Biotechnology Bulletin ›› 2023, Vol. 39 ›› Issue (5): 103-111.doi: 10.13560/j.cnki.biotech.bull.1985.2022-1085
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LI Xin-yi1,3(), JIANG Chun-xiu1,3, XUE Li1,3, JIANG Hong-tao1,3, YAO Wei1,3, DENG Zu-hu1,2,3, ZHANG Mu-qing1,3, YU Fan1,3()
Received:
2022-09-02
Online:
2023-05-26
Published:
2023-06-08
Contact:
YU Fan
E-mail:lixinyi1776@163.com;yufanky@163.com
LI Xin-yi, JIANG Chun-xiu, XUE Li, JIANG Hong-tao, YAO Wei, DENG Zu-hu, ZHANG Mu-qing, YU Fan. Enhancing Hybridization Signal of Sugarcane Chromosome Oligonucleotide Probe via Multiple Fluorescence Labeled Primers[J]. Biotechnology Bulletin, 2023, 39(5): 103-111.
引物名称 Primer name | 引物序列 Primer sequence |
---|---|
T7-Cy3 | /iCy3dT/ACGACTCACTATAGGGAGA |
D9-Cy3 | /iCy3dA/GTGTGTACGGACTAATAAT |
T7-FAM | /iFAMdT/ACGACTCACTATAGGGAGA |
D9-FAM | /iFAMdA/GTGTGTACGGACTAATAAT |
T7-Cy3-3 | /iCy3dT/ACGAC/iCy3dT/CAC/iCy3dT/ATAGGGAGA |
D9-Cy3-3 | /iCy3dA/G/iCy3dT/G/iCy3dT/GTACGGACTAATAAT |
T7-FAM-3 | /iFAMdT/ACGAC/iFAMdT/CAC/iFAMdT/ATAGGGAGA |
D9-FAM-3 | /iFAMdA/G/iFAMdT/G/iFAMdT/GTACGGACTAATAAT |
Table 1 Amplification sequences of oligo probe fluoresc-ence labeled primers
引物名称 Primer name | 引物序列 Primer sequence |
---|---|
T7-Cy3 | /iCy3dT/ACGACTCACTATAGGGAGA |
D9-Cy3 | /iCy3dA/GTGTGTACGGACTAATAAT |
T7-FAM | /iFAMdT/ACGACTCACTATAGGGAGA |
D9-FAM | /iFAMdA/GTGTGTACGGACTAATAAT |
T7-Cy3-3 | /iCy3dT/ACGAC/iCy3dT/CAC/iCy3dT/ATAGGGAGA |
D9-Cy3-3 | /iCy3dA/G/iCy3dT/G/iCy3dT/GTACGGACTAATAAT |
T7-FAM-3 | /iFAMdT/ACGAC/iFAMdT/CAC/iFAMdT/ATAGGGAGA |
D9-FAM-3 | /iFAMdA/G/iFAMdT/G/iFAMdT/GTACGGACTAATAAT |
Fig. 2 Comparison of flow before and after optimization of FISH Blue area: Pre-optimization hybridization step; red area: post-optimization hybridization step; yellow marker: improvement step; dashed part: pre-hybridization step area
Fig. 3 Signal capture of Cy3 and FAM with different exposure gradients based on hybridization system optimization a: Imaging of the FAM probe at different exposure duration in the unoptimized hybridization system; b: imaging of the FAM probe at different exposure duration in the optimized hybridization system; c: imaging of the Cy3 probe at different exposure duration in the unoptimized hybridization system; d: imaging of the Cy3 probe at different exposure duration in the optimized hybridization system; e: comparison of fluorescence density before and after optimization system, the red background is marked with Cy3, the green background is marked with FAM.IntDen: Intensity Image. bar=10 μm
