different restriction enzymes have to be used to isolate larger upstream fragments. In this paper, we present the advantageous use of partial and size-selected DNA as templates for genome walking, in isolating larger upstream fragments. We have successfully tested this approach to isolate larger upstream fragments using the FailSafe™ PCR System. Use of partial digestion and size selection can provide better chances in obtaining larger flanking regions of known DNA sequence, when compared to use of total digested DNA.

Original language	English (US)
Pages (from-to)	9-15
Number of pages	7
Journal	Journal of Biotechnology
Volume	111
Issue number	1
DOIs	https://doi.org/10.1016/j.jbiotec.2004.03.008
State	Published - Jul 1 2004

Bibliographical note

Funding Information:
Project funded by Sota Tec Fund (Grant No. S2001-0003), USDA-CSREES (Grant No. 2001-34158-11118), and Minnesota State Special appropriations to UMD-NRRI. The authors thank Mr. V. Thompson for help in graphic design.

Copyright:
Copyright 2008 Elsevier B.V., All rights reserved.

Keywords

FailSafe™ PCR
Genome walking
Long-distance PCR
Promoter
Upstream elements

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10.1016/j.jbiotec.2004.03.008

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title = "Genome walking of large fragments: An improved method",

abstract = "The PCR-based genome walking method has been commonly used to isolate upstream regions from known cDNA sequences. The limitation of this technique is based on the location of the restriction site upstream to the gene-specific primer in the genome; hence, different restriction enzymes have to be used to isolate larger upstream fragments. In this paper, we present the advantageous use of partial and size-selected DNA as templates for genome walking, in isolating larger upstream fragments. We have successfully tested this approach to isolate larger upstream fragments using the FailSafe{\texttrademark} PCR System. Use of partial digestion and size selection can provide better chances in obtaining larger flanking regions of known DNA sequence, when compared to use of total digested DNA.",

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AU - Nelson, Neil D

AU - Goyal, Arun

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N2 - The PCR-based genome walking method has been commonly used to isolate upstream regions from known cDNA sequences. The limitation of this technique is based on the location of the restriction site upstream to the gene-specific primer in the genome; hence, different restriction enzymes have to be used to isolate larger upstream fragments. In this paper, we present the advantageous use of partial and size-selected DNA as templates for genome walking, in isolating larger upstream fragments. We have successfully tested this approach to isolate larger upstream fragments using the FailSafe™ PCR System. Use of partial digestion and size selection can provide better chances in obtaining larger flanking regions of known DNA sequence, when compared to use of total digested DNA.

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KW - Promoter

KW - Upstream elements

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Genome walking of large fragments: An improved method

Abstract

Bibliographical note

Keywords

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