甲基化特异性PCR过程中的影响因素分析

DNA甲基化是表观遗传学的重要研究内容之一。它是指在DNA甲基转移酶的作用下，将甲基添加在DNA分子中的碱基上,常见的DNA甲基化发生在DNA链上的胞嘧啶第五位碳原子和甲基闯的共价结合，即形成5甲基胞嘧啶（5mC），哺乳动物基因组中约70％的5mC存在于CpG二联核苷。DNA甲基化影响着基因的转录表达．因此它在基因的表达调控、细胞增殖、分化发育等方面起着重要作用，

利用自制纸质教具促学生掌握PCR反应过程

多聚酶链式反应(PCR)实验是分子水平的实验。对于高中生而言,学习PCR过程需要具备一定的生物学基础和空间想像能力,这给学生的学习带来了一定的困难。为此,可利用纸片、纸条等材料借助魔术表演的方式展示PCR反应过程,从而让学生在魔术表演的游戏操作中轻松掌握PCR反应过程。

用M13噬菌体作探针进行人的DNA限制性片段长度多态性分析

用M13噬菌体作为探针,可以检测人和动物基因组DNA中高度可变的小卫星区域,其有效顺序是该噬菌体基因Ⅲ中的两组15bp的串联重复。本文介绍的是采用随机引物与PCR过程相结合的方法标记M13噬菌体,得到高比活性的探针,与经HinfⅠ消化的人DNA杂交,产生具有高度多态性的DNA图谱,并与33.15探针杂交的DNA图谱进行了比较,证明M13噬菌体是进行DNA多态性分析的一种非常有用的探针。

单基因遗传性疾病的胚胎种植前遗传学诊断

因在绝大多数的单基因遗传性疾病(SGD)中基因突变是异质性的,所以对大多数SGD病例行胚胎种植前遗传学诊断(SGD-PGD)时都需个别设计。为此,设计就成为SGD-PGD的重要一环。由于SGD的遗传模式(常染色体显性和隐性,性染色体连锁显性和隐性)和突变种类(点突变,大小片段插入和丢失,重复顺序数超常等)不同,应有相应的不同设计技巧和策略。笔者通过设计实例,向读者介绍了不同的设计技巧和策略。现今SGD-PGD的最基本实验室操作还是用聚合酶链反应(PCR)把单细胞(卵裂细胞)靶DNA片段扩增,然后诊断扩增后所得片段。设计最重要的一步就是对PCR所用引物的设计。设计还应包括单细胞PCR的特殊操作规定,DNA限制性内切酶酶切点设计(因在点突变时,常需用限制性酶内切后片段长度多态性分析方法作诊断)做诊断结果分析预估,确定最后一轮PCR的两个引物之一为荧光标记引物。成功的SGD-PGD工作需要有权威性的领导核心组织,协调,对团队的统管和支持,这也是成批地开展SGD-PGD的先决条件。而且,只有在遗传流行病学工作者、各医院儿科、各专门疾病研究所和分子生物学基因诊断科室的密切配合下,才能对多种SGD全面开展SGD-PGD。

Expression Level of a Transcription Factor Gene Mdhd-Zip Up-regulated during Apple Fruit Senescence

[Objective] This study aimed to explore the role of one apple transcription factor of homeodomain-leucine zipper(MdHD-Zip) during apple fruit senescence.[Method] Postharvest Red Fuji fruits(Malus domestica Borkh ‘Red Fuji') were stored at room temperature(18 ℃-20 ℃) and cold condition(0 ± 1 ℃) separately.Fruit firmness and ethylene production during storage process were analyzed.Transcript level of Md HD-Zip was detected by real-time quantitative PCR during apple fruit storage under room temperature and cold condition.[Result] Expression level of Md HD-Zip was found up-regulated in later stage of apple fruit senescence at room temperature,while it showed a peak level after one month of cold storage.[Conclusion] The results of the present study suggest that Md HD-Zip may play a role in regulating apple fruit senescence.

实时荧光定量PCR检测技术及其在病原微生物检测中的应用

实时荧光定量PCR（Real-time PCR,RT-PCR）技术于1996年由美国Applied Biosystems公司推出,通过对PCR过程的实时监控,可专一、灵敏、快速、可重复地精确定量起始模板浓度,真正实现了PCR从定性到定量的飞跃。该技术拥有特异性强、灵敏度高、重复性好、定量准确、速度快、全封闭反应等优点,已成为了分子生物学研究的重要工具。本文对RTPCR技术的原理、检测方法、定量方法及其应用进行了综述。

Cloning and Expression Analysis of vasa During the Reproductive Cycle of Korean Rockfish, Sebastes schlegeli

Vasa, which is a conserved member of the DEAD-box protein family, plays an indispensable role in primordial germ cell proliferation. However, the expression ofvasa gene during the reproductive cycle in ovoviviparous fish has not been documented. In this study, the full-length sequence of vasa was obtained from the ovary of Korean rockfish （Sebastes schlegeli） using reverse transcription-PCR and rapid amplification of cDNA ends. The Vasa with a mature protein of 650 amino acids showed greatest homology （84%） with giant gourami （Osphronemus gorarny） and Pacific bluefin tuna （Thunnus orientalis）. The expression of vasa mRNA in Korean rockfish was detected in gonads only, suggesting its specific role in gonadal development. In addition, seasonal changes in the vasa expression levels were examined in gonads by quantitative real-time PCR. The vasa transcript levels in adult testis were found higher during spermatogenesis than during spermiation. The vasa transcript levels remained relatively high at the early ovary stage but declined during ovary maturation in adult female fish. These results suggest that the vasa gene play an important role in spermatogenesis and early oogenesis during the reproductive cycle of Korean rockfish.

Identification and characterization of a Dna J gene from red alga Pyropia yezoensis(Bangiales, Rhodophyta)

Members of the Dna J family are proteins that play a pivotal role in various cellular processes, such as protein folding, protein transport and cellular responses to stress. In the present study, we identified and characterized the full-length Dna J c DNA sequence from expressed sequence tags of Pyropia yezoensis( Py Dna J) via rapid identification of c DNA ends. This c DNA encoded a protein of 429 amino acids, which shared high sequence similarity with other identified Dna J proteins, such as a heat shock protein 40/Dna J from Pyropia haitanensis. The relative m RNA expression level of Py Dna J was investigated using real-time PCR to determine its specific expression during the algal life cycle and during desiccation. The relative m RNA expression level in sporophytes was higher than that in gametophytes and significantly increased during the whole desiccation process. These results indicate that Py Dna J is an authentic member of the Dna J family in plants and red algae and might play a pivotal role in mitigating damage to P. yezoensis during desiccation.

叠加通气模式联合心肺复苏机对心肺脑复苏的影响

心搏骤停（cardiac arrest，CA）是严重的心脏急症，无论心源性还是非心源性，病死率都极高。如何在心肺脑复苏（cardiopulmonary-cerebral resuscitation，CPCR）救治过程中提高成功率，成为当今急救医学研究的热点。本研究在CPCR过程中应用Thumper1007型心肺复苏机（美国密执安仪器公司生产）和PB760型呼吸机（美国PB公司生产）采用叠加通气模式和Thumper心肺复苏机联用，并与VCV通气模式和徒手标准复苏进行分析比较，现报道如下。