焦磷酸测序技术的流程
英文回答：
Sequencing technologies have revolutionized the field
of genomics, allowing us to unravel the genetic code of organisms. One of the widely used sequencing techniques is the Sanger sequencing method, also known as the chain termination method or dideoxy sequencing. This technique relies on the incorporation of chain-terminating dideoxynucleotides (ddNTPs) during DNA synthesis.
The process of Sanger sequencing involves several steps. First, the DNA sample of interest is isolated and purified. This can be done from various sources, such as blood, tissue, or cultured cells. Once the DNA is extracted, it is fragmented into smaller pieces to facilitate the sequencing process.
Next, the DNA fragments are amplified using the polymerase chain reaction (PCR). PCR is a technique that
allows for the amplification of specific DNA sequences. It involves multiple cycles of DNA denaturation, primer annealing, and DNA synthesis. During PCR, primers specific to the target DNA sequence are used to initiate DNA synthesis.
After PCR amplification, the sequencing reaction is set up. This involves mixing the amplified DNA fragments with a primer, DNA polymerase, and a mixture of normal deoxynucleotides (dNTPs) and small amounts of chain-terminating ddNTPs. The ddNTPs lack the 3'-OH group necessary for DNA chain elongation, resulting in the termination of DNA synthesis at specific positions.
The sequencing reaction mixture is then subjected to capillary electrophoresis. In this step, the DNA fragments are separated based on their size and charge as they migrate through a gel-filled capillary under the influence of an electric field. The fragments are detected by a fluorescent dye attached to the ddNTPs, which emits a signal when excited by a laser.
The data obtained from the capillary electrophoresis
are processed and analyzed using specialized software. The software assigns a nucleotide base to each peak in the electropherogram, which represents the sequence of the DNA fragment. The sequence is determined by analyzing the order of the peaks corresponding to the different nucleotides.
Once the sequence is obtained, it can be compared to known reference sequences or analyzed further for various purposes, such as identifying genetic variations or
studying gene expression patterns.
中文回答：
焦磷酸测序技术（Sanger测序）已经彻底改变了基因组学领域，使我们能够解读生物体的遗传密码。
焦磷酸测序是一种广泛使用的
测序技术，也被称为链终止法或二脱氧测序。
该技术依赖于DNA合
成过程中链终止的二脱氧核苷酸（ddNTPs）的引入。
焦磷酸测序的过程包括几个步骤。
首先，需要提取和纯化感兴
趣的DNA样品。
这可以从血液、组织或培养细胞等不同来源进行。
一旦DNA被提取，就会将其断裂成较小的片段，以便于测序过程。
接下来，使用聚合酶链式反应（PCR）对DNA片段进行扩增。
PCR是一种允许特定DNA序列扩增的技术。
它涉及多个DNA变性、
引物退火和DNA合成的循环。
在PCR过程中，使用与目标DNA序列
特异性的引物来启动DNA合成。
经过PCR扩增后，设置测序反应。
这涉及将扩增的DNA片段与
引物、DNA聚合酶以及一种混合物（包含正常的脱氧核苷酸（dNTPs）和少量的链终止的ddNTPs）混合。
ddNTPs缺乏DNA链延伸所需的
3'-OH基团，从而导致DNA合成在特定位置终止。
然后，将测序反应混合物进行毛细管电泳。
在这一步中，DNA
片段根据其大小和电荷在带有凝胶的毛细管中受电场影响而迁移分离。
片段通过与ddNTPs结合的荧光染料进行检测，该染料在激光激
发下发出信号。
从毛细管电泳获得的数据使用专门的软件进行处理和分析。
该
软件为电泳图中的每个峰分配一个核苷酸碱基，代表了DNA片段的
序列。
通过分析对应不同核苷酸的峰的顺序，可以确定序列。
一旦获得序列，可以将其与已知的参考序列进行比对，或进一
步分析以进行各种目的，例如识别基因变异或研究基因表达模式。