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Objective To study the polymorphisms of short tandem repeat (STR) loci in intron 1, 24, 13 and 22 (STR 1,24, 13, 22) of factor Ⅷ (FⅧ) gene in Chinese population, and establish single tube multiple fluorescent PCR method for rapid diagnosis of haemophilina A(HA). Methods Four STRs from genomie DNA of 220 females without blood relationship were amplified in a single tube using quadri-fluorescence PCR. Capillary electrophoresis was analyzed in ABI PRISM 310 Genetic Analyzer. DNA sequencing was used to assay the number of dinueleotide repeats. Gene diagnosis were performed in 96 HA families. Results It was observed that 7, 9, 10 and 7 different alleles were found in STR1, 24, 13 and 22, respectively. The PIC (polymorphism information contents) were 0. 3789, 0. 4055, 0. 5239 and 0. 4713 in STR1,24, 13 and 22, respectively, and the HR (heterozygesity rate) were 34. 55% (76/220), 38. 18% (84/220), 49. 55% (109/220) and 43.64% (96/220). In 96 HA families, the diagnosis rate of STR1, 24, 13 and 22 were 38. 54% (37/96), 38. 54% (37/96), 54. 17% (52/96), 42. 71% (41/96), respectively. Whereas it achieved 79. 17% (76/96) when combining the four STR markers. Conciusion The single tube multiple fluorescent PCR of four STR loci is an effective, simple, quick method for linkage analysis and gene diagnosis of haemophilia A.
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Objective To test whether multiplex ligation-dependent probe amplification(MLPA)could be used for the prenatal detection of the most common aneuploidies of chromosomes 13,18,21,X,and Y.Methods 34 cases including 22 blood samples(12 with trisomy 21,1 with monosomy X,one male witll extra Y and 8 healthy persons),4 cord blood samples with Down syndrome and 8 amniotic fluid samples ( 1 with trisomy 21 and 7 normal fetuses)were recruited into this study.All samples were confirmed by karvotype analysis. DNA was extracted from blood and amniotic lysate was incubated with proteinase K.MLPA was used to determine the relative copy numbers.Results The resuhs were available within 48 h and were concordant with karyotype analysis in all but one case of amniotic fluid that was suggested to be triploid sample 69,XXY by MLPA or contaminated by maternal blood.This sample actually was found containing a number of red blood cells after centfifugation in test. In total,the concordance rate with clinical characteristics was 97.1%.The Ratio values of 13,18,21,X in normal samples were approaching 1.0 except chromosome Y having slightly higher variation in relative copy number.The difference of ratio means between the normal and trisomy 21 samples was statistically significant by one-way ANOVA(F=298.906.P=0.000).Conclusion Computer assisted MLPA with high sensitivity is a rapid,simple,automatic and reliable method for detection of common chromosomal aneuploidies.
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Objective To develop a technique for the detection of genotypes of ?-thalassemia, which was rapid and automatical with high through-put.Methods The Real Time PCR and dissociation curve analysis (D.C) were carried out by using SYBR Green1 on ABI7000.Positive results of the 3 SYBR-Q-PCRs were defined by the -dF/t℃≥cut off value of the peak at the specific Tm for each right PCR product.Three PCR products were recombined with the T-vector.The correct positive clones were selected by sequencing.Standard curves of the CT vs. log values of copies were done from a serious dilutions of the recombinant plasmid DNA which served as the template in SYBR-Q-PCRs.Results The conditions of the PCR including concentrations of primers, thurmocycler program etc.were optimized.Specific Tm values for the 3 SYBR-Q-PCRs were 82.5?1℃, 83.0?℃ and 84.0?1℃ respectively. The standard curves for p800 bp and p206 bp DNA showed a good linear relationship between CT and log value of the copies of the template DNA ranging from 10~5 copies to single copy. Sensitivity of the technique were at least 10~1 to 10~2 times higher than that of the regular PCR plus gel electrophoresis method.The techniques and reagents showed very good reproducibility and stability besides the high sensitivity. The ?-thalassemia 1(??/--SEA), Bart′s Hydrops Syndrom (--SEA/--SEA), deletion type and non-deletion type of HbH diseases, and homozygote of ?-thalassemia-2 were diagnosed by the methods.Conclusion The genotypes of the ?-Thalassemia could be diagnosed by the Real-time PCR with SYBR Green1 combined with the melting curve analysis.rapidly, automatically, accurately with high throughput and without dual fluorescent labeled probe.
