Shot gun method is a method of obtaining directly from biological cell genomeTarget geneMethod.The shotgun methodorganismWhole genome or singlechromosomeCut into suitable sized DNA fragments, connect them to the carrier DNA, and transform themReceptor cell, forming a set of reorganizationclone, and screen out the purpose containing the target geneRecombinant[1-2]。This method is simple, but the workload is large[3]。
Chinese name
Shotgun method
Foreign name
Shotgun method
Alias
Shotgun technique
Purpose
DNA is randomly processed into fragments of different sizes
The shotgun method is the most commonly used method to obtain target genes directly from biological cell genomes.It uses restriction enzymes to cut the DNA in donor cells into many segments, load these segments into transport vectors, and then transfer them into differentReceptor cellMake all segments of DNA (i.e. foreign DNA) provided by donor cells replicate in a large number in each recipient cell, find out the cells containing the target gene, and then separate the DNA segments with the target gene by certain methods[3]。This method is simple to operate, but the workload is large.
Its advantage is that it is fast, and the biological cell genome can be obtained in a very short time (for example, 95% of the human genome sequence).The idea of this method is unique. It seems that there is a large group of birds in the forest, and many people shoot indiscriminately. In a very short time, most of the birds in the forest can be shot.The shotgun method is similar to the jigsaw puzzle that people play.The jigsaw puzzle is to divide a complete picture into disorderly pieces, and then reassemble.The "shotgun method" is to first disrupt the whole genome and cut it into random fragments, then determine the sequence of each small fragment, and finally use computers to sort and assemble these slices, and determine their correct positions in the genome.
principle
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"Shotgun method" is a method of extracting target genes from biological genomes.Firstly, physical methods (such as shearing force, ultrasound, etc.) or enzymatic chemical methods (such asRestriction endonuclease）Biological cellsChromosome DNACut intogeneAnd then combine these fragments with the appropriate vector, transfer the recombinant DNA into the receptor bacteria for amplification, and obtainAsexual reproductionOfGene library, combined with screening methodsTransformantStrainsThe strains containing a certain gene are selected from the strains, and the recombinant DNA is separated and recovered.
This method is to use genetic engineering technology to isolate target genes, which is characterized by bypassing the difficulty of directly isolating genesGenome DNA LibraryThe target gene was screened out from.It can be said that this is to "hit" a gene by using the principle of "stray bullet shooting".becauseTarget geneIt is too small in the whole genome, and to a considerable extent, it depends on "luck", so people call this method "shotgun method" or "shotgun method".
Basic process
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Preparation of objective genomic DNA fragments
It is separated and purified from biological cells as donors by conventional methodsChromosome DNAUnder general conditions, due to the physical shearing effect in the separation and purification operation, the average size of the prepared chromosome DNA fragments is about 10-200 kb.Then, the chromosome DNA is cut into fragments by the following methods for in vitro recombination with the carrier molecule[1]。
(1) Mechanical cutting.The donor chromosome DNA can be randomly cut into double stranded flat end segments by mechanical methods (such as ultrasonic treatment). The cut DNA segments can be controlled within a certain range by taking appropriate ultrasonic treatment intensity and time. The upper limit is the maximum load of the vector, and the lower limit should be greater than the length of the target gene, otherwise the complete target gene cannot be obtained in a recombinant clone.For one amine, the gene length of prokaryotes is mostly within 2kb. The gene length of eukaryotes varies greatly, and the maximum gene can reach more than 1001kb. Therefore, it is always correct to treat the foreign DNA fragments to a length slightly less than the upper limit of the carrier loading capacity, because the larger the foreign DNA fragments contained in each recombinant gradient, the smaller the scale of subsequent selection.WhenChromosome DNAOf the target gene regionRestriction digestion mapWhen unknown, machine cutting is the preferred method for preparing DNA fragments to be cloned. However, because these DNA fragments have random flat ends, they must be inserted into the flat end restriction enzyme cutting site of vector DNA, and it is difficult to completely remove cloned foreign DNA fragments from recombinant molecules.
(2)Restriction endonucleasePartial solution.Partial degradation by restriction endonucleases (such as MbolSaa3A, Alal, etc.) with recognition sequence of four broken base pairsChromosome DNA, you can also get large pieces ofDNA molecule。Because the recognition sequence of these restriction endonucleases frequently appears in any biological genome, random DNA fragments of certain length can also be obtained as long as appropriate partial enzymolysis conditions are taken, and the DNA fragments obtained by partial enzymolysis have sticky ends, which can be directly spliced with carrier molecules.
