OBJECTIVE: To explore the change rules of peak area ratio of STR loci to Amelogenin (AMEL) locus (STR/AMEL), a sex-determining gene in DNA degradation, and to evaluate the application of STR/AMEL value in the estimation of DNA degradation degree. METHODS: DNA was extracted from iliopsoas, and the variations of STR/AMEL value (Penta E/AMEL, Penta D/AMEL, FGA/AMEL) were analyzed after the artificial degradation was made by DNase I, and the changes of these three ratios of the iliopsoas naturally degraded in an outdoor environment were also analyzed. The regression curves were analyzed using the periods of DNA degradation and outside the body as the independent variable (x) and the STR/AMEL value as the dependent variable (y) and three curve equations under two conditions were established. RESULTS: Both under the conditions of artificial and natural degradation, STR/AMEL value had a negative relationship with the degradation time. The relationship between STR/AMEL and degradation time can be well simulated by the cubic function. R2 was over 0.99 under controlled degradation condition and over 0.86 under natural degradation condition. CONCLUSION The STR/AMEL value (Penta E/AMEL, Penta D/AMEL, FGA/AMEL) is negatively related with the DNA degradation degree, which follows mathematical regression models strictly, and it might be applied to evaluate the DNA degradation degree.
Amelogenin/genetics* ; DNA Damage/genetics* ; DNA Primers ; Humans ; Microsatellite Repeats ; Regression Analysis ; Time Factors

OBJECTIVE: To identify 14 bacteria by sequencing the 16S rRNA gene and establish the basis for clinical application in the future. METHODS: DNA samples of the 14 bacteria were extracted. The 16S rRNA genes were amplified by PCR and sequenced with common primers. The sequences of the 16S rRNA genes were aligned by online software Blastn in nucleotide database. The bacteria were identified according to the homology of their 16S rRNA genes. RESULTS: Twelve bacteria were classified to species, the other 2 bacteria were classified to genus. CONCLUSION 16S rRNA gene sequence analysis is useful in identifying pathogenic bacteria.
Bacteria ; classification ; DNA Primers ; DNA, Bacterial ; genetics ; Polymerase Chain Reaction ; RNA, Ribosomal, 16S ; genetics ; Sequence Analysis, DNA

UNLABELLED: To ensure the consistency of genotype results for PowerPlex 21 kit and Goldeneye 20A kit. METHODS: The STR loci were amplified in DNA samples from 205 unrelated individuals in Beijing Han population. And consistency of 19 overlap STR loci typing were observed. The genetic polymorphism of D1S1656 locus was obtained. RESULTS: All 19 overlap loci typing showed consistent. The proportion of peak height of heterozygous loci in two kits showed no statistical difference (P > 0.05). The observed heterozygosis of D1S1656 was 0.878. The discrimination power was 0.949. The excluding probability of paternity of triplet was 0.751. The excluding probability of paternity of diploid was 0.506. The polymorphism information content was 0.810. CONCLUSION PowerPlex 21 kit and Goldeneye 20A kit present a good consistency. The primer design is reasonable. The polymorphism of D1S1656 is good. The two kits can be used for human genetic analysis, paternity test, and individual identification in forensic practice.
DNA/analysis* ; DNA Fingerprinting ; DNA Primers ; Genotype ; Heterozygote ; Humans ; Microsatellite Repeats ; Paternity ; Polymerase Chain Reaction ; Polymorphism, Genetic

