Location: Cereal Crops ResearchTitle: Nested loop PCR (NL-PCR) for amplification of large DNA fragments with complex structures in wheat [Abstract]
|LONG, YUNMING - North Dakota State University|
|SUN, QING - North Dakota State University|
|STEUERNAGEL, BURKHARD - John Innes Center|
|CAI, XIWEN - North Dakota State University|
|HARRIS, MARION - North Dakota State University|
|WULFF, BRANDE - John Innes Center|
|LAGUDAH, EVANS - Commonwealth Scientific And Industrial Research Organisation (CSIRO)|
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.
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.