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基因敲除经典方法

基因敲除经典方法
基因敲除经典方法

J OURNAL OF B ACTERIOLOGY,

0021-9193/97/$04.00?0

Oct.1997,p.6228–6237Vol.179,No.20 Copyright?1997,American Society for Microbiology

Methods for Generating Precise Deletions and Insertions in the

Genome of Wild-Type Escherichia coli:Application to Open

Reading Frame Characterization

ANDREW J.LINK,1?DERETH PHILLIPS,1AND GEORGE M.CHURCH1,2*

Department of Genetics1and Howard Hughes Medical Institute,2Harvard Medical School,

Boston,Massachusetts02115

Received6March1997/Accepted8August1997

We have developed a new system of chromosomal mutagenesis in order to study the functions of unchar-

acterized open reading frames(ORFs)in wild-type Escherichia coli.Because of the operon structure of this

organism,traditional methods such as insertional mutagenesis run the risk of introducing polar effects on

downstream genes or creating secondary mutations elsewhere in the genome.Our system uses crossover PCR

to create in-frame,tagged deletions in chromosomal DNA.These deletions are placed in the E.coli chromosome

by using plasmid pKO3,a gene replacement vector that contains a temperature-sensitive origin of replication

and markers for positive and negative selection for chromosomal integration and https://www.sodocs.net/doc/3711304753.html,ing kanamycin

resistance(Kn r)insertional alleles of the essential genes pepM and rpsB cloned into the replacement vector,we

calibrated the system for the expected results when essential genes are deleted.Two poorly understood genes,

hdeA and yjbJ,encoding highly abundant proteins were selected as targets for this approach.When the system

was used to replace chromosomal hdeA with insertional alleles,we observed vastly different results that were

dependent on the exact nature of the insertions.When a Kn r gene was inserted into hdeA at two different

locations and orientations,both essential and nonessential phenotypes were https://www.sodocs.net/doc/3711304753.html,ing PCR-generated

deletions,we were able to make in-frame deletion strains of both hdeA and yjbJ.The two genes proved to be

nonessential in both rich and glucose-minimal media.In competition experiments using isogenic strains,the

strain with the insertional allele of yjbJ showed growth rates different from those of the strain with the deletion

allele of yjbJ.These results illustrate that in-frame,unmarked deletions are among the most reliable types of

mutations available for wild-type E.coli.Because these strains are isogenic with the exception of their deleted

ORFs,they may be used in competition with one another to reveal phenotypes not apparent when cultured

singly.

With the completion of the Escherichia coli K-12genome sequence(https://www.sodocs.net/doc/3711304753.html,/and http://mol.genes .nig.ac.jp/ecoli/),a variety of tools will be required to deter-mine the functions of the vast array of uncharacterized open reading frames(ORFs)found within the genome.Even in an organism as well studied as E.coli,over58%of the putative coding regions remain without a recognized function,and many others are only partially understood.To study these regions,we devised a system for creating in-frame deletions of any desired sequence in wild-type E.coli.

Gene replacements in E.coli have generally relied on spe-ci?c genetic backgrounds as starting strains,such as polA,recD, strR,sup?,or F?(15,21,37,39,44).After replacement of a wild-type sequence with an in vitro-altered sequence in a mu-tant background,the altered chromosomal region must then be transduced into a wild-type genetic background.Unfortu-nately,these methods often require the transduction of a marker along with the mutant allele.This marker can obscure the phenotype of the mutant allele because it may itself cause a mutant phenotype.

Bacterial genes needed in a particular pathway tend to be grouped in cotranscribed clusters or operons(32).Insertional, frameshift,nonsense,or antisense disruption of an ORF within an operon can affect upstream and downstream gene expres-sion in addition to the gene targeted for inactivation.These polar effects could confuse the assignment of a mutant pheno-type to the disrupted gene.At the other extreme,point mu-tants can leave signi?cant parts of the gene intact.To reduce these problems,we developed methods for creating precisely engineered deletions of E.coli ORFs by using a procedure known as crossover PCR(18,19).To integrate these PCR-generated deletions into the genome of wild-type E.coli,we constructed a new gene replacement vector,pKO3. Hamilton et al.have described a method for gene replace-ment in wild-type E.coli that uses homologous recombination between the bacterial chromosome and a plasmid carrying cloned chromosomal sequences whose replication ability is temperature sensitive(16).At the nonpermissive temperature, cells maintain drug resistance only if the plasmid integrates into the chromosome by homologous recombination between the cloned fragment and the bacterial chromosome.Excision of the integrated plasmid is allowed at the permissive temper-ature.Depending on the position of the second recombination event that excises the plasmid,the chromosome retains either the wild-type sequence or the altered sequence from the plas-mid.Although this method can be applied to wild-type strains, there is no selection for loss of the excised plasmid.The Ba-cillus subtilis gene sacB encodes levansucrase,an enzyme that catalyzes the hydrolysis of sucrose and levan elongation(12). When expressed in E.coli growing on media supplemented

