OBJECTIVETo evaluate the fidelity of multiple displacement amplification (MDA) from small number of cells (1-10 cells) by 10K 2.0 SNP mapping array.

METHODSA fibroblast cell line (Tri-18; GM02732, 47, XY, +18) was used as the template, and 6 groups were set up in the study. Groups A and B were positive and negative control, respectively; groups C-F were experimental groups involving the MDA products from 1, 2, 5 and 10 cells respectively. In combination of single nucleotide polymorphism (SNP) array, the product of each group was assessed based on the genome coverage, loss of heterozygosity (LOH) rate and allele dropout (ADO) rate.

RESULTSThe nonspecific product of negative control presented an average call rate of 3.2%. The genome coverage of the MDA product increased from 86.4% to 96.4% with the increasing number of template from 1 to 10 cells, while the LOH rate and ADO rate decreased significantly (P<0.05).

CONCLUSIONMDA is a highly efficient and reliable method for whole genome amplification. The fidelity of MDA will be improved significantly with the increasing number of template cells. 10K 2.0 SNP mapping array is a quick, accurate and comprehensive method to evaluate the fidelity of amplified DNA products, but the ADO SNPs should be distinguished from those of preferential amplification from the LOH loci to avoid errors.

Cell Line ; Cells ; cytology ; DNA ; genetics ; Humans ; Loss of Heterozygosity ; Nucleic Acid Amplification Techniques ; methods ; Polymorphism, Single Nucleotide ; Templates, Genetic

Even when DNA sequencing of purified DNA template failed under the optimal condition, it can be generally contributed to high GC content. GC-rich region of template causes a secondary structure to produce shorter readable sequence. To solve this problem, the sequencing reaction was modified by using dimethyl sulfoxide (DMSO). It was found that 5% (v/v) of DMSO in the reaction mixture recovers sequencing signal intensity with reduced frequency of ambiguous bases. When DMSO was added to sequencing reaction of DNA template with normal GC content, it did not show any adverse effect. Sequencing accuracy and unambiguous base frequency were significantly improved at concentration of 2% to 5% (v/v) DMSO in GC-rich DNA template. DMSO has been empirically introduced to enhance the efficiency of PCR in GC-rich templates. However, the underlying mechanism of improved cycle sequencing by DMSO is unknown. Thus, cycle sequencing reaction was remodified with other additives such as N-methyl imidazole, N-methyl2-pyrrolidone, N-methyl-2-pyridone and glycerol, possessing the similar chemical properties as DMSO. Most of methyl nitrogen ring-containing chemicals did not improve sequencing accuracy, whereas only glycerol mimicked the positive effect of DMSO by the same extent. In the present study, we suggest that the treatment of DMSO improve cycle sequencing by the alteration of structural conformation of GC-rich DNA template.
Base Composition ; DNA/chemistry* ; Dimethyl Sulfoxide/pharmacology* ; Plasmids/genetics ; Polymerase Chain Reaction/methods* ; Sequence Analysis, DNA* ; Solvents/pharmacology ; Solvents/chemistry ; Templates

A protocol for enrichment and adsorption of karyocyte from whole blood by using magnetic nanometer beads as solid-phase absorbents was presented. The PCR amplification could be accomplished by using the nanobeads with karyocyte as template directly and the PCR products were applied on an oligonucleotide array to do gene typing. The HLA-A PCR amplification system and a small HLA-A oligonucleotide microarray were applied as the platform and an experiment protocol of separating karyocyte from whole blood using the magnetic nanometer beads (Fe2O3) were set up. The experimental conditions were also discussed. It showed that pH level of PBS eluent, Taq enzyme quantity and fragment length of products could influent the amplification results, and the magnetic nano-beads could succeed in sample preparation in microarray to provide a promising way in automatic detection and lab-on-a-chip.
Adsorption ; Cell Separation/instrumentation ; Cell Separation/methods ; Genotype ; Leukocytes/*cytology ; *Magnetics ; Microchemistry/instrumentation ; Microchemistry/*methods ; Nanotechnology ; Oligonucleotide Array Sequence Analysis/*methods ; Polymerase Chain Reaction ; Templates, Genetic

A protocol for enrichment and adsorption of karyocyte from whole blood by using magnetic nanometer beads as solid-phase absorbents was presented. The PCR amplification could be accomplished by using the nanobeads with karyocyte as template directly and the PCR products were applied on an oligonucleotide array to do gene typing. The HLA-A PCR amplification system and a small HLA-A oligonucleotide microarray were applied as the platform and an experiment protocol of separating karyocyte from whole blood using the magnetic nanometer beads (Fe2O3) were set up. The experimental conditions were also discussed. It showed that pH level of PBS eluent, Taq enzyme quantity and fragment length of products could influent the amplification results, and the magnetic nano-beads could succeed in sample preparation in microarray to provide a promising way in automatic detection and lab-on-a-chip.
Adsorption ; Cell Separation ; instrumentation ; methods ; Genotype ; Leukocytes ; cytology ; Magnetics ; Microchemistry ; instrumentation ; methods ; Nanotechnology ; Oligonucleotide Array Sequence Analysis ; methods ; Polymerase Chain Reaction ; Templates, Genetic

