Improved Glass Bead-Vortex Oscillation Method for DNA Extraction from Diatom / 法医学杂志

OBJECTIVES@#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).@*CONCLUSIONS@#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.

Application Progress of High-Throughput Sequencing Technology in Forensic Diatom Detection / 法医学杂志

Diatom detection is an important method for identifying drowning and throwing corpses after death and inferring the drowning sites in forensic examination of corpses in water. In recent years,high-throughput sequencing technology has achieved rapid development and has been widely used in research related to diatom taxonomic investigations. This paper reviews the research status and prospects of high-throughput sequencing technology and its application in forensic diatom detection.

Diagnosis of amniotic fluid embolism with blood samples by liquid-based cytology technique / 法医学杂志

OBJECTIVE@#To establish the diagnosis of amniotic fluid embolism with blood samples by liquid-based cytology technique and to study the validity of method.@*METHODS@#The blood samples were collected from patients who suffered from amniotic fluid embolism. The components of amniotic fluid in blood samples were examined with blood smear by two direct smear methods (supernatant smear, sediment smear) and two liquid-based cytology methods (automatic smear, manual smear). The positive detection rate of each method was calculated.@*RESULTS@#The positive detection rates of two liquid-based cytology methods (84.6% and 92.3%, respectively) were much higher than those of two direct methods (53.8% and 61.5%, respectively).@*CONCLUSION@#The liquid-based cytology technique could improve the positive detection rate of amniotic fluid embolism.

Degenerate oligonucleotide primed-PCR technology establishment and its sensitivity test analysis / 法医学杂志

OBJECTIVE@#To establish a whole genome amplification testing system based on degenerate oligonucleotide primed-PCR (DOP-PCR) and to explore its reliability and sensitivity.@*METHODS@#DOP-PCR amplified production was detected by fluorescent labeled multiplex STR amplification and capillary electrophoresis detection system to determine reliability and sensitivity of DOP-PCR system.@*RESULTS@#DOP-PCR system was successfully established and the detection sensitivity reached 5 cells (30 pg) by pretreatment of DOP-PCR and then detection of STR genotyping.@*CONCLUSION@#The system established in this study is reliable and more testing sensitive for forensic trace evidence.

Forensic application of 9 Y-STRs fluorescence-labeled multiplex amplification system / 法医学杂志

OBJECTIVE@#In order to increase significantly the discriminatory potential of Y-STR systems available to the forensic community, we have developed a system capable of simultaneously amplifying 9 Y-STR loci by fluorescence-labeled multiplex PCR technique.@*METHODS@#Primers of STR loci DYS434, GATA-A10, DYS438 and DYS439 were labeled with 6-FAM, primers of STR loci DYS531, DYS557, DYS448 were labeled with HEX, and primers of STR loci DYS456, DYS444 were labeled with TAMRA, respectively. PCR products were analyzed using capillary electrophoresis and GeneScan Software on the ABI Prism310 DNA Analyzer. Series experiments were carried out to evaluate the useful value in forensic application such as the sensitivity, male specificity and genotyping DNA different tissues of the same individual.@*RESULTS@#9 Y-STR loci were exactly determined following optimization of the polymerase chain reaction. In a sample of 120 males, a total of 105 different haplotypes was identified, 97 of them being unique. Overall, haplotype diversity was 0.996 8. It was proved that genotyping of these 9 Y-STR loci in some sexual crime should be prior to that of automosomal STR.@*CONCLUSION@#The results suggest that the newly constructed 9-plex will be very powerful for establishing Y-STR database, population genetic studies and mixture stains identification.

Constructing of STR slippage model by optimizing some factors / 法医学杂志

OBJECTIVE@#To construct STR slippage model and study factors involved in this procedure.@*METHODS@#DNA samples were amplified with the technology of Degenerate oligonucleotide- primed PCR, then their products were taken as later DNA template and their STR genotype were analyzed by optimizing several factors.@*RESULTS@#STR slippage model was constructed.@*CONCLUSION@#Several factors were involved in the produce of STR slippage, such as amount of modulate DNA, concentration of MgCl2, property of DNA polymerase, motif sequence of STR loci, sample, etc.

