The extraction and purification of nucleic acids is one of the key technologies for forensic DNA bioassay. The most commonly used methods in the laboratory include the spin column magnetic bead method and the clean-up system. The magnetic bead method is easy and fast to operate. The high-purity nucleic acid DNA is extracted and can be automated, thus becoming the main means of nucleic acid extraction and purification in the forensic DNA laboratory. However, due to the imbalance of the magnetic molecules adsorbed by the magnetic beads and the breakage and loss of the nucleic acid during the subsequent elution process, the PCR method fails in the verification process of the difficult samples, and the DNA test is unsuccessful. Happening.
At present, in the forensic material certificate test, the difficult samples mainly include the following characteristics:
1. Low-copy DNA, how to improve the efficiency of DNA extraction in low-copy (LCN) samples such as contact DNA during the test, and improve the success rate of detection;
2, the inhibitor content is high and there are many types, how to effectively remove the inhibitors contained in textiles, hair, tissues, bones, soil and other samples, and improve the success rate of PCR amplification;
3, highly degraded, how to improve the DNA detection rate of highly degraded samples;
It is understood that a research project funded by the US Department of Justice uses SCODA (Synchronous Rotating Electric Field Technology) to effectively remove various types of inhibitors in the nucleic acid extraction process , and related papers have been published in the International Journal of Forensic Medicine. The new DNA purification technology, synchronous rotating electric field technology (Aurora, provided by Boreal Genomics, Canada), the author selected a variety of inhibitors that affect PCR amplification, SCODA technology and current mainstream DNA The performance of the purification kits was compared, and the purified samples were subjected to STR typing for further study of the effects of inhibitors on late typing. The paper included the following common inhibitors:
- Melanin: found in hair and tissues
- Collagen and calcium phosphate: found in bones and tissues
- Humic acid: present in the soil
- Heme: present in fresh blood
- Tannins and dyes: found in clothing and various textiles
The following figure compares the removal effects of different inhibitors using SCODA technology and common purification kits;
Figure a: Purification effect of adding melanin and humic acid to DNA samples (using SCODA technology)
Figure b: Purification effect of an internationally renowned brand purification kit after adding melanin to DNA samples
Figure c Purification effect of an internationally renowned brand purification kit after adding humic acid to the DNA sample
The results show that for the samples containing severe inhibition of PCR, the SCODA electrophoresis extraction and purification technology is used, and the purification effect of the samples is much better than the traditional magnetic beads method and solid phase adsorption technology. The relevant test results are as follows:
1, SCODA technology (Aurora) and a purification kit to remove different inhibitors, the final STR typing site acquisition rate and signal strength comparison results
2. The sample containing 600ug of humic acid was purified by SCODA technology (Aurora) and a kit, and then STR was typed. Aurora could obtain a good peak signal, while the sample signal purified by the traditional kit was very weak.
In summary, the advantages of SCODA electrophoresis extraction and purification technology over traditional purification kits are obvious, including:
- Improve the extraction amount and purity of low-copy DNA samples, and achieve higher PCR amplification success rate
- Efficient removal of inhibitors and contaminants from samples: Effectively removes inhibitors and contaminants from various materials such as textiles, blood, hair, tissues, bones, soil, etc., and removes contaminants more than conventional techniques. Thorough, can be directly used for subsequent amplification, cloning and analysis
- Maintain DNA fragment integrity without mechanical fragmentation
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