genetic engineeringThe upstream works ofTarget genePreparation andAsexual reproduction。Specifically, it refers to the preparation of target genes or artificial synthesis of target genes from tissues, organs or cells of organismsDNASpliceRecombinantMolecular introductionReceptor cell, screening and asexual reproduction.This process can be called gene cloning.[1]
Gene cloning was developed in the 1970srevolutionary characterThe research technology of "," can be summarized as: dividing, cutting, connecting, turning, and selecting.The ultimate goal is to use corresponding technical means toTarget geneImportHost cell, onhost cellThe target genes are copied in large quantities.
Genes are intracellularDNAThere aregenetic effect Specific tonucleotide sequenceThe generic term ofDNAMolecular fragments.Gene controlprotein synthesisIt is the fundamental reason why different species and different individuals of the same species show different traits, namely“As you sow, you reap"," a mother bornNine sonsThe nine sons are differentDNA replicationandcell divisionholdgenetic informationIt is passed on to the next generation, and the genetic information is expressed by controlling the synthesis of proteins.
MinutesIt refers to the separation and preparation of qualified DNA to be operated, includingCarrierAnd the target DNA to be cloned."cut"Refers to the sequence specificRestriction endonucleaseCut the carrier DNA, or cut outTarget gene；"even"It refers to the use of DNA ligases to connect the target DNA with the carrier DNA to form recombinant DNA molecules;"turn"It means that the recombinant DNA molecules are sent intohost cellReplication and amplification;"choose"It is to select individuals carrying recombinant DNA molecules from the host population.genetic engineeringThe two most basic characteristics of technology are molecular level operation and cell level expression, and molecular level operation is the process of in vitro recombination, actually usingtool enzymeDNA molecule“surgical operation"。
Using recombinant DNA technologyDNA moleculeIn vitro, it was specifically cut, reconnected and assembled into a new hybrid DNA molecule.On this basis, this hybrid molecule canhost cellThe process is called gene cloning.
history
The idea of gene cloning technology first appeared at a scientific conference on plasmid in Honolulu in November 1972.(Plasmids are DNA rings found in bacteria. Plasmids carry certain antibioticsDrug resistance)Stanley Cohen, who has been studying plasmids, is very interested in the introduction of specific parts of DNA molecules cut by bacterial enzymes on Herbert Boyer's website.Visiting a room in Waikiki late at nightDeliThe two scientists met and talked aboutscientific knowledgeCombined, we conceived an experiment to create a modern biotechnology industry.At the beginning of 1973, they launched a series of experiments to get methods to select specificforeign geneReplicate in bacteria.[2]
Cloning process
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DNA cloning means that in vitroTarget geneOr other meaningful DNA fragmentsSelf replicationAnd then transfer it intohost cellOr the process of molecular manipulation of receptor organisms for expression or further research, soDNA cloningalso calledmolecular cloning，Gene manipulationOr recombinant DNA technology.DNA cloning involves a series ofMolecular Biology Technology, such as obtaining target DNA fragments, selecting vectorstool enzymeSelection, in vitro recombination, host cell introduction andRecombinantScreening technology and so on.
Acquisition of target DNA fragments
The first step of DNA cloning is to obtainTarget geneA group ofDNA moleculeThese DNA molecules may come from target organismsGenomic DNAOr from target cell mRNAReverse transcriptionSyntheticDouble chainCDNA molecule.Because the genomic DNA is large and not conducive to cloning, it is necessary to process it into small DNA fragments suitable for cloning. The common methods areMechanical cuttingAnd nucleic acidRestriction endonucleaseDigestion.If the gene sequence is known and relatively small, it can be directly synthesized by artificial chemistry.If the sequence at both ends of the gene is known, design according to the known sequenceprimerFrom genomic DNA or cDNAPCR technologyThe target gene can be obtained.