Genetic diversity of Lilium sargentiae was detected in this paper by sequence-related amplified polymorphism (SRAP) marker. One hundred wild samples were collected from 10 places, and 15 SRAP primer combinations were used for determination. NTSYS-pc2.1 and POPGEN1.32 were used for data analysis. The results showed that a total of 170 clear DNA bands were amplified, 163 of which were polymorphic. The proportion of polymorphic loci was 90.58% on the level of species. Nei's (1973) gene diversity (H) was 0.2631, Shannon's Information index was 0.3661, the G(st), was 0.3672, and the genetic distance ranged from 0.2021 to 0.5749. All materials could be clustered into four groups by UPGMA. The results demonstrated that the genetic diversity of L. sargentiae was rich on the level of species, and the genetic diversity within populations exceeded among populations. The correlations of genetic diversity and distribution were significant in L. sargentiae.
Cluster Analysis ; Conservation of Natural Resources ; DNA Primers ; genetics ; Genetic Markers ; genetics ; Lilium ; genetics ; Polymorphism, Genetic ; genetics

The molecular maker system TRAP was utilized to develop a novel more accurate tool to identify the variety and establish the evolutionary relationship of different kind of Salvia miltiorrhiza Bge. PopGene 32 software was applied to conduct the cluster analysis and genetic dendrogram establishment. The results showed that 203 different fragments were amplified with 6 pair primers using the TRAP marker system. The polymorphic fragments number is 43, which takes up to 21.1%. The cluster analysis shows that the 4 materials we used in this study can be classified into 2 main groups and 3 subgroups. The genetic identity is 0.0788 and the average genetic distance is 0.9245 among the four materials in this study. A new tool using the TRAP marker system is more accurate and can be used to identify different kind of Salvia miltiorrhiza Bge at molecular level.
Cluster Analysis ; DNA Primers ; Plants, Medicinal ; classification ; genetics ; Polymorphism, Genetic ; Salvia miltiorrhiza ; classification ; genetics

OBJECTIVE: To develop a multiplex allele-specific PCR (AS-PCR) assay with three-color fluorescence labeling for mitochondrial DNA (mtDNA) SNP typing. METHODS: Based on the principle of AS-PCR, the primer sets were designed for 20 SNP located on the coding region of mtDNA and divided into 2 groups labeled with FAM and HEX fluorescence, respectively. A primer set included two forward (reverse) allelic specific primers with different sizes and a generic reverse (forward) primer. Blood samples from 200 unrelated individuals were analyzed by AS-PCR and capillary electrophoresis. Three random samples at least for each SNP site were examined and verified by direct sequencing. The haplotype frequency was investigated. RESULTS: Distinct electropherograms of 200 blood samples were obtained successfully. The typing results of direct sequencing were identical to those obtained from AS-PCR. The minimum detectable DNA concentration was 0.2 pg under the system of 10 microL. The sensitivity of the DNA concentrations ranged from 0.5 to 5 pg. The 200 individuals were assigned into 15 haplotype, and the haplotype diversity was 0.906 0. CONCLUSION AS-PCR is a simple, rapid and efficient method for mtDNA SNP typing, and can be applied to forensic practice.
Alleles ; DNA ; DNA Primers ; DNA, Mitochondrial/analysis* ; Electrophoresis, Capillary ; Haplotypes ; Humans ; Mitochondria ; Polymerase Chain Reaction/methods* ; Polymorphism, Single Nucleotide ; Sequence Analysis, DNA

OBJECTIVETo identify a novel HLA allele DRB1 * 16:36 from a Uygur woman.

METHODSPCR-SBT technology was applied to the extracted DNA for genotyping, and a possible new gene was sequenced by using sequence specific primers and single stranded SBT. This novel allele was compared with known most homologous gene sequences and their difference was analyzed.

RESULTSThis novel allele was different from HLA alle DRB1 * 16:23, and had highest similarity in 2 nucleotides at position 227 A→T and 236 T→C in exon 2, resulting in 3 amino acid changes from Tyr to Phe at codon 47 and Val to Ala at codon 50. The sequence of this novel allele had been submitted to GenBank.

CONCLUSIONThis HLA allele DRB1 * 16:23 has been confirmed to be a novel allele, and has been officially named DRB1 * 16:36 by the World Health Organization (WHO) Nomenclature Committee in May 2015.

