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ARS Home » Plains Area » Fargo, North Dakota » Edward T. Schafer Agricultural Research Center » Cereal Crops Research » Research » Publications at this Location » Publication #365734

Research Project: Improvement of Biotic Stress Resistance in Durum and Hard Red Spring Wheat Using Genetics and Genomics

Location: Cereal Crops Research

Title: Nested loop PCR (NL-PCR) for amplification of large DNA fragments with complex structures in wheat [Abstract]

item LONG, YUNMING - North Dakota State University
item SUN, QING - North Dakota State University
item STEUERNAGEL, BURKHARD - John Innes Center
item CAI, XIWEN - North Dakota State University
item Faris, Justin
item HARRIS, MARION - North Dakota State University
item WULFF, BRANDE - John Innes Center
item LAGUDAH, EVANS - Commonwealth Scientific And Industrial Research Organisation (CSIRO)
item Xu, Steven

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 5/16/2019
Publication Date: 7/21/2019
Citation: Long, Y., Sun, Q., Steuernagel, B., Cai, X., Faris, J.D., Harris, M.O., Wulff, B.B., Lagudah, E.S., Xu, S.S. 2019. Nested loop PCR (NL-PCR) for amplification of large DNA fragments with complex structures in wheat [Abstract]. 1st International Wheat Congress. July 21-26, 2019. Saskatoon, Saskatchewan, Canada. Poster No. P048.

Interpretive Summary:

Technical Abstract: Polymerase chain reaction (PCR) is a powerful tool in molecular biology. However, it is still a challenge to amplify large DNA fragments with complex secondary structures such as forward and/or reverse repeats and interlaced GC-rich regions. Here we describe a novel PCR protocol named nested loop PCR (NL-PCR). In this protocol, the reaction mixtures were supplemented with betaine and bovine serum albumin to enhance amplification efficiency and specificity and to overcome hindrance caused by trace amounts of contaminants. Usually, a high temperature and/or a high concentration of the additives can be applied to dissociate the secondary structure with high stability in templates for strand extension. However, Taq polymerase activity is limited to temperatures below 68 °C in the betaine-containing PCR mixtures, while a higher additive concentration may melt the newly-generated double-stranded DNA at GC-poor regions causing PCR failure. To remedy this, the NL-PCR protocol uses a temperature loop instead of the constant extension temperature used in normal PCR programs. The effectiveness of NL-PCR was confirmed by sequencing a tiling path of amplicons with an average size of 7.5 kb spanning a 120-kb contiguous sequence in a highly-repetitive region in Aegilops tauschii. Among these amplicons, one contained three forward repeats, two reverse repeats, and a region with interlaced GC-rich (> 85%) and GC-poor (< 15%) fragments. NL-PCR will facilitate genomic research in wheat and other species with large and complex genomes. It will be particularly useful for the targeted amplification of genes or special fragments and development of DNA markers for genomic regions hitherto untapped due to the restrictions of conventional PCR amplification.