*Corresponding author.Mailing address:Department of Genetics, Warren Alpert Building,Room513,Harvard Medical School,200 Longwood Ave.,Boston,MA02115.Phone:(617)432-7562.Fax:(617) 432-7266.E-mail:church@https://www.sodocs.net/doc/3711304753.html,.

?Present address:Department of Molecular Biotechnology,Uni-versity of Washington,Seattle,WA98195.

6228

with sucrose,the sacB gene is lethal(14).Blom?eld et al. developed a system for using a temperature-sensitive plasmid and a counterselectable sacB marker in the chromosome to facilitate allelic exchange(5).We have reduced this system to one component by incorporating the sacB gene into a gene replacement plasmid(pKO3)and have developed a protocol for introducing altered alleles into wild-type E.coli strains.By combining the crossover PCR and gene replacement methods, we demonstrate a system for creating precise deletions that eliminate gene function without introducing polar affects on expression of distal genes in an operon.

When making a survey of the most abundant proteins in E. coli,we found two poorly understood genes,yjbJ and hdeA, that encode unexpectedly abundant proteins in the cell(25). The E.coli yjbJ gene,with sequence similarity to the unchar-acterized ORF ywmH in B.subtilis,encodes a small69-amino-acid protein that is highly abundant during early stationary phase in rich media.HdeA is a121-amino-acid protein with a 23-amino-acid signal peptide whose expression is affected by mutations that eliminate the protein HU-1(45,46).The HdeA protein is abundant during growth in minimal media and dur-ing stationary phase in rich media(25).To determine if mutant alleles of yjbJ and hdeA have signi?cant phenotypes,we re-placed the chromosomal genes with both insertion and dele-tion alleles by using the pKO3gene replacement protocol.In the following text,we will discuss the advantages and disad-vantages of both insertional and deletion methods.In light of the completion of the genomic sequences of several free-living organisms,the results of these gene replacements are dis-cussed as paradigms for addressing the function of chromo-somal sequences.

MATERIALS AND METHODS

Strains.All plasmid constructions were electroporated and propagated in E. coli DH5?[F???endA1hsdR17hsdM?supE44thi1recA1gyrA96relA1?(argF lacZYA)U169?80d?(lac Z)M15].The gene replacement experiments used the recombination pro?cient wild-type K-12strain EMG2(F???).

Media and growth conditions.All strains were grown in LB medium(1% Bacto Tryptone,0.5%yeast extract,0.5%NaCl)with the appropriate selection. For antibiotic selection,the concentrations of antibiotics were50mg/ml(ampi-cillin and kanamycin)and20mg/ml(chloramphenicol).For selection against sacB,LB medium was supplemented with sucrose to a?nal sucrose concentra-tion of5%(wt/vol).

DNA puri?cation.Plasmid DNA was puri?ed by the alkaline lysis method(4). Genomic DNA was puri?ed by previously described methods(10).

Partial digestion.All partial digestion of genomic and plasmid DNA used serial dilution of the restriction enzyme and a constant1-h incubation time(26). The reactions were stopped by adding0.25M EDTA(pH8)to a?nal concen-tration of50mM.

Blunt-end reactions.Unless stated otherwise,T4DNA polymerase and de-oxynucleoside triphosphates(dNTPs)were used to create all blunt-ended DNA fragments.

Ligation.Ligations were performed overnight at room temperature,using a DNA concentration of10?g/ml and an insert-to-vector molar ratio of1:1or an oligonucleotide-to-insert molar ratio of160:1.The ligation buffer used for the reactions contained66mM Tris-HCl(pH7.5),5mM MgCl2,50mM dithiothre-itol,1mM ATP,and0.05Weiss units of T4DNA ligase/?l.The ligated DNA was ethanol precipitated,washed with70%ethanol,vacuum dried,and resuspended either in10mM Tris-HCl(pH8)–1mM EDTA or40?l of electroporation-competent E.coli cells(for immediate transformation).

