Evaluating the Efficiency of REPLI-g® Single Cell Kit for Trace DNA Amplification / 法医学杂志

Objective To evaluate the efficiency of REPLI-g® Single Cell Kit for sample DNA amplification, and explore its application value in forensic trace DNA amplification. Methods Three DNA extraction kits were selected to extract DNA from peripheral blood of 10 unrelated individuals. The DNA yield and purity of the three DNA extraction kits were compared. According to the results of comparison, one DNA sample was selected to concentrate and dilute, then used as the initial sample of whole genome amplification （WGA）. REPLI-g® Single Cell Kit was used to amplify the initial sample at the whole genome level. The amplification yield and amplification times were calculated, and the distribution of DNA fragments was detected by agarose gel electrophoresis. Goldeneye® DNA ID System 20A Kit was used to perform the STR typing of the initial sample and DNA samples amplified at the whole genome level to evaluate the performance of REPLI-g® Single Cell Kit in trace DNA amplication in terms of purity and yield as well as the success rate of STR typing. Results After comparison, one DNA sample was selected from QIAsymphony® DNA Investigator® Kit extracts to concentrate and dilute as the initial sample of WGA. After amplifying the whole genome of a series of initial samples by REPLI-g® Single Cell Kit, the lowest average of amplification yield reached 8.77×103 ng, while the average of the corresponding amplification times reached 1.40×106. DNA fragments were large and concentrated. The STR typing success rate of WGA samples became lower with the decrease of initial samples used, but when the amount of samples was lower than 0.5 ng, the STR typing success rate of samples after DNA WGA was higher than that of samples without DNA WGA. Conclusion REPLI-g® Single Cell Kit can increase the yield of template DNA. Especially for trace DNA, the STR typing success rate can be improved to a certain extent.

Application of Multiple Displacement Amplification in Samples with Inhibitors / 法医学杂志

OBJECTIVES@#To explore the ability of inhibition resistibility of multiple displacement amplification （MDA） in samples with inhibitors. To explain the application and value of MDA in forensic medicine by comparing with using magnetic beads methods （MBM） to purify sample.@*METHODS@#Different concentrations of hemoglobin and humid acid （HA） mixed with DNA samples and then divided the samples into MDA group, MBM group and control group. D3S1358 locus was amplified and detected by polyacrylamide gel electrophoresis detection system and AmpFℓSTR® Identifiler™ Plus Kit-capillary electrophoresis detection system.@*RESULTS@#When hemoglobin concentrations exceed 1 ng/μL or HA concentrations exceed 0.1 ng/μL, amplification products could not be obtained by single-locus system in control group. When hemoglobin concentration exceeds 100 ng/μL or HA concentrations exceed 1 ng/μL, the samples could not be amplified by MBM. Inhibitors in different concentrations were amplified successfully in MDA group without any influence from inhibitors.@*CONCLUSIONS@#MDA has the capability to remove the inhibition of hemoglobin and HA, which is better than MBM and has a certain value in forensic practices.

Application of Multiple Displacement Amplification in Samples with Inhibitors / 法医学杂志

ObjectiveTo explore the ability of inhibition resistibility of multiple displacement amplification (MDA)in samples with inhibitors. To explain the application and value of MDA in forensic medicine by comparing with using magnetic beads methods(MBM)to purify sample.MethodsDifferent concentra-tions of hemoglobin and humid acid(HA)mixed with DNA samples and then divided the samples into MDA group, MBM group and control group.D3S1358locus was amplified and detected by polyacry-lamide gel electrophoresis detection system and AmpF?STR? IdentifilerTM Plus Kit-capillary electrophore-sis detection system.ResultsWhen hemoglobin concentrations exceed 1 ng/μL or HA concentrations ex-ceed 0.1 ng/μL, amplification products could not be obtained by single-locus system in control group. When hemoglobin concentration exceeds 100 ng/μL or HA concentrations exceed 1 ng/μL, the samples could not be amplified by MBM. Inhibitors in different concentrations were amplified successfully in MDA group without any influence from inhibitors.ConclusionMDA has the capability to remove the inhibi-tion of hemoglobin and HA, which is better than MBM and has a certain value in forensic practices.