(3) Complete enzymolysis with specific restriction enzymes.If there are known restriction endonuclease recognition sites on both sides of the target gene in chromosome DNA, and the distance between the two does not exceed the upper limit of the carrier load, it may be more advantageous to use one (or two) restriction endonuclease to fully enzymatically hydrolyze chromosome DNA fragments. The generated DNA fragments are non random, which can simplify subsequent recombination and screening operations to some extent.At the same time, the inserted fragments of the recombinant molecules can be completely cut down by the same restriction enzyme, which makes it possible to directly screen the target recombinants by using the restriction enzyme map method.
Full cloning of foreign DNA fragments
The vector of attenuation is determined according to the nature and size of the end of the foreign DNA fragment. The shotgun method generally selects plasmid or DNA as the vectorCloning vector，Receptor cellMost choicesEscherichia coli, only when the subsequent screening must use external sourcesgene expressionThe corresponding receptor systems that can make foreign genes express should be selected for product detection[1]。
Screening of target recombinants
The most effective way to quickly detect the target recombinant from numerous shotgun clones is colony (plaque)In situ hybridizationThe former requires an ideal probe, while the latter relies on the establishment of a simple screening type.As previously mentioned, if you clone amylase, protein orAntibiotic resistance geneIt is the best choice to screen the target recombinants by the function test of foreign gene products.In the absence of probes and difficulty in establishing a rapid screening model, the obtained recombinant clones can also be screened in batches by restriction enzyme digestion map.For example, as the target gene is known to be located in the 2.8kb ECORI DNA fragment, ECORI can be used to enzymatically hydrolyze all recombinant molecules, preliminarily determine the recombinant clone containing the 2.8kb restricted insert fragment, and then conduct the second round of restriction screening according to the characteristic restriction enzyme digestion site inside the target gene, finally find the target recombinant[1]。
Localization of target gene
In most cases, the target recombinants obtained by shotgun method are only DNA fragments containing the target gene. The target gene must be accurately located on the cloned DNA fragments through secondary cloning, and then the target gene is sequenced to search for its coding sequence and possible epigenetic DNA regulatory sequence.It is conceivable that the amount of work required to clone the target gene by shotgun method is large. The more detailed you know about the nature of the target gene and its encoded product, the less work required[1]。
application
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The "shotgun method" was originally used to determine microorganismsgenome sequence。In recent years, Drosophila melanogaster andhuman genomeAnd proved its feasibility and effectiveness in determining large genomes[4]。
In July 2001, China's hybrid rice genome project was officially launched. First, pure indica rice 93-11, the male parent of hybrid rice, was taken as the research object.Different from the International Rice Genome Project, this study uses the whole genome "shotgun" strategy for sequencing[4]。
characteristic
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The "shotgun method" has the advantages of fast speed, simplicity and low cost[4]。However, it is not easy to assemble the final sequencing results.Chinese scientists have designed a sequence assembly software that can effectively overcome the "shotgun method"whole genome sequencing Difficulties during assembly[4]。
In the study, they firstRice GenomeGenerate many DNA of known length on（Deoxyribonucleic acid）Slice and arrange them according to the overlapping area of DNA sequence.The number of these sections is enough to cover the rice genome four times.Scientists then determine the base pair sequence of each slice, and use computer programs to assemble it into longer fragments, and then sort and assemble these fragments into more than 100000 larger components called scaffolds[4]。
The software they designed focuses on assembly by approaching at the scaffold level, and adopts a unique repetitive sequence processing algorithm, which can identify and temporarily block about 40% of the rice genomeRepeating sequence。The advantage of this is that it can not only reduce the amount of calculation, but also minimize the possibility of wrong splicing.
U.S.APurdue UniversityPlant geneticist Bennett Zeen commented that therice genome project"Provides an excellent example of the speed and efficiency of shotgun sequencing"[4]。
Scope of application
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Eukaryotes ingene expressionWhen,Structural geneThe introns in cannot guide protein synthesis.So eukaryoticTarget geneIt cannot be obtained by "shotgun method".EukaryoteGenes are mostly composed of coding sequences - exons and non coding sequences——IntronAnd the ratio is 1:4.Shotgun Cloning stayGenome DNA LibraryFiltered inTarget gene, the same as the natural gene in the chromosome, contains intronsStructural geneThe junction region at both ends also has transcriptionRegulatory sequenceFragments, that isRegulatory gene。Such genes can providegene expressionRegulation analysis.Because of the existence of introns, the structural genes obtained in this way should not beProkaryoteIt is expressed in host tissue.Therefore, in order to obtain the target gene of eukaryotes, it is necessary to obtain the target gene of eukaryotesCoding sequenceThe shotgun method should not be used.