Electroporation.Electroporation-competent cells(40?l;1011CFU/ml)were mixed with1to3?l of DNA solution in an ice-cold microcentrifuge tube and transferred to a0.2-cm electroporation cuvette(Bio-Rad,Inc.).The cells were electroporated at2.5kV with25?F and resistance of200-ohms.Immediately after electroporation,1ml of SOC medium(2%Bacto Tryptone,0.5%yeast extract,10mM NaCl,2.5mM KCl,10mM MgCl2,10mM MgSO4,20mM glucose)was added to the cuvette.The cells were transferred to a17-by100-mm polypropylene tube and allowed to recover for1h at either30°C(for tempera-ture-sensitive plasmids)or37°C with shaking at250rpm before plating on selective media.

PCR.All PCRs were performed in a Perkin-Elmer9600thermal cycler.PCR buffer(28)consisted of30mM tricine(pH8.4),2mM MgCl2,5mM?-mercap-toethanol,0.01%(wt/vol)gelatin,0.1%(wt/vol)Thesit,200?M each dNTP,600?M each primer,and1U of Taq polymerase(Boehringer Mannheim,Inc.). After addition of template DNA,the PCR mixture was denatured at94°C for3 min before addition of the Taq polymerase.The thermal cycle pro?le was15s at 94°C,15s at55°C,and30s at72°C.All experiments used30cycles and a?nal 5-min72°C hold step.

Analysis of PCR products.PCR products were analyzed on2%high-strength agarose–1%NuSieve agarose gels(FMC,Inc.)or1%high-strength agarose gels cast in0.5?Tris-borate-EDTA with ethidium bromide.

pKO3plasmid construction.The gene replacement vector pKO3was con-structed as follows.First,the1.6-kb Eco NI fragment from pMAK700(16)con-taining the temperature-sensitive pSC101replication origin and the1.6-kb Bbv II-Bsu36I fragment of pMAK700containing the cat gene were blunt ended and ligated together to create pKO1.

Second,the1.35-kb Not I-Nru I fragment from pBS-TS(2a)containing the sacB gene and the5.6-kb Sph I-linearized pMAK705plasmid(16)were blunt ended and ligated to create pMAK705s.The following Not I polylinker was then ligated into the Bam HI site of pMAK705s to create pMAK705so:

5?-GATCGCGGCCGCGGACCGGATCCTCTAGAGCGGCCGC-3?

3?-CGCCGGCGCCTGGCCTAGGAGATCTCGCCGGCGCTAG-5?

The550-bp Bgl I-Bsm AI fragment from pBluescript II SK?(Stratagene,Inc.) containing the M13origin of replication and Hin dIII-linearized plasmid pMAK705so were blunt ended and ligated to create pMAK705som.The single Pst I site in plasmid pMAK705som was deleted by using T4DNA polymerase and dNTPs to create pMAK705somp.

Finally,the2.4-kb Ecl136II-Eco RV fragment from pMAK705somp containing the polylinker,M13origin of replication,and sacB gene was blunt ended and ligated to Age I-linearized,blunt-ended plasmid pKO1to create pKO3. Crossover PCR deletions and subcloning.Crossover PCR deletion products were constructed in two steps,as illustrated in Fig.4.In the?rst step,two different25-?l asymmetric PCRs were used to generate fragments to the left and right of the sequences targeted for deletion.The PCR conditions were as de-scribed above except that the primer pairs were in a10:1molar ratio(600?M outer primer and60?M inner primer).In the second step,the left and right fragments were annealed at their overlapping region and ampli?ed by PCR as a single fragment,using the outer primers.Speci?cally,1?l of each of the two asymmetric PCR mixtures and600?M each of the two outside primers were mixed together and PCR ampli?ed.The fusion products were phenol-chloroform extracted,ethanol precipitated,washed with70%ethanol,vacuum dried,resus-pended in50?l of1?Bam HI restriction buffer containing40U of Bam HI restriction enzyme,and digested overnight at37°C.The fusion products were gel puri?ed,ligated into Bam HI-digested and phosphatase-treated pKO3vector, electroporated into E.coli,and plated on chloramphenicol plates at30°C.The recombinant colonies were screened for inserts with PCR,using primers pKO3-L and pKO3-R(described below).

To construct the286-bp deletion of yjbJ by crossover PCR,the following set of oligonucleotide primers was used:yjbJ-No,5?-CGCGGATCCTCACCTTTAC CGCCTATGCG-3?;yjbJ-Ni,5?-CCCATCCACTAAACTTAAACACCGTCA CGTTGCGGCAAACC-3?;yjbJ-Co,5?-CGCGGATCCTTGCGCCTGATGAG TCTGCAGG-3?;and yjbJ-Ci,5?-TGTTTAAGTTTAGTGGATGGGGTGGAT TGGGAAACCCGC-3?.

To construct the deletion of hdeA,the following set of primers was used: hdeA-No,5?-CGCGGATCCGAAATTATGACTGCGGTTGC-3?;hdeA-Ni,5?-CCCATCCACTAAACTTAAACAGCCTAATACTTTTTTCATCG-3?;hdeA-Co,5?-CGCGGATCCTACTCCTTTTTACTTGCACC-3?;and hdeA-Ci,5?-TG TTTAAGTTTAGTGGATGGGAAAGGCGAATGGGACAAAAT-3?.

DNA sequencing.DNA sequencing was performed as previously described with the Stratagene Cyclist sequencing kit H(27).Sequencing products were labeled with[?-32P]dATP and resolved on a4.5%wedge-gradient sequencing gel.Sequencing primers used for the pKO3left and right vector-insert junctions were pKO3-L(5?-AGGGCAGGGTCGTTAAATAGC-3?)and pKO3-R(5?-T TAATGCGCCGCTACAGGGCG-3?).Sequencing primers used to prime from multiplex tag04(10)were CP-04(5?-AGTGTGAGGTTTAAATATTG-3?)and CE-04(5?-TGTTTAAGTTTAGTGGATGG-3?).Sequencing primers used to prime from multiplex tag01(10)were CP-01(5?-TGATTAGTTGTAATGAA AGG-3?)and CE-01(5?-TAGTATGATTTTATTGGGGG-3?).

Gene replacement.Mutant alleles cloned into the pKO3gene replacement vector were electroporated into EMG2and allowed to recover for1h at30°C. The cells were plated on prewarmed chloramphenicol-LB plates and incubated at43and30°C.The integration frequency was calculated as the ratio of colonies at43°C to colonies at30°C.From the43°C plate,one to?ve colonies were picked into1ml of LB broth,serially diluted,and immediately plated at30°C on either 5%(wt/vol)sucrose or5%sucrose-kanamycin plates and at43°C on chloram-phenicol plates.The excision frequency is the ratio of30°C-grown sucrose-resistant colonies to43°C-grown chloramphenicol-resistant colonies.The5% sucrose plates were replica plated to chloramphenicol plates at30°C to test for loss of the replacement vector.The gene replacement was con?rmed by PCR using primers?anking the targeted ORF.

Construction of multiplex interposons.To construct the kanamycin resistance (Kn r)interposon,the1.3-kb Dra III-Bam HI fragment from pNK2859(23)con-taining the kan gene was blunt ended and ligated to the following Bst XI linkers:

V OL.179,1997PRECISE E.COLI GENOME ENGINEERING6229

5?-TCTAGACCACCTGC -3?3?-AGATCTGGTG -5?

The kan fragment with the attached Bst XI linkers was ligated to the 2.5-kb Bst XI fragment from multiplex vector plex.04B containing multiplex tags 04(10)to create plasmid pplexkan04B.The Kn r fragment with the attached linkers was similarly inserted into multiplex vectors 01,02,07,09,10,11,14,16,17,18,and 19(10)to construct a series of plexkan interposons.

To add a 5?-CG-3?overhang on the Kn r interposon,the 1.4-kb Not I fragment from plasmid pplexkan.04B was ligated with the following adapters and gel puri?ed:

Dap15?-CGCCCCCTGCAGGA -3?Dap23?-GGGGGACGTCCTCCGG -5?

Constructing pepM ,rpsB ,yjbJ ,and hdeA insertion mutations.A gel-fraction-ated genomic library of 3-to 7-kb Sau 3A inserts prepared from EMG2genomic DNA was ligated into the phosphatase-treated Bam HI site of pKO3.This library was electroporated into E.coli DH5?,plated on chloramphenicol plates,and overlaid with nylon membranes to create colony lifts essentially as previously described (26).To identify recombinant plasmids carrying the desired genomic inserts,the colonies were screened by hybridization using oligonucleotides la-beled at the 5?end with [?-32P]ATP and T4polynucleotide kinase at a probe concentration of 1nM,overnight at 42°C (10,26).The probes used were com-plementary to the 5?ends of pepM (5?-TTCTGTCCATCAGCGTCGGTG-3?),yjbJ (5?-GCCGGCTTCATCTTTATTCAT-3?),and hdeA (5?-CCACCAAGAAT AACGCCTAAT-3?).The pepM oligonucleotide was used to screen for rpsB clones simultaneously.

To identify clones with at least 1kb of genomic DNA ?anking each side of the desired genes,positive clones were screened by a combination of restriction mapping and DNA sequencing across the vector-insert junctions.These results were compared to physical maps of the regions.To create lesions in pepM and rpsB ,a positive clone was partially digested with a mixture of ?ve four-base recognition site restriction enzymes that create a 5?-CG-3?overhang (Aci I,Hpa II,Hin pI,Mae II,and Taq I).The singly cut,linearized plasmid was gel puri?ed and ligated with the Kn r interposon plexkan04,using adapters Dap1and Dap2.For yjbJ and hdeA ,positive clones (p1.7[yjbJ ]and p15.3[hdeA ])were linearized by partial digestion with restriction enzymes with unique sites in the ORFs (e.g.,Nae I for yjbJ and Pst I or Pvu II for hdeA ).The singly cut,linearized plasmids were gel puri?ed and ligated to a blunt-ended multiplex interposon (e.g.,yjbJ ::plexkan04,hdeA ::plexkan01[Pst I site],and hdeA ::plexkan04[Pvu II site]).Before performing the gene replacement,we characterized the in vitro -altered insert by DNA sequencing across both the vector-insert junctions (using the primer sites in the vector as primer sites)and the interposon-insert junctions (using the interposon’s multiplex tags as primer sites).

Screening for gene replacements.PCR was used to screen for gene replace-ments of yjbJ and hdeA .The yjbJ gene replacement was con?rmed by using the primers yjbJ-Nout and yjbJ-Cout ?anking the gene.The hdeA gene replacement was con?rmed by PCR using primer pair hdeA-Nout1plus hdeA-Cout1,hdeA-Nout2plus hdeA-Cout2,or hdeA-Nout3plus hdeA-Cout3?anking the gene.Sequences of the primers are as follows:yjbJ-Nout,5?-AGGTGAAAAAGAA ACCGCGTT-3?;yjbJ-Cout,5?-TGGTTTGCCGCAACGTGACGG-3?;hdeA-Nout1,5?-CGCGGATCCCATATACAGAAAACC-3?;hdeA-Cout1,5?-CGCG GATCCTTTTAAAGAAGATAT-3?;hdeA-Nout2,5?-CTGATGCATCTGTAA CTCATT-3?;hdeA-Cout2,5?-AACGCAGATTGTGCGTTCACC-3?;hdeA-Nout3,5?-GGATGAAGAAATAGCCGATC-3?;and hdeA-Cout3,5?-CTTCCC ATGCCAATTAATAC-3?.

Competition https://www.sodocs.net/doc/3711304753.html,petition experiments were performed by cocul-turing equal concentrations of two strains in rich media and then sampling the population density of each strain at various time points.Equal optical densities at 600nm of diluted overnight cultures of the various strains were mixed in the following combinations and sampled at various time points.EMG2yjbJ ::ple-kan04and EMG2hdeA ::plekan01strains were each cocultured with the wild-type EMG2strain.In a second competition experiment,EMG2yjbJ ::plexkan04was cocultured with EMG2?yjbJ .Each mixed culture was grown aerobically in a 250-ml Erlenmeyer ?ask containing 50ml of LB medium at 37°C shaking at 250rpm (New Brunswick Scienti?c G2platform).Since each culture contained both a marked and unmarked strain,the survival ratios could be determined by plating on both LB and kanamycin plates at various time points and counting the colonies surviving on each

plate.

FIG.1.Gene replacement vector and protocol.(A)The pKO3vector used in the gene replacement experiments.The cloning region is enlarged.Arrows in the circular plasmid indicate the direction of transcription and the direction of M13replication.The arrows in the enlarged region are the DNA primer sites.Unique restriction sites are shown (B,Bam HI;N,Not I;S,Sal I;Sm,Sma I).ori,origin of replication.(B)Protocol used for replacing wild-type sequences on the chromo-some with in vitro-altered sequences.The gene replacement vector carrying in vitro-altered sequences is transformed into E.coli and plated at the nonpermis-sive temperature of the plasmid replicon.An integration event allows replication of plasmid sequences by the chromosomal origin.When shifted to 30°C,the plasmid is excised from the chromosome at either crossover point 1or 2.The counterselectable sacB marker is used to select for loss of plasmid sequences.The sucrose-resistant colonies are screened for loss of vector sequences by replica plating to chloramphenicol plates and then for the gene replacement event by PCR.The “mutated sacB ?”in the left panels indicates loss of sacB gene

function by some unknown mechanism.The wavy,thin line represents the gene replacement vector sequences.The straight,thin line represents the E.coli chromosome.The boxes represent homologous sequences cloned into the vector (open)and located in the E.coli chromosome (striped).The black box within the homologous vector sequence could represent any type of sequence alteration (insertion,deletion,single-base change,etc.).

6230LINK ET AL.

J.B ACTERIOL .

RESULTS

Developing an improved gene replacement method.We con-structed a gene replacement vector for creating null mutations in the chromosomal sequences of wild-type E.coli strains as described in Materials and Methods and illustrated in Fig.1A. The plasmid is derived from a previously described gene re-placement vector and has the lac sequence removed to elimi-nate homologous recombination at the lac region in the E.coli chromosome(16).The repA(Ts)replication origin is derived from pSC101and has a permissive temperature of30°C but is inactive at42to44°C.The cat gene(encoding chloramphenicol resistance)is used as a marker to select for chromosomal integrates and as a marker for cells harboring vector sequences after plasmid excision.The sacB gene is used to counterselect vector sequences by growing cells harboring the plasmid on medium supplemented with5%sucrose.The M13replication origin facilitates generation of single-stranded copies of the plasmid by using helper phage(a feature not used in this study).Finally,the primer sites pKO3-L and pKO3-R?anking the cloning site enable screening the vector for inserts by PCR or for DNA sequencing across the vector-insert junctions. Figure1B diagrams the protocol that we used to perform gene replacements in E.coli.The in vitro-altered sequences carried in the vector pKO3are transformed into E.coli,and the transformed cells are allowed to brie?y recover at the permissive temperature.The cells are then plated on chloram-phenicol plates at the nonpermissive temperature to select for chromosomal integrates.This was more effective for obtaining the?nal gene replacement event than plating cells at30°C and shifting them to43°C.We found that integrates could also be obtained by serially diluting cells harboring the plasmid at30°C and plating them at43°C.To select cells in which the plasmids are excised and lost,we picked and suspended colonies from the43°C plates,diluted the suspension,and plated the cells on LB plates containing5%sucrose at30°C.Only cells that have excised the plasmid sequences and lost sacB’s counterselect-able function should grow under these conditions.We found this procedure worked better for getting the?nal gene replace-ment event than simply replica plating colonies from43°C to sucrose plates at30°C.Finally,the sucrose-resistant and chlor-amphenicol-sensitive colonies are screened for the desired gene replacement event by using PCR and primers to the genomic DNA?anking the altered sequences or by Southern hybridization.

Replacing yjbJ with an insertional allele.Suspecting that the null allele of yjbJ would be lethal,we decided to disrupt yjbJ by inserting a specialized Kn r selectable marker,or interposon, into the gene(29).A5.5-kb DNA fragment from a genomic library containing yjbJ was cloned into pKO3,and a Kn r gene (plexkan04)was inserted at the unique Nae I site in the gene (see Materials and Methods).Before doing the gene replace-ment,we sequenced both the vector-insert junctions and the insertion site of the Kn r gene and showed that the insertional allele had at least1kb of chromosomal sequence?anking both sides of the interposon(Fig.2A).When the yjbJ replacement vector was transformed into E.coli and plated at43°C,the integration frequency was10?2of the plated cells.Several integrates were picked,serially diluted,and plated at30°C on various selective media to induce the plasmid excision and loss (Fig.2B).These different master plates were then replica plated to chloramphenicol plates and kanamycin plates to identify colonies that retained the Kn r gene and not the vector (Fig.2B).When the integrate cells were plated on kanamycin medium without sucrose at30°C,most of the sucrose-resistant colonies were still chloramphenicol resistant,indicating that the cells retained the vector sequences(Fig.2B,row a).When

integrate cells were plated on kanamycin–5%sucrose medium,

more than98%of the sucrose-resistant colonies were chlor-

amphenicol sensitive,indicating loss of plasmid sequences and

a probable gene replacement event(Fig.2B,row b).When the

integrate cells were plated on rich medium containing5%

sucrose,48%of the sucrose-resistant colonies were chloram-

phenicol sensitive and kanamycin resistant,indicating loss of

the plasmid and a probable gene replacement event(Fig.2B,

row c).We veri?ed the structure of the initial43°C integration

and the replacement of yjbJ with the insertional allele by

screening colonies via PCR using primers?anking yjbJ(Fig.

2C).These results proved that the pKO3replacement system

worked and showed that yjbJ is a nonessential gene under these

environmental conditions.

Lethal gene replacement phenotype.To observe the results of the gene replacement protocol when trying to replace an

essential E.coli gene with an insertional allele,we tested two

known essential genes,pepM and rpsB.The pepM(map)gene

encodes methionine aminopeptidase,and rpsB encodes the

ribosomal protein S2(7,9).Each gene was cloned into the

gene replacement vector pKO3,and insertion mutations were

constructed by using the Kn r gene(see Materials and Meth-ods).DNA sequencing and restriction enzyme mapping

showed that both inserts had at least1kb of genomic DNA

?anking each side of the insertion site.

The pKO3plasmids carrying the insertionally disrupted es-

sential genes were electroporated into E.coli and plated at

43°C to select for integration.The integration frequency was

approximately10?2to10?3,similar to that for yjbJ.Ten inte-

grate colonies were picked,suspended in medium,serially di-

luted,and plated at30°C on5%sucrose–kanamycin plates.We

found the sucrose resistance frequencies for both the pepM and

rpsB integrates were approximately10?8,compared to a fre-

quency of10?2to10?4for the insertional disrupted nonessen-

tial yjbJ gene replacement.For both pepM and rpsB,all of the

sucrose-resistant,kanamycin-resistant colonies remained chlor-

amphenicol resistant,indicating that plasmid sequences were

still present in the cell.In addition,the colonies had a mucoid

phenotype compared to colonies that had lost the plasmid

sequences.It is unknown whether sacB’s activity had been

directly compromised by a mutation in the gene or if a sec-

ondary mutation in the genome conferred sucrose resistance.

These results showed the phenotype expected when one tries

to replace an essential gene with a disrupted https://www.sodocs.net/doc/3711304753.html,ing the

pKO3gene replacement procedure,Brown et al.have shown

that an essential gene,murA,can be replaced on the E.coli

chromosome with a deletion allele,as long as the deletion is

complemented by another copy of the essential gene(8).

Paradoxical phenotypes of different hdeA insertional alleles. Speculating that hdeA might be an essential gene,we con-

structed two different insertional alleles of hdeA(Fig.3).Both

were made using the same chromosomal insert cloned into the

vector pKO3.In one allele,the inserted Kn r gene was cloned into the Pst I site of the hdeA gene;in the second allele,the Kn r

gene was cloned into the Pvu II site in the opposite orientation

(see Materials and Methods).

In the?rst step of the gene replacement procedure,the two

plasmids transformed and integrated at similar frequencies.

However,when resolving the integrates,we found that the

Pvu II insertional allele had a sucrose resistance frequency of ?10?8,compared to approximately10?3for the Pst I allele.All of the sucrose-resistant,kanamycin-resistant colonies with the

Pvu II insertional allele were chloramphenicol resistant,indi-cating that the plasmid sequences were still present.This?nd-ing suggested that the Pvu II allele is a lethal mutation.How-

V OL.179,1997PRECISE E.COLI GENOME ENGINEERING6231

ever,the replacement of the wild-type gene with the Pst I insertion was con?rmed by PCR screening of colonies with primers ?anking hdeA .The latter ?nding suggested that the Pst I allele is nonlethal.These Pst I and Pvu II results together illustrated the dif?culty of classifying hdeA as either an essen-tial or a nonessential gene because the phenotype varied ac-cording to the insertion site of the marker and/or its orienta-tion in the disrupted allele.

Engineering in-frame deletions to minimize polar effects.To avoid problems associated with insertional mutations,we de-veloped a system that replaces ORFs with in-frame deletions.Figure 4shows how we used crossover PCR to create a

dele-

FIG.2.Replacement of yjbJ with an insertional allele.(A)The top panel shows a genomic insert containing yjbJ cloned into pKO3and mutagenized with the plexkan04interposon.The interposon insertion at the Nae I site was con?rmed by sequencing across the interposon-genomic insert junction by using primers CE-04and CP-04,which ?ank the interposon.The insert was mapped on the E.coli chromosome by sequencing across the vector-insert junctions by using primers pKO3-L and pKO3-R.The physical map of the wild-type chromosomal region is shown.The arrows ?anking the Nae I sites are the PCR primer sites yjbJ-Nout and yjbJ-Cout used to identify the yjbJ allele.The expected sizes of the PCR products are 0.4kb for yjbJ and 1.8kb for yjbJ ::plexkan04alleles.The thick lines represent chromosomal fragment sequences.The thin line represents the vector pKO3.The open box represents the interposon.(B)Resolution of yjbJ ::plexkan04integrates when plated at 30°C under different selection conditions.Five 43°C integrates were picked,serially diluted,and plated on the master plates shown at the left.These master plates were replica plated ?rst to chloramphenicol plates and then to kanamycin plates at 30°C to detect loss of plasmid sequences (chloramphenicol sensitive)and retention of the interposon (kanamycin resistant).(C)Use of PCR to verify the replacement of yjbJ with the insertional allele.The gel shows the products of the PCRs using primers yjbJ-Nout and yjbJ-Cout to detect either the wild-type (wt)or the disrupted yjbJ allele.The sources of the template DNA are plasmid p1.7(yjbJ )(lane 1),plasmid p1.7(3)(yjbJ ::plexkan04)(lane 2),EMG2genomic DNA (lane 3),genomic DNA from 43°C integrate of p1.7(3)(yjbJ ::plexkan04)into the EMG2(lane 4),EMG2yjbJ ::plexkan04genomic DNA from a sucrose-resistant and chloramphenicol-sensitive colony shown in row b (lane 5),and EMG2yjbJ ::plexkan04genomic DNA from a sucrose-resistant and chloramphenicol-sensitive colony shown in row c (lane 6).The size marker is a 123-bp ladder (lane M).

6232LINK ET AL.J.B ACTERIOL .

tion of any E.coli ORF (18,19).Complementary oligonucle-otide primers and asymmetric PCR are used to generate two DNA fragments having overlapping ends.The two fragments are combined in a fusion reaction in which the overlapping ends annealed and served as primers for 3?extension of the complementary strand.This fusion molecule is then ampli?ed by PCR using the outer primers.

To construct the deletions,we developed the following rules for designing the oligonucleotides to ensure suf?cient homol-ogy for recombination during gene replacement and to mini-mize disruption of ?anking sequences.The lengths of the two fragments ?anking the deletion are at least 500bp.The deci-sion to use 500bp is based on published integration frequen-cies for various lengths of chromosomal regions cloned into similar gene replacement vectors.The predicted integration frequency should be approximately 10?4(5,16).The two com-plementary oligonucleotides (C and B)have at least a 21-base complementary region to allow the products from the asym-metric ?rst and second PCRs to anneal and extend (Fig.4).The primers were designed so that the deletion maintained the original translational reading frame of the ORF and the added bases provided unique sequences for tracking the deletion in a population of different E.coli deletion strains.To minimize potential affects on expression of neighboring genes,we engi-neered the deletion of the ORF to begin 18bp downstream of the translation start site and end 36bp upstream of the stop codon.The oligonucleotides (A and D)have Bam HI restric-tion sites in the 5?end to allow ef?cient cloning of the fusion product.

Deletion of hdeA.To further investigate the null phenotype of the hdeA mutation,we engineered a deletion of the gene (Fig.5A).Using the above-speci?ed rules,we deleted a 279-bp region,or 84%of the coding region of hdeA and replaced it with a 21-bp in-frame sequence tag,using the crossover PCR protocol (see Materials and Methods).Figure 5B shows the two complementary PCR products and the ?nal crossover PCR deletion product.The deletion fragment was cloned into the vector pKO3,and chromosomal deletions were introduced into the chromosome by using our pKO3gene replacement protocol.The deletion plasmid had an integration frequency of 1.8?10?3.Integrates were serially diluted and plated at 30°C on 5%sucrose plates to select for excision and loss of

the

FIG.3.Paradoxical phenotypes of replacing hdeA with insertional alleles.Physical maps of the DNA fragment containing hdeA and the chromosomal region before and after gene replacement with the insertional alleles are shown.The unique Pvu II and Pst I sites in hdeA are the insertion sites for the plexkan01and plexkan04interposons,respectively.The arrows ?anking the Pst I and Pvu II sites are the PCR primer sites hdeA-Nout1and hdeA-Cout1?anking the hdeA gene.The construct marked with an “X”could be integrated but not resolve to the replacement allele.Details are as described in the legend to Fig.2A.

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plasmid sequence.Figure 5C shows the results of screening a fraction of the resolved colonies by PCR with primers ?anking hdeA .Approximately 7%of the sucrose-resistant and chlor-amphenicol-sensitive resolved integrate colonies had the dele-tion replacing the wild-type hdeA sequence.Why the resolