Horticultural Science & Technology (원예과학기술지)

Korean Society of Horticultural Science (한국원예학회)
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Development of an Effective PCR Technique for Analyzing T-DNA Integration Sites in Brassica Species and Its Application

배추과에서 T-DNA 도입 위치 분석을 위한 효과적인 PCR 방법 개발 및 이용

  • Lee, Gi-Ho (Department of Horticultural Biotechnology, Kyunghee University) ;
  • Yu, Jae-Gyeong (Department of Horticultural Biotechnology, Kyunghee University) ;
  • Park, Young-Doo (Department of Horticultural Biotechnology, Kyunghee University)
  • 이기호 (경희대학교 생명과학대학 원예생명공학과) ;
  • 유재경 (경희대학교 생명과학대학 원예생명공학과) ;
  • 박영두 (경희대학교 생명과학대학 원예생명공학과)
  • Received : 2014.05.09
  • Accepted : 2014.06.08
  • Published : 2015.04.30
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Abstract

Insertional mutagenesis induced by T-DNA or transposon tagging offers possibilities for analysis of gene function. However, its potential remains limited unless good methods for detecting the target locus are developed. We describe a PCR technique for efficient identification of DNA sequences adjacent to the inserted T-DNA in a higher plant, Chinese cabbage (Brassica rapa ssp. pekinensis). This strategy, which we named variable argument thermal asymmetric interlaced PCR (VA-TAIL PCR), was designed by modifying a single-step annealing-extension PCR by including a touch-up PCR protocol and using long gene-specific primers. Amplification efficiency of this PCR program was significantly increased by employing an autosegment extension method and linked sequence strategy in nested long gene-specific primers. For this technique, arbitrary degenerate (AD) primers specific to B. rapa were designed by analyzing the Integr8 proteome database. These primers showed higher accuracy and utility in the identification of flanking DNA sequences from individual transgenic Chinese cabbages in a large T-DNA inserted population. The VA-TAIL PCR method described in this study allows the identification of DNA regions flanking known DNA fragments. This method has potential biotechnological applications, being highly suitable for identification of target genomic loci in insertional mutagenesis screens.

기능 유전체 연구에서 이동유전자 또는 T-DNA 도입을 이용한 삽입돌연변이체 분석은 미지의 유전자 기능을 분석할 수 있게 한다. 이에 따라 본 연구에서는 배추(Brassica rapa ssp. pekinensis)와 같은 고등 식물에서 genomic DNA조각을 분리할 수 있는 효과적인 PCR 방법을 개발하였다. 본 연구에서 개발한 variable argument thermal asymmetric interlaced PCR(VA-TAIL PCR)은 single-step annealin-gextension PCR 방법과 길게 구성된 유전자 특이적 primer 및 touch-up PCR 방법이 적용되었으며, 증폭 효율을 증가시키기 위하여 autosegment extension 증폭 방법이 적용되었다. 또한 개발된 VA-TAIL PCR 방법은 배추에 특화된 변성 primer를 Integr8 단백질체 데이터베이스를 분석하여 작성하였으며 대량의 배추 T-DNA 삽입 돌연변이 집단에서 각각의 T-DNA 인접 염기 서열을 분석하는 데 매우 정확하고 효과적인 것으로 분석되었다. 따라서 본 연구에서 개발된 VA-TAIL PCR 방법은 기존에 확인된 염기 서열을 이용하여 양쪽 방향을 모두 분석할 수 있기 때문에, 유전체 연구에서 T-DNA 또는 기 밝혀진 염기 서열에 인접한 미지의 염기서열을 확인하는데 매우 효과적일 것으로 기대된다.

Keywords

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