Constructing standard allelic ladders for four short tandem repeat loci and employing them in a population study on Han Nationality of Chengdu in China / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To solve the problems in the accuracy and standardization of short tandem repeats-polymerase chain reaction (STR-PCR) typing, the authors adopted the molecular clone technology in producing the standard allelic ladders of D1S1676, D2S2735, D11S1977 and D22S444 loci and applied them in a population study on the Hans in Chengdu, China.</p><p><b>METHODS</b>PCR was used to produce several different allelic fragments of these loci. PCR products were eluted from the gel and re-amplified by PCR. The purified allelic fragments were then blunt-end subcloned individually into the pGEMR-T plasmid vectors and the recombinant were transfected into competent E.coli DH5alpha TM cells. The results of sequencing confirmed that the size and the construction of the inserts were correct. The recombinant plasmids DNA with the inserts were then used as template for re-amplification to generate the four loci standard ladders.</p><p><b>RESULTS</b>The authors succeeded in producing large quantity of standard allelic ladder of these four loci, with which the genetic polymorphisms of these loci in Chengdu Han population of China were studied.</p><p><b>CONCLUSION</b>This method is of high value for forensic DNA typing to construct standard ladders. D1S1676, D2S2735 loci are robust for forensic analysis in Chinese Han population, whereas the value of D11S1977 and D22S444 loci is limited.</p>

Genetic polymorphisms of four STR loci on chromosome X and their forensic applications in a Chinese Han population / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To add DXS7133, GATA198A10, DXS9896 and DXS6797 to the panel of forensically validated X chromosome markers, and apply the multiplex amplification system to a population study and forensic analysis on the Hans of Chengdu.</p><p><b>METHODS</b>The PCR products were detected by the polyacrylamide gel electrophoresis and silver staining method. Hardy-Weinberg equilibrium of females was tested and every forensically interested value was calculated.</p><p><b>RESULTS</b>Sequencing revealed that their common sequence motifs were tetranucleotide repeats. Population genetic data were obtained by analyzing 120 unrelated females and 100 males from Chengdu Han ethnic group. In this population, DXS7133, GATA198A10, DXS9896 and DXS6797 exhibited 6, 6, 11, 8 distinguishable alleles respectively. Chi-square test demonstrated that genotype frequencies in females did not depart from Hardy-Weinberg equilibrium. Power of discrimination for female samples for the four loci were 0.7962, 0.8021, 0.9675, and 0.9444. The parentage testing in 32 cases revealed a typical X-linked inheritance and no mutations.</p><p><b>CONCLUSION</b>DXS7133, GATA198A10, DXS9896 and DXS6797, which are highly polymorphic in Chengdu Han population, are appropriate for individual identification and paternity testing involving a female child.</p>

Practice value of whole genome amplification technology to be used in forensic science and analysis of its result / 法医学杂志

Genetic analysis from forensic microsamples is a urgent, difficult task in forensic science, because it is frequently limited by the amount of specimen available in forensic practice, much effort has been carried out to resolve this difficulty. Whole genome amplification (WGA) technology, which was developing quickly in these years, has been thought to be a powerful, reliable and efficient strategy in analysis of minute amount DNA on many fields. In this review, we discuss its application in forensic science.

DNA samples preparation from single cell and its application in sensitivity test / 法医学杂志

OBJECTIVE@#To establish a reliable, exact and practical method to prepare DNA samples for sensitivity-test purposes.@*METHODS@#The micromanipulation method was employed to prepare exact quantity DNA samples used to study the sensitivity of Profiler Plus Kit-ABI PRISM 310 system.@*RESULTS@#We succeed in establishing a micromanipulation method to prepare groups of DNA samples, which contain 1-11 cells in turn, and also succeed in using them to study the sensitivity of Profiler Plus Kit-ABI310 system.@*CONCLUSION@#The method we have established is proved to be a reliable, exact and practical way to prepare DNA samples for sensitivity-test purposes.