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This article in CS

  1. Vol. 52 No. 3, p. 1229-1234
     
    Received: Oct 20, 2011


    * Corresponding author(s): phil.bregitzer@ars.usda.gov
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doi:10.2135/cropsci2011.10.0560

An Efficient Method for Flanking Sequence Isolation in Barley

  1. Ryan H. Browna,
  2. Lynn Dahleenb and
  3. Phil Bregitzer *a
  1. a USDA-ARS, National Small Grains Germplasm Research Facility, 1691 S. 2700 W., Aberdeen, ID 83210
    b USDA-ARS, Northern Crop Science Laboratory, 1605 Albrecht Blvd, Fargo, ND 58102. Mention of trade names or commercial products in this publication is solely to provide specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. USDA is an equal opportunity employer

Abstract

A polymerase chain reaction (PCR)-based adapter ligation method was developed to determine native barley (Hordeum vulgare L.) sequences flanking Dissociation (Ds) insertions and barley expressed sequence tags (ESTs). This method is simple and efficient, with the majority (∼70%) of queries returning valid sequence information. The success of this method can be traced to experimental protocols that control sample cross contamination and that segregate specific products from nonspecific products. This report describes the protocol in detail, quantifies its efficiency to enable comparisons to future modifications, and discusses specific problems that may be addressed to enable further improvements to this method.

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Copyright © 2012. Copyright © by the Crop Science Society of America, Inc.