Alleles ; Base Sequence ; DNA Primers ; Exons ; Female ; Genotype ; HLA-DRB1 Chains ; genetics ; Humans ; Polymerase Chain Reaction ; Sequence Analysis, DNA

OBJECTIVE: To investigate the feasibility and clinical significance of high resolution melting(HRM) curve analysis to detect SLC4A1 gene D38A and K56E mutations in the patients with hereditary spherocytosis(HS). METHODS: Peripheral blood was collected from 23 cases of HS for routine tests and their genomic DNA was extracted by routine technique. Specific primers of mutation sites D38A and K56E of SLC4A1 gene were designed. The HRM method was used to analyze all the samples, and then the results of HRM were verified with DNA sequencing technology. RESULTS: Among 23 specimens of HS patients, 6 cases of heterozygous mutant gene were detected by HRM technology, including 3 cases of D38A mutation and 3 cases of K56E mutation, which were confirmed by DNA sequencing. CONCLUSION The HRM technology can correctly detect 2 common mutation sites including D38A and K56E in SLC4A1 gene in an efficient, fast, and reliable way, which not only can be used for clinical diagnosis, but also expected to be a new method for clinical researchers to define gene mutation spectrum in HS patients.
Anion Exchange Protein 1, Erythrocyte ; genetics ; Base Sequence ; DNA Mutational Analysis ; DNA Primers ; Heterozygote ; Humans ; Mutation ; Spherocytosis, Hereditary ; genetics

The different growing habitats of Bupleurum chinense were investigated in Donglin mountain & Wuling mountain areas, the saikosaponin a and d in samples of B. chinense collected from different habitats were determined by HPLC. Results showed that B. chinense distributed in various habitats, such as meadow, understory and brushy. Significant differences of saikosaponin contents were observed. The higher saikosaponins contents were showed in samples from meadow habitats, while the lower saikosaponins contents in samples from understory and brushy habitats. The ventilation situation and light condition showed positive correlation with the saikosaponins accumulation in B. chinense. It could be concluded that growing habitats play an important role in accumulation of saikosaponins in B. chinense.
Bupleurum ; chemistry ; China ; Chromatography, High Pressure Liquid ; DNA Primers ; genetics ; Ecosystem ; Oleanolic Acid ; analogs & derivatives ; analysis ; Saponins ; analysis

Objective Quantitative analysis and comparison of the expression of ribonucleic acid （RNA） from frozen organs and formaldehyde-fixed and paraffin-embedded （FFPE） tissues. Methods Frozen specimens of human brain, myocardium and liver tissues as well as FFPE samples at different postmortem intervals were collected and mass concentration of RNA was extracted and detected. Reverse transcription-quantitative polymerase chain reaction （RT-qPCR） technology was used to analyze the amplification efficiency and relative expression of each RNA marker. Results The mass concentration and integrity of RNA extracted from FFPE samples were relatively low compared with frozen specimens. The amplification efficiency of RNA markers was related with RNA species and the length of amplification products. Among them, glyceraldehyde-3-phosphate dehydrogenase （GAPDH） and β-actin （ACTB） with relatively long amplification products failed to achieve optimal amplification efficiency, whereas 5S ribosomal RNA （5S rRNA） achieved ideal amplification efficiency and showed quite stable expression across various tissues, therefore it was chosen as internal reference marker. The expression quantity of GAPDH and ACTB in frozen specimens with longer postmortem intervals and in FFPE samples with relatively long amplification products was decreased. The expressions of tissue-specific microRNAs （miRNAs）, GAPDH and ACTB with relatively short amplification products had consistency in the same tissues and FFPE samples. Conclusion Through standardizing the RT-qPCR experiment, selecting the appropriate RNA marker and designing primers of appropriate product length, RNA expression levels of FFPE samples can be accurately quantified.
DNA Primers ; Formaldehyde ; Gene Expression Profiling ; Humans ; MicroRNAs/analysis* ; Myocardium ; Paraffin Embedding ; RNA/analysis* ; Real-Time Polymerase Chain Reaction/standards*