Selection of carrier
The vector of genetic engineering should have some basic properties: (1)host cellIn order to makeExogenousThe recombinant DNA fragments were amplified.(2) The molecular weight should be as small as possible to facilitate more copies in the host cell and to facilitate the combination of larger foreign DNA fragments.At the same timeExperimental operationIt is also not easy to be damaged by mechanical shearing.(3) It is better to have more than two easily detectedgenetic marker(e.gDrug resistanceMarker gene）To endow host cells with differentphenotypeCharacteristics (such as resistance to antibiotics).(4) The carrier itself should preferably have as manyRestriction enzymeA single cut point provides a wider selection range to avoid interference of restriction enzyme sites in foreign DNA fragments.If the singleRestriction siteIt is located in the marker gene of the detected phenotype, which can causeInsertion inactivationEffect is more beneficialRecombinantFilter for.
In vitro recombination is the process of connecting the target fragment with the carrier molecule in vitro.Most nucleic acidsRestriction endonucleaseAble to cutDNA moleculeFormed withViscous end, using the same enzyme orHomotailaseCutting the appropriate carrierPolyclonal siteThe same viscous ends can be obtained, and the viscous ends are annealed with each other through T4DNA ligaseCan be formedRecombinant, this is sticky end connection.When the target DNA fragment isFlat end, can be directly connected to the carrier with flat end, this isFlat endConnection, but connection efficiency ratioSticky endThe connection is poor.Sometimes for different cloning purposes, such as inserting flat ended DNA molecules into the expression vector with sticky ends to achieve expression, the flat ended DNA molecules need to be modified through some modifications, such as adding tails to polymersCatcherorsynthetic adapter，PCR methodThe introduction of enzyme digestion sites, etc., can obtain the corresponding sticky ends, and then connect them. This is the modified sticky end connection.
The carrier DNA molecule hasProkaryoteHost cell recognizedReplication start site, so you canProkaryotic cellFor example, replication in Escherichia coliTarget geneIt is amplified together with the vector, and a large number of identical recombinations are finally obtainedDNA molecule。
Introduction of foreign recombinant DNA molecules into prokaryoteshost cellMethods of transformation and transfection（transfection）, transduction.Recombinant plasmidThrough transformation technology, it can be imported into host cells, and also reorganizedphageDNA can be introduced by transfection technology.The transfection efficiency is not high, so phage DNA or Coss will be recombinedplasmidExternal packagingInfectiousBacteriophage particleWith the help of these phage particles, recombinant DNA molecules can be introduced into host cells. The efficiency of this transduction technology is higher than that of transfection.
Screening of recombinants
The transformants obtained from different recombinant DNA molecules were identified to containTarget geneThe transformant ofPositive cloneThe process is screening.The developed mature screening methods are as follows:
The foreign DNA fragment is inserted into the screening marker gene (antibiotic gene orβ-Galactosidase gene)Polyclonal siteAfter, it will cause markingGene inactivation, showing the correspondingAntibiotic resistanceVanishing or color change of transformant, through which it can be preliminarily identified that the transformant isRecombinantOr non recombinants.Commonly usedβ-GalactosidaseColor development method, i.e. blue and white screening method (whitecolony of bacteriaIs a recombinant plasmid).
Extract theRecombinant DNAMolecule is used as template, and specific design is made according to the known sequence at both ends of the target geneprimerThe positive clones were screened by PCR technology.The positive clones screened by PCR were usedRestriction endonucleaseFurther identification by enzyme digestionInsert ClipSize of.
preparationTarget geneIdiosyncraticNucleic acid probeThe target clone was screened from many transformants by nucleic acid molecular hybridization.The target gene specific nucleic acid probe can be part of the obtained purposegene segment, or purposegene expressionPartial sequence of proteinBacksteppingA group of oligonucleotides obtained, or those of other speciesHomologous gene。
Purposegene expressionImmunological screening method can be used to obtain the target gene clone.These antibodies can purify the target gene expression protein antibody from the organism itself, and can also be obtained from the target gene partORFFragment cloning inexpression vector Antibodies against the expressed protein were obtained in.
The positive clone obtained by the above method should be carried out finallySequencing analysisFor final confirmationTarget gene。