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<p><b>OBJECTIVE</b>To explore the expression of WT1 gene in leukemia patients and its clinical implications.</p><p><b>METHODS</b>Expression of WT1 mRNA was detected in two leukemia cell lines (K562 and HL-60), 49 acute leukemia (AL) patients, 33 chronic myeloid leukemia (CML) patients and 25 healthy subjects by reverse trans-criptase-nested polymerase chain reaction (RT-Nested PCR).</p><p><b>RESULTS</b>WT1 gene was expressed in all subtype of AL including K562 and HL-60 cell lines, 21/29 newly diagnosed and relapsed AL patients, 1/20 complete remission (CR) AL patients, 15/18 CML blastic crisis patients, 1/5 CML patients in accelerated phase, and 1/10 CML patients in chronic phase. WT1 gene was undetectable in 25 healthy subjects. The expression level of WT1 gene was related to the prognosis of AL, patients with relative level >/= 1.0 had lower CR rates and disease-free survival. For CML patients, WT1 gene expression was associated with the clinical phase, it increased with disease progressed.</p><p><b>CONCLUSION</b>WT1 gene expression is associated with pathogenesis of leukemia. It is a prognostic factor and a marker for the detection of minimal residual disease in AL and may used as an indicator for diagnosing CML blastic crisis.</p>
Subject(s)
Humans , Disease-Free Survival , Gene Expression , HL-60 Cells , Neoplasm, Residual , Diagnosis , RNA, Messenger , Genetics , Reverse Transcriptase Polymerase Chain Reaction , WT1 Proteins , GeneticsABSTRACT
<p><b>OBJECTIVE</b>To establish a kind of molecular biology clinical detective method to cariogenic S. mutans.</p><p><b>METHODS</b>Using the coamplification of target and reference genes. One pair of specific primers were designed according to a portion of the dextranase (dexA) gene of S. mutans. The reference gene was plasmid pET23b DNA. The saliva samples of 196 children were quantitative detected. The PCR method was compared with the routine culture method.</p><p><b>RESULTS</b>The rate of S. mutans counts >/= 10(8) CFU/L (colony-forming unit per millilitre) saliva by quantitative PCR was 91.3%. The results of coincidence rate between the new method and the routine way was 94.9%.</p><p><b>CONCLUSIONS</b>The new quantitative detective method is fast and provides with high scoincidence rate and high specificity, so have extensive clinical practice foreground.</p>
Subject(s)
Humans , DNA Primers , Polymerase Chain Reaction , Saliva , Sensitivity and Specificity , Streptococcus mutans , Genetics , Streptococcus sobrinus , GeneticsABSTRACT
<p><b>OBJECTIVE</b>To investigate the frequency of p16 and p15 gene methylation in multiple myeloma (MM), and its relationship with bone marrow cell apoptosis and clinical outcome.</p><p><b>METHODS</b>Twenty-two patients with MM were studied to detect p16 and p15 gene methylation. Methylation-specific polymerase chain reaction (MSP) was used to detect gene methylation, and terminal transferase-mediated dUTP nick end-labeling (TUNEL) was used to detect cell apoptosis.</p><p><b>RESULTS</b>p16 and/or p15 gene methylatoin was detected in 10 of 22 patients (45.4%). There were 3 patients with p16 gene methylation, 9 patients with p15 gene methylation, and 2 patients with both genes methylation. The incidence of methylation of p15 gene was higher than that of p16 gene (P < 0.05). The patients with p16 and/or p15 gene methylation had a delayed cell apoptosis, poor response to chemotherapy, and a short over-all survival (OS).</p><p><b>CONCLUSION</b>The methylation of p16 and/or p15 gene plays a key role in MM apoptosis pathogenesis. The patients with both p16 and p15 gene methylation had a poor prognosis.</p>
Subject(s)
Humans , Apoptosis , Cell Cycle Proteins , Cyclin-Dependent Kinase Inhibitor p15 , Cyclin-Dependent Kinase Inhibitor p16 , Genetics , DNA Methylation , DNA, Neoplasm , Genetics , Gene Silencing , Genes, p16 , Multiple Myeloma , Genetics , Pathology , Prognosis , Transcription Factors , Genetics , Tumor Suppressor ProteinsABSTRACT
