Improved Glass Bead-Vortex Oscillation Method for DNA Extraction from Diatom

doi:10.12116/j.issn.1004-5619.2021.410801

Abstract

Abstract: Objective

To examine the effect of improving diatom DNA extraction by glass bead - vortex oscillation method.

Methods

The DNeasy PowerSoil Pro kit was used as control, two plant DNA extraction kits with different principles （New Plant genomic DNA extraction kit and Plant DNA Isolation kit） and one whole blood DNA extraction kit （whole blood genomic DNA extraction kit） were selected to extract diatom DNA from lung tissue and water sample of the same drowning case. The combination of mass ratio of glass beads with different sizes and vortex oscillation time was designed, and the optimal DNA extraction conditions were selected with the addition of glass beads oscillation. The extracted products of the conventional group and the modified group were directly electrophoretic and detected by diatom specific PCR. Finally, all the extracts were quantified by qPCR, and the Ct values of different groups were statistically analyzed.

Results

When the frequency of vortex oscillation was 3 000 r/min, the optimal combination of DNA extraction was vortex oscillation for 4 min, and the mass ratio of large glass beads to small glass beads was 1∶1. The DNeasy PowerSoil Pro kit was used as a reference, and the Ct value of 10 mL water sample was greater than that of 0.5 g tissue. The Ct values of the other three kits used for plant DNA extraction decreased after the glass beads-vortex oscillation method was used, and the Ct values of the tissues before and after the improvement were statistically significant （P<0.05）. The whole blood genomic DNA extraction kit used in this study could successfully extract diatom DNA, the extraction of water samples was close to DNeasy PowerSoil Pro kit, after the modified method was applied to tissue samples, the difference in Ct value was statistically significant （P<0.05）. However, when the three kits were used to extract diatom DNA from water samples, Ct values before and after the improvement were only statistically significant in New Plant genomic DNA extraction kit group （P<0.05）.

Conclusion

The improved glass bead-vortex oscillation method can improve the extraction efficiency of diatom DNA from forensic materials, especially from tissue samples, by plant and blood DNA extraction kits.

Key words: forensic pathology, drowning, DNA extraction, diatom, glass bead-vortex oscillation improving method

CLC Number:

DF795.1

Jie CAI, Bo WANG, Sun-lin HU, Yi-hong QU, Tao SONG, Jian-hua CHEN, Jian-qiang DENG. Improved Glass Bead-Vortex Oscillation Method for DNA Extraction from Diatom[J]. Journal of Forensic Medicine, 2022, 38(1): 119-126.

Figures/Tables 5

Tab. 1 Combination of different glass bead proportion and vortex oscillation time

振荡时间/min	大小玻璃珠混合质量比
振荡时间/min	1∶2	1∶1	2∶1
2	①	②	③
3	④	⑤	⑥
4	⑦	⑧	⑨
5	⑩	?	?
6	?	?	?

Fig. 1 The electrophoresis result of different glass bead mass proportions and vortex oscillation time combinations

Fig. 2 Electrophoresis results of PCR amplification products extracted from three kits extracts

Fig. 3 Electrophoresis results of PCR amplification products extracted from four kits

Tab. 2 Comparison of Ct values of water samples and tissues before and after improvement with three kits

试剂盒	水样		组织
试剂盒	常规组	改良组	常规组	改良组
PowerSoil	24.06±0.91	-	16.97±0.60^1）	-
TIANGEN	25.71±0.63^2）	24.33±0.58^3）	26.56±0.30^2）	20.97±1.38^2）3）
FOREGENE	22.16±0.32^2）	21.86±0.32^2）	25.98±1.26^2）	19.60±0.89^2）3）
BioTeKe	23.67±1.49	21.94±0.40^2）	26.56±1.07^2）	19.38±0.77^2）3）

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