Nanocavity biomaterials with recognition specificity imprinted by using proteins as templates may successful serve as substitutes for antibodies, enzymes, and other native biological structures as well as cell bracket materials. It has numerous applications in biotechnology, medicine and so on. In this paper, the principle of template imprinting is introduced briefly, the specialty of template imprinting of proteins is analyzed, and the methods of template imprinting of proteins including protein entrapment, microbead surface imprinting, flat surface imprinting as well as the epitope are reviewed in details.
Animals ; Biocompatible Materials ; chemical synthesis ; Biotechnology ; Genomic Imprinting ; Humans ; Macromolecular Substances ; Nanotechnology ; Peptides ; chemical synthesis ; Polymers ; Proteins ; chemical synthesis ; Templates, Genetic

OBJECTIVE: To detect low copy number of DNA samples by using a newly launched commercial miniSTR detection kit (MiniFiler) in forensic practice. METHODS: Low concentration and/or challenged forensic DNA samples were analyzed according to protocols provided by the manufacturer (Applied Biosystems, Foster City, USA). RESULTS: DNA samples as low as 10 pg could be amplified by MiniFiler kit, and the optimal DNA quantity was 40 pg or above. CONCLUSION MiniFiler kit can be used for analysis of low copy number STR.
Alleles ; DNA Fingerprinting/methods* ; Forensic Genetics/methods* ; Genotype ; Humans ; Polymerase Chain Reaction/methods* ; Reagent Kits, Diagnostic ; Sensitivity and Specificity ; Tandem Repeat Sequences ; Templates, Genetic

OBJECTIVE: To explore the appropriate amount of template DNA for Sinofiler Kit. METHODS: The DNA samples with ideally genotyped results by Sinofiler Kit were detected by real-time quantitative PCR assay. RESULTS: It was shown that 1.29-1.51 ng of template DNA in 12.5 microL reaction volume was optimal for STR genotyping with Sinofiler Kit. CONCLUSION Real time quantitative PCR is an accurate and necessary technique for detection of appropriate amount of template DNA for different kits.
DNA/analysis* ; Forensic Medicine/methods* ; Hair/chemistry* ; Humans ; Microsatellite Repeats ; Reagent Kits, Diagnostic ; Reverse Transcriptase Polymerase Chain Reaction/methods* ; Templates, Genetic

Low template DNA (LTDNA) has been widely applied in the field of forensic science in recent years. However, the application of low copy number(LCN) analysis is still controversial in certain forensic. This paper focus on the definition of LCN and LTDNA, casework because of its inherent limiting factors. the validity and application of LCN in forensic science, methods of typing, quality control, replicate analysis, detection thresholds and then reviews the latest development of LCN in forensic science.
DNA/genetics* ; DNA Fingerprinting/methods* ; Forensic Genetics/methods* ; Gene Dosage ; Genome, Human ; Genotype ; Humans ; Microsatellite Repeats/genetics* ; Polymerase Chain Reaction/methods* ; Sensitivity and Specificity ; Templates, Genetic

OBJECTIVETo investigate the optimization system of SRAP-PCR molecular marker technology in the analysis on Pinellia ternata.

METHODSRAP-PCR reaction system for P. ternata was optimized by L16 (5(4)) orthogonal design with five elements (dNTPs, Mg2+, the template DNA, primers, Taq enzyme) and four standards.

RESULTThe most suitable forward primer for SRAP for Pinellia ternata was 5'-TGAGTCCAAACCGGAAG-3', while the reverse primer was 5'-GACTGCGTACGAATTACG-3'. The optimized reaction system contained 70 ng DNA template, 0.9 micromol x L(-1) primer, 0.20 mmol x L(-1) dNTP s, 1.5 - 2.0 mmol x L(-1) Mg2+, and 2 U Taq enzyme.

CONCLUSIONSRAP-PCR system for P. ternata is established to lay a foundation for future construction of SRAP genetic map of P. ternata.

China ; DNA Primers ; genetics ; DNA, Plant ; genetics ; Electrophoresis ; Magnesium ; metabolism ; Nucleic Acid Amplification Techniques ; methods ; Nucleotides ; genetics ; Pinellia ; genetics ; Polymerase Chain Reaction ; methods ; Reproducibility of Results ; Taq Polymerase ; metabolism ; Templates, Genetic

OBJECTIVE: To optimize low copy number (LCN) DNA analysis methods for forensic STR genotyping. METHODS: Two groups of DNA sample, extracted using either Magnetic bead method or Chelex-100 methods, were previously amplified with a Identifiler PCR Amplification kit, but no genotype was detected. The DNA samples were concentrated using either a drying method or the Microcon-100 method, then amplified using an miniFiler PCR Amplification kit and genotyped. RESULTS: Among the 127 DNA samples, 47 samples, previously extracted using the Magnetic bead method, were genotyped with 36% success rate. Eighty samples, previously extracted using the Chelex-100 method, were genotyped with 30% success rate. CONCLUSION The application of sample concentration methods and miniFiler kit can improve the success rate of LCN STR analysis.
Blood Stains ; DNA/analysis* ; DNA Fingerprinting/methods* ; DNA Primers ; Forensic Genetics/methods* ; Genotyping Techniques/methods* ; Humans ; Microsatellite Repeats ; Polymerase Chain Reaction/methods* ; Saliva/chemistry* ; Sensitivity and Specificity ; Specimen Handling/methods* ; Templates, Genetic