Fig. 4 Signal capture of Cy3 and FAM with different exposure gradients based on different fluorophore group probes a: Imaging of FAM probe at different exposure duration under single fluorescence modification; b: imaging of FAM probe at different exposure duration under three fluorescence modifications; c: imaging of Cy3 probe at different exposure duration under unoptimized hybridization system; d: imaging of Cy3 probe at different exposure duration under three fluorescence modifications; e: comparison graph of fluorescence density under different number of fluorescence modifications, red background for Cy3 marker, green background is FAM marker; IntDen is gray scale value; scale bar is 10 μm
oligo探针 oligo probes | 高粱染色体 Sorghum chromosome | ||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|
1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | ||
Chr1 | 18 400 | 63 | 120 | 79 | 51 | 84 | 68 | 46 | 55 | 66 | |
Chr2 | 73 | 12 522 | 55 | 65 | 29 | 55 | 30 | 80 | 34 | 42 | |
Chr3 | 45 | 46 | 15 771 | 61 | 54 | 47 | 41 | 30 | 125 | 41 | |
Chr4 | 28 | 47 | 48 | 11 448 | 46 | 42 | 29 | 28 | 18 | 65 | |
Chr5 | 27 | 34 | 26 | 107 | 3 680 | 29 | 16 | 53 | 25 | 44 | |
Chr6 | 85 | 20 | 70 | 93 | 48 | 7 959 | 32 | 39 | 41 | 47 | |
Chr7 | 75 | 81 | 54 | 33 | 40 | 36 | 7 760 | 30 | 39 | 95 | |
Chr8 | 29 | 34 | 21 | 65 | 143 | 25 | 25 | 5 218 | 24 | 40 | |
Chr9 | 28 | 39 | 43 | 15 | 25 | 33 | 15 | 26 | 8 592 | 57 | |
Chr10 | 86 | 112 | 47 | 28 | 46 | 21 | 33 | 60 | 46 | 9 573 |
Table 2 Number statistics of sorghum genome aligned by S. officinarum oligos probes
oligo探针 oligo probes | 高粱染色体 Sorghum chromosome | ||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|
1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | ||
Chr1 | 18 400 | 63 | 120 | 79 | 51 | 84 | 68 | 46 | 55 | 66 | |
Chr2 | 73 | 12 522 | 55 | 65 | 29 | 55 | 30 | 80 | 34 | 42 | |
Chr3 | 45 | 46 | 15 771 | 61 | 54 | 47 | 41 | 30 | 125 | 41 | |
Chr4 | 28 | 47 | 48 | 11 448 | 46 | 42 | 29 | 28 | 18 | 65 | |
Chr5 | 27 | 34 | 26 | 107 | 3 680 | 29 | 16 | 53 | 25 | 44 | |
Chr6 | 85 | 20 | 70 | 93 | 48 | 7 959 | 32 | 39 | 41 | 47 | |
Chr7 | 75 | 81 | 54 | 33 | 40 | 36 | 7 760 | 30 | 39 | 95 | |
Chr8 | 29 | 34 | 21 | 65 | 143 | 25 | 25 | 5 218 | 24 | 40 | |
Chr9 | 28 | 39 | 43 | 15 | 25 | 33 | 15 | 26 | 8 592 | 57 | |
Chr10 | 86 | 112 | 47 | 28 | 46 | 21 | 33 | 60 | 46 | 9 573 |
Fig. 5 S. officinarum Oligos sequences distribute on the chromosome of sorghum a: The hot map of Oligo probe distribution. b: The statistics of oligo probe density
Fig. 6 Oligo probe located on sorghum chromosome a: Probe Chr1 and Chr2 located on sorghum chromosome; b: probe Chr3 and Chr4 located on sorghum chromosome; c: probe Chr5 and Chr6 located on sorghum chromosome; d: probe Chr7 and Chr8 located on sorghum chromosome; e: probe Chr9 and Chr10 located on sorghum chromosome; Cy3 is red, FAM is green; bar=10 μm
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