利用PCR方法鉴定hiTAIL-PCR扩增产物中的质粒骨架片段

doi:10.11983/CBB16216

摘要/Abstract

摘要： T-DNA突变体是研究基因功能的重要资源。高效热不对称交错PCR (hiTAIL-PCR)是克隆突变体中T-DNA插入位点侧翼序列的常用方法。然而我们发现, 利用hiTAIL-PCR克隆到的一些侧翼序列并不对应于宿主的染色体DNA序列, 而是质粒的骨架DNA片段。通过设置1组RB-S4/AC1或者LB-A4/AC1对照反应, 用PCR方法鉴定了hiTAIL-PCR扩增产物中位于T-DNA侧翼的质粒骨架片段。在后续分析中, 通过排除这些片段, 提高了利用hiTAIL-PCR获得宿主染色体DNA片段的效率。同时, 通过调整反应程序, 使得整个PCR的反应时间也大为缩短。在拟南芥(Arabidopsis thaliana) T-DNA突变体drf1侧翼序列的克隆实例中, 对照反应的引入将hiTAIL-PCR中需鉴定的22条扩增产物降至4条, 效率提高了81.8%。

关键词: hiTAIL-PCR, T-DNA突变体, 侧翼序列, 质粒骨架, 克隆

Abstract: T-DNA mutants are important resources for research of gene function. High-efficiency thermal asymmetric interlaced PCR (hiTAIL-PCR) is widely used for cloning the flanking sequence near the T-DNA insertion sites. However, we found that some cloned flanking fragments in hiTAIL-PCR products corresponded not to the host genomic DNA but to the plasmid backbone DNA. In this study, with a control of the RB-S4/AC1 or LB-A4/AC1 product, we amplified PCR fragments from the plasmid backbone DNA. By excluding them from further analysis, we amplified fragments from the unknown genomic DNA more effectively. Meanwhile, by adjusting the PCR programs, the whole PCR time was greatly shortened. In cloning the flanking sequence of Arabidopsis thaliana T-DNA mutant drf1, our method with hiTAIL-PCR reduced the total 22 DNA bands required for further checking to 4 bands, which improved the efficiency by 81.8%.

Key words: hiTAIL-PCR, T-DNA mutant, flanking sequence, plasmid backbone, cloning

曹媛, 杨云, 徐化全, 刘洋, 王丹阳. 利用PCR方法鉴定hiTAIL-PCR扩增产物中的质粒骨架片段. 植物学报, 2018, 53(1): 104-109.

Yuan Cao, Yun Yang, Huaquan Xu, Yang Liu, Danyang Wan. PCR Used to Find Plasmid Backbone Fragments in the Products of hiTAIL-PCR. Chinese Bulletin of Botany, 2018, 53(1): 104-109.

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链接本文: https://www.chinbullbotany.com/CN/10.11983/CBB16216

https://www.chinbullbotany.com/CN/Y2018/V53/I1/104

图/表 4

表1 根据T-DNA序列设计的特异性引物以及随机引物

Table 1 Specific primers designed according to T-DNA sequence and random primers

Primers	Sequence (5'-3')
Specific primers
RB-S1	GTTATCCGCTCACAATTCCACA
RB-S2	TCGGGAAACCTGTCGTGCCA
RB-S3	AGAGGCGGTTTGCGTATTGGG
RB-S4	AAGTCGCTGTATGTGTTTGTTTGAGA
LB-A1	GGCGGACCGCTATCAGGACAT
LB-A2	TTGGCTACCCGTGATATTGCTG
LB-A3	GACCGCTTCCTCGTGCTTTA
LB-A4	GTTACACCACAATATATCCTGCCAAGAT
AC1	ACGATGGACTCCAGAG
DRF1-S	ACAACAGAAACAACCAAAAATAATG
QRT-S	TGTGCAGGAGACATCATTCC
QRT-A	TTTCGCATTGCCAAAGAT
Random primers
LAD1-1	ACGATGGACTCCAGAVNVNNNGGAA
LAD1-2	ACGATGGACTCCAGABNBNNGGTT
LAD1-3	ACGATGGACTCCAGAVVNVNNNCCAA
LAD1-4	ACGATGGACTCCAGABDNBNNNCGGT
LAD1-QA	ACGATGGACTCCAGAGWWWWHWWACCT
LAD1-HS	ACGATGGACTCCAGAGWWWWWWDYAGG
LAD1-A	ACGATGGACTCCAGAGVNVNNNGGCC
LAD1-B	ACGATGGACTCCAGAGBNBNNGGGG
LAD1-C	ACGATGGACTCCAGAGVVNVNNNCCGG
LAD1-D	ACGATGGACTCCAGAGBDNBNNNCCCC
LAD1-E	ACGATGGACTCCAGAGVNVNNNCAGA
LAD1-F	ACGATGGACTCCAGAGVNVNNNAGAT

图1 设计原理(A) T-DNA质粒RB和LB处区域; (B) T-DNA质粒与染色体DNA整合后RB和LB处区域; (C) 当hiTAIL-PCR扩增质粒骨架时, RB-S2/AC1、RB-S3/AC1与RB-S4/AC1都将产生相应扩增; (D) 当hiTAIL-PCR扩增基因组DNA时, RB-S4/AC1将不产生相应扩增(虚线框示未扩增)

Figure 1 The principle of design(A) The regions of RB and LB of T-DNA plasmid; (B) The regions of RB and LB which integrate with chromosomal DNA; (C) When hiTAIL-PCR amplifies the backbone of plas- mid, the three PCR groups containing the RB-S2/AC1, RB-S3/AC1 and RB-S4/AC1 primer pairs, respectively, will all produce the positive bands; (D) When hiTAIL-PCR amplifies the genomic DNA, RB-S4/AC1 will not produce the positive bands (dashed frame)

图2 拟南芥突变体drf1 T-DNA插入位点侧翼序列的克隆(A) 用RB-S系列引物扩增的第2轮PCR的结果; (B) 用LB-A系列引物扩增的第2轮PCR的结果。* 代表非特异性扩增结果; ☆代表潜在的特异性扩增结果; ** 代表来自RB之前或LB之后的T-DNA片段扩增结果。

Figure 2 The cloning of flanking sequence at the T-DNA insertion site of Arabidopsis mutant drf1(A) The second round results amplified by PCR with RB-S serial primers; (B) The second round results amplified by PCR with LB-A serial primers. * display the non-specific amplification results; ☆ represent the potential specific amplification results; ** show the amplification results from the T-DNA region before RB or after LB.

图3 拟南芥突变体drf1侧翼序列的PCR验证(A) 部分测序结果; (B) 用引物对DRF1-S/LB-A2 (1,3)及DRF1-S/LB-A3 (2,4)扩增野生型(WT)与drf1突变体基因组的结果; (C) 用引物对QRT-S/QRT-A扩增野生型(WT)与drf1突变体基因组的结果

Figure 3 The PCR confirmation for Arabidopsis mutant drf1 flanking sequence(A) The partial sequencing result; (B) The results amplifying the genomic DNA from wild type (WT) and drf1 mutant with primer pairs of DRF1-S/LB-A2 (1,3) or DRF1-S/LB-A3 (2,4); (C) The PCR results with primer pairs of QRT-S/QRT-A to amplifying WT and drf1 mutant genomic DNA

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