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The magic of amplified fragments: How to find key clues in genetic research?
In molecular biology, an amplicon is a segment of DNA or RNA that is produced by an amplification or replication event. These fragments can be artificially generated using various methods such as polymerase chain reaction (PCR) or ligase chain reaction (LCR), or they can be the result of natural gene duplications. Amplification as referred to herein includes the production of multiple copies of a genetic segment or target sequence, and particularly refers to the amplified segment itself.
Artificial amplification has applications in research, forensics, and medicine for purposes including detecting and quantifying infectious pathogens, identifying human remains, and extracting genotypes from human hair.
Natural gene duplications play an important role in evolution and have been linked to several forms of human cancer, including primary mediastinal B-cell lymphoma and Hodgkin lymphoma. Therefore, in this context, amplicon can refer not only to specific chromosomal DNA segments, but also to segments that have been excised, amplified, and reinserted elsewhere in the genome, as well as extrachromosomal DNA segments known as diminigenes. , each fragment can be composed of one or more genes.
The amplification of the genes encoded by these amplified fragments usually increases the transcription efficiency of these genes, ultimately leading to an increase in the amount of related proteins.
Structure of the amplified fragment
The amplicon is usually a direct repeat (head-to-tail) or inverted repeat (head-to-head or tail-to-tail) of a genetic sequence and can be linear or circular in structure. The structure of the circular amplicon consists of imperfect inverted repeats that conditionally coalesce to form a circle, presumably formed from a precursor linear amplicon. When amplifying artificially, the length of the amplified fragment depends on the goal of the experiment.
Advances in Amplification Technology
The analysis of amplified fragments has been made possible thanks to the development of amplification methods such as PCR, and more and more high-quality and high-throughput DNA sequencing technologies have emerged, such as ion semiconductor sequencing technology, which is often referred to as The developer's brand is called Ion Torrent. These sequencing technologies have made it possible to study amplicon fragments in the fields of genome biology and genetics, including cancer genetics research, phylogenetic studies, and human genetics. Take the 16S rRNA gene as an example. This gene is part of the genome of every bacteria and archaea and is highly conserved. By comparing the sequence of the amplified fragment with the known sequence, bacteria can be classified.
Almost all amplified fragment sequencing requires quantification of the amplified product, which usually involves a capture step and a detection step, and the specific implementation methods vary.
Application of amplified fragments
PCR can be used to determine the sex of a human DNA sample. By selecting the Alu element insertion site for amplification and evaluating the size of the fragment, the gender test uses AluSTXa to correspond to the X chromosome and AluSTYa to correspond to the Y chromosome. This design can effectively reduce the possibility of error. When the homologous region is amplified, the inserted chromosome will produce larger fragments. Males can be distinguished as having two DNA amplification fragments, while females only have one amplification fragment. The method package for this method includes a pair of primers for amplification sites and selective polymerase chain reaction reagents. In the diagnosis of tuberculosis, LCR has also been used as a test, with the target sequence containing protein antigen B being targeted by four oligonucleotide primers for the sense and reverse strands, respectively.
The amplified products or fragments are evaluated through various detection and capture steps. With the development of amplicon sequencing, the various amplicon fragments generated from a common sample are connected and sequenced, followed by quality control classification, and finally the number of the same type is calculated to reflect their relative abundance in the sample. Richness.
With the advancement of technology, amplified fragments have become an indispensable part of today's genetic research. But in this rapidly evolving field, do we fully understand the potential of amplified fragments?