A Fosmid Cloning Strategy for Detecting the Widest Possible Spectrum of Microbes from the International Space Station Drinking Water System

In this study, fosmid cloning strategies were used to assess the microbial populations in water from the International Space Station (ISS) drinking water system (henceforth referred to as Prebiocide and Tank A water samples). The goals of this study were: to compare the sensitivity of the fosmid cloning strategy with that of traditional culture-based and 16S rRNA-based approaches and to detect the widest possible spectrum of microbial populations during the water purification process. Initially, microbes could not be cultivated, and conventional PCR failed to amplify 16S rDNA fragments from these low biomass samples. Therefore, randomly primed rolling-circle amplification was used to amplify any DNA that might be present in the samples, followed by size selection by using pulsed-field gel electrophoresis. The amplified high-molecular-weight DNA from both samples was cloned into fosmid vectors. Several hundred clones were randomly selected for sequencing, followed by Blastn/Blastx searches. Sequences encoding specific genes from Burkholderia, a species abundant in the soil and groundwater, were found in both samples. Bradyrhizobium and Mesorhizobium, which belong to rhizobia, a large community of nitrogen fixers often found in association with plant roots, were present in the Prebiocide samples. Ralstonia, which is prevalent in soils with a high heavy metal content, was detected in the Tank A samples. The detection of many unidentified sequences suggests the presence of potentially novel microbial fingerprints. The bacterial diversity detected in this pilot study using a fosmid vector approach was higher than that detected by conventional 16S rRNA gene sequencing.

The usefulness of multiple displacement amplification in the molecular studies in Hansen's disease / 나학회지

Appreciable amounts of high-quality DNA are frequently required for use in molecular diagnostic and epidemiology studies. The limited availability of DNA can be a critical obstacle to meeting research and clinical needs. And molecular methods have not been routinely applied to the obligate intracellular organism Mycobacterium leprae because of the difficulty of obtaining a genomic DNA template from clinical material. The MDA (multiple displacement amplification) reaction is increasingly the method of choice for many applications because of its extensive coverage of the genome, the generation of extremely long DNA products compared with older whole genome amplification methods and the high DNA yields, even from exceedingly low amounts of starting material. In the study, the author evaluated the usefulness of this method for the molecular studies in Hansen's disease.

Detection of Aneuploidy from Single Cells by Array-Comparative Genetic Hybridization / 中山大学学报(医学科学版)

[Objective] To set up an optimized protocol for aneuploidy detection from single cells through Array- Comparative Genetic Hybridization (CGH).[Method] Two cell lines,trisomy 18 (Tri-18;GM02732,47,XY,+18) and chromosome 4 segment deletion [sDel-4;GM00343,46,XY,4(del) (qter ＞ p14)],were used in the study.In combination of 10 k 2.0 SNP mapping array platform and multiple displacement amplification (MDA),the diagnostic accurate rates of MDA product from single cells of the two cell lines using gDNA and single-cell MDA product as reference were compared.[Result] An extremely lower call rate (3.2 ± 1.2)% in the negative control group was observed compared to the experiment groups.When the single-cell MDA product was used as reference,the standard deviations of Log2 (signal intensity ratio) were significantly decreased in both groups,compared with when the gDNA as reference (P ＝ 0.004).Through CNAT analytic software,some specific chromosomes (16,17,19,and 22) presented obvious preferential amplification (PA) when the gDNA was used as reference,but this PA could be eliminated when single-cell MDA product was used as reference.[Conclusion] 10 k 2.0 SNP mapping array in combination with MDA could be a quick,highly efficient and accurate method to detect aneuploidy in single cells.