[Sequence analysis of a novel human leukocyte antigen allele B*5827].

OBJECTIVE: To investigate the molecular basis for a novel human leukocyte antigen (HLA) allele B*5827. METHODS: DNA from the proband was analyzed by polymerase chain reaction-sequence specific oligonucleotide (PCR-SSO) typing. The amplified product was sequenced bidirectionally. RESULTS: Abnormal HLA-B locus was observed and its nucleotide sequence was different from the known HLA-B allele sequences, with highest homology to HLA-B*5820 allele. It differs from HLA-B*5820 by 8 nucleotide substitutions in exon 3, i.e., nt 290 (G > C), nt 346 (T > A), nt 390 (A > C), nt 404 (G > C), nt 413 (C > G), nt 471 (A > G), nt 486 (A > G) and nt 487 (C > A), resulting in an amino acid change from ser > arg at nt 97, phe >tyr at nt 115, ser > arg at nt 130, thr > ala at nt 157 and thr > glu at nt 162. Nucleotide differences of nt 404 (G > C) and nt 413( C > G) did not change amino acid. CONCLUSION: The sequences of the novel allele have been submitted to GenBank (access No.GU071234). A novel HLA class I allele B*5827 has been officially assigned by the WHO HLA Nomenclature Committee in Jan. 2010.

[Study on mitochondrial DNA gene tRNA(Leu(UUR)) A3243G mutation in type 2 diabetes mellitus].

OBJECTIVE: To explore the prevalence and the clinical characteristics of mitochondrial gene mutation A3243G (mt tRNA(Leu(UUR)) 3243 A-->G) in patients with type 2 diabetes mellitus (DM2) in China. METHODS: Four hundred and twenty-eight cases of DM2 patients were selected randomly. One hundred and eighty-eight individuals were healthy controls. The mutation was assayed by PCR-restriction fragment length polymorphism technique. The target fragments of PCR were digested with restriction endonuclease Apa I. RESULTS: mt tRNA(Leu(UUR)) 3243A-->G gene mutation was found in 2 of 428 patients with DM2, but not found in the controls. Further investigation of the relatives of the 2 patients' families revealed that 3 members were the carriers of mt tRNA A3243G gene mutation and the patients with diabetes. In addition, one proband and her son were characterized with the syndrome of mitochondrial encephalomyopathy with lactic acidosis. The diabetes of these patients is frequently accompanied by hearing impairment or deafness with maternal inheritance. CONCLUSION: The prevalence of the mitochondrial gene A3243G mutation is 0.47% in DM2 patients in China. The data acquired in this study suggest that the clinical phenotype of these patients with A3243G should be heterogeneous.

[A new method of processing quantitative PCR data].

Today standard PCR can't satisfy the need of biotechnique development and clinical research any more. After numerous dynamic research, PE company found there is a linear relation between initial template number and cycling time when the accumulating fluorescent product is detectable.Therefore,they developed a quantitative PCR technique to be used in PE7700 and PE5700. But the error of this technique is too great to satisfy the need of biotechnique development and clinical research. A better quantitative PCR technique is needed. The mathematical model submitted here is combined with the achievement of relative science,and based on the PCR principle and careful analysis of molecular relationship of main members in PCR reaction system. This model describes the function relation between product quantity or fluorescence intensity and initial template number and other reaction conditions, and can reflect the accumulating rule of PCR product molecule accurately. Accurate quantitative PCR analysis can be made use this function relation. Accumulated PCR product quantity can be obtained from initial template number. Using this model to do quantitative PCR analysis,result error is only related to the accuracy of fluorescence intensity or the instrument used. For an example, when the fluorescence intensity is accurate to 6 digits and the template size is between 100 to 1,000,000, the quantitative result accuracy will be more than 99%. The difference of result error is distinct using same condition,same instrument but different analysis method. Moreover,if the PCR quantitative analysis system is used to process data, it will get result 80 times of accuracy than using CT method.

[The kinetic and mathematical model of PCR amplification experiment].

The PCR technique has been set up for nearly twenty years and is becoming more and more ripe. But because of the multiple influencing factors and complicated reaction procedures,no mathematical method that can describe the PCR reaction has been given. On the basis of its elementary principle,we suggested a kinetic equation to describe the reaction procedure,Wamp=[Ntarg x (1+P)n1+0.5 x Cenz x U x P x Ceactive x (n-nl)-Ntarg x (1+n x P)] x Cu x M. This equation can describe correctly the accumulation rule of PCR product and thus build up the kinetic-mathematical model of PCR reaction. The predicted CT value of PE 7700 by the kinetic-mathematical model was in accordance with the real value detected by the machine. This kinetic-mathematical model accompanied by proper detecting equipment and computer could make an automatic PCR instrument, which would produce much better result. A laboratory can predict the amount of PCR product by this model and provide accurate information for further handling of PCR product according to its own condition. In this model,the molecular basis that PCR reaction is doomed to change from exponential amplification to linear amplification had been clarified.