Objective To discuss the enrichment method of fetal DNA from maternal plasma on size-fractionated separation.Methods Antecubital venous blood (5 ml from each pregnant woman) was collected in EDTA-containing tubes.DNA was extracted from plasma and white blood cells, separately.The DNA was fractionated by agarose gel electrophoresis.Three sections of
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Objective To develop techniques based on polymerase chain reaction(PCR) which can detect 2 of most common deletional ? thalassemia ? 3.7 deletion and ? 4.2 deletion in China accurately and speedily. Methods Two groups of primers were designed and synthesized. PCR conditions were optimized. The PCR pro ducts were analysed by 1.0% agarose gel electrophoresis. The gel was stained by EB and photographed using an UVP gel documentation. Results PCR product of a 1 700 bp DNA fragment with primers A′, B′, C3 indicates the ? 3.7 deletion while a 1 900 bp fragment indicates a normal or wild type of ? globin gene. Occurrence of the 1 700 bp and 1 900 bp simultaneously indicates a heterozygous of the ? 3.7 deletion. Neither of the 2 bands was presented, indicating a homozygous of South East Asia type of deletion (-? SEA ). According to patterns of 1 580 bp and 1 180 bp hand amplified by a PCR with primer G′, E, F′, we detected the ? 4.2 deletion and distinquished its heterozygous and homozygous. Conclusions The 2 PCR based techniques developed in our laboratory are accurate, simple, well reproducible and easy to use for screening of the 2 deletion types of ? thalassemia determinants.
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An eukaryotic expression plasmid Rc/CMV GHcDNA containing CMV late promoter and hGH cDNA was constructed and introduced into mouse skeletal muscle by means of direct injection with or without the cationic liposome (Lipofectin and Lipofectamine reagent) intramuscularly. The expression of the gene was detected through both RT-PCR and IRMA (Immunoradiometric assay) at the level of transcription and protein respectively. The gene expression can be detected even after 90 days of single plasmid injection. Compared with the mice injected with the plasmid itself, the expression levels of mice injected with the plasmid-cationic liposome complex seem to be higher, and the expression period longer. Furthermore, the effect of Lipofectamine seems to be even better than that of Lipofectin. The results indicate that direct intramuscular injection with recombinant expression plasmid or plasmid-cationic liposome complex is a simple, efficient and economical way for foreign gene transfer and expression in experimental mice in vivo.
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Objective To investigate and evaluate a novel fluorotyping procedure for HLA-DRB1 locus. Methods On the basis of low resolutation SSP technique,the fluorogenic probe was used to establish the fluorotyping procedure of HLA-DRB1 which was carried out for 46 samples. Results The fluorotyping has been successful for all the 46 samples and was completely coincident to the SSP results.As compared to the serological typing of DR locus,the coincidence rate was 66.3%(61/92) whereas the coincidence rate of both the 2 specific genes was 43.5%(20/46). Conclusions Fluorotyping is accurate,sensitive,reproducible,depends less on manual manipulation and eliminates the problems related to contamination.
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The kinetic changes in morphology and cellular density of epidermal Langerhanscells (LC) in the guinea pig were observed by ATPase cytochemical staining techniqueafter repeated skin application of 5%,3% and 1% of benzalkonium bromide (BB,primary irritant) and 0.05% dinitrochlorobenzene (DNCB,allergen) respectively.We have found that there was a significant difference in the density and morpho-logical change of LC between BB and DNCB application.Treatment with 5% BBcould induce a reversible decline in LC density and changes in cell processes,andwith 0.05% DNCB,the number of LC was decreased and the ATPase activity wasweakened only in the late stage of treatment.The significance of using the kineticchanges in morphology and cellular density of LC as the criteria for the safetyevaluation of weak allergens and weak primary irritants as well as cosmetics wasdiscussed.