Shotgun Protein Sequencing Method

Introduction

The most common method for obtaining a gene of interest directly from the genome of a biological cell is the "shotgun method", also known as the "shotgun method." The "shotgun method" is to separate the genomes by chromosomes, scramble them all, cut them into pieces, perform random sequencing, and then splicing them together after sequencing. It has the advantage of being fast and in a short time to get the genome of a biological cell (for example, 95% of the human genome). The idea of the law is unique. It seems that a large group of birds have been parked in the woods. Many people shoot and shoot, and in a short period of time, most of the birds in the forest can be hit. The "shotgun" is a bit like a jigsaw puzzle played by people. The jigsaw puzzle divides a complete picture into disorganized pieces and then reassembles them. The "shotgun method" first scrambles the entire genome, cuts it into random fragments, then measures each small fragment sequence, and finally uses a computer to sort and assemble the slices and determine their correct position in the genome.

The shotgun protein sequencing method is a sequencing method in which the target DNA is randomly processed into fragments of different sizes, and the sequences of these fragments are connected together, belonging to the first generation sequencing technology.

Application

The "shotgun method" was originally used primarily to determine the microbial genome sequence. In recent years, the biological genius Craig Venter and its company, Celera, have used the improved genome-wide “shotgun method” to complete the sequencing of the fruit fly and human genome, demonstrating its ability to determine large genomes. Feasibility and effectiveness

Features

Advantages

The advantage of shotgun method is that it is fast, simple and easy to operate, and the cost is low. But it is not easy to use for sequencing service, and the final assembly result is not easy. Chinese scientists have designed a sequence assembly software that can effectively overcome the "shotgun method". Difficulties in the whole genome sequencing monoclonal antibodies assembly process.

In the study, they first generated a number of DNA (deoxyribonucleic acid) slices of known length across the entire rice genome and then arranged them in overlapping regions of the DNA sequence. The number of these slices is sufficient to cover the rice genome 4 times. The scientists then determined the base pair sequence for each slice and assembled it into longer pieces using a computer program, then sorted and assembled the pieces into more than 100,000 larger components called stents.

The software they designed focused on assembly at the level of the scaffold, and adopted a unique repetitive sequence processing algorithm that identified and temporarily blocked approximately 40% of the repeats of the rice genome. This has the advantage of reducing the amount of computation and minimizing the possibility of false stitching.

Bennett Zeen, a plant geneticist at Purdue University in the United States, commented that the Chinese scientist's rice genome project "provides an excellent example of the speed and efficiency of shotgun sequencing."

Limitation

When eukaryotes express genes, introns in structural genes cannot guide protein synthesis. Therefore, the target gene of eukaryotes cannot be obtained by the "shotgun method".

The "shotgun method" is a method of extracting a gene of interest from a biological genome. First, cleavage of biological cell chromosomal DNA into many fragments at the gene level using physical methods (such as shear, ultrasound, etc.) or enzymatic chemistry (such as restriction endonucleases), and then combining these fragments with appropriate vectors, The recombinant DNA is transferred into a recipient strain to obtain a gene library of asexual reproduction, and a strain containing a certain gene is selected from a plurality of transformant strains, and the recombinant DNA is separated and recovered therefrom.

This method is to use genetic engineering technology to isolate the target gene, which is characterized by bypassing the difficulty of directly isolating the gene and screening the target gene in the genomic DNA library. It can be said that this is to use the "slack shot" principle to "hit" a certain gene. Because the target gene is too small and too small in the whole genome, and to a certain extent depends on "take a chance", people call this method "shotgun" or "shotgun" experimental method.

Conclusion

Most of the eukaryotic genes consist of a sequence with coding function, an exon and a sequence without coding function, and the ratio of the two is 1:4. The target gene selected by the shotgun cloning method in the genomic DNA library is the same as the natural gene in the chromosome, and contains an intron. The junction region at both ends of the structural gene also has a transcriptional regulatory sequence fragment, that is, a regulatory gene. Such genes are available for regulatory analysis of gene expression. Because of the presence of introns, the structural genes thus obtained are not suitable for expression in prokaryotic host tissues. Therefore, in order to obtain the target gene of eukaryotes, it is not appropriate to use the shotgun method to obtain the coding sequence of the gene for the eukaryote.