Method for recovery of sperm nucleic acid from forensic samples
A sample and sperm technology, applied in the field of sperm nucleic acid recovery from forensic samples, can solve the problems of sperm cell loss, limit the precise identification of criminals, hinder accurate sperm analysis, etc., and achieve the effect of reducing the number
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Embodiment 1
[0113] Example 1 - Comparative Differential Extraction Procedure: Prior Art Wash Solution (1×PBS) vs. MgCl 2 washing solution
[0114] 500 μL MgCl 2 Wash solution (260 mM final concentration in 1X PBS) or 1X PBS was added to the cell suspension sample containing 50 μL of a heterogeneous cell suspension containing approximately 10,000 epithelial cells, 1,000 sperm cells and impurities including residual DNA. The samples and wash solutions were then transferred to spin filter tubes (CorningspinXCostar8160, pore size 220nm). The tubes were centrifuged at 14,000 RPM in a Beckman Coulter Microfuge 18 centrifuge after which time the filtrate was discarded.
[0115] will contain 875mM NaCl 2 and 85 mMdTT, 200 μL of selective sperm lysis buffer diluted in 1×PBS were added to the spin filter tube. The sperm cells were then lysed by mixing for about 5 minutes. The tubes were centrifuged at 14,000 RPM in a Beckman Coulter Microfuge 18 centrifuge to separate sperm lysate from intact...
Embodiment 2
[0120] Example 2 - MgCl 2 Wash solution reduces exogenous epithelial DNA from epithelial cell samples
[0121] It has been found that MgCl in the concentration range of 0-260mM 2 Efficient removal of surface-bound DNA, more so than when using the classic wash solution 1X PBS (Figure 3). Epithelial cell samples were first treated with MgCl 2 Wash, dilute with 1x PBS, and mix. Wash buffer is removed and cells are incubated with selective sperm lysis buffer. Lysis was performed for 5 minutes, after which time the samples were filtered, and the DNA in the filtrate was purified and quantified. Because the lysis buffer is selective, what remains in the filtrate is residual epithelial cell DNA that was originally present in the sample. As can be seen from Figure 3, compared with the 1×PBS control, with as low as 30mM MgCl 2 Concentration reduces the amount of residual DNA. It is evident from Figure 3 that the best MgCl 2 The concentration is between 160mM and 260mM.
Embodiment 3
[0122] Example 3 - Comparing Differential Extraction Steps: DNase-Free Wash vs. DNase Wash
[0123] 500 μL MgCl 2 Wash solution (260 mM final concentration in 1×PBS) was added to the cell suspension samples containing 50 μL of the post-coitus samples. After mixing and incubation at room temperature for 5 minutes, the samples and wash solutions were transferred to spin filter tubes (CorningspinXCostar 8160, pore size 220 nm), the tubes were centrifuged at 14,000 RPM in a Beckman Coulter Microfuge 18 centrifuge, after which the filtrate was discarded.
[0124] 100 μL of DNase wash solution containing 10 units of Turbo DNase (AM2238, Applied Biosystems, Foster City, CA) was added to one of the two samples in the spin filter tube. A DNase wash was performed at 37°C for 5 minutes. The tubes were centrifuged at 14,000 RPM in a Beckman Coulter Microfuge 18 centrifuge after which time the filtrate was discarded. Two washing solutions (MgCl 2 and DNase) corresponding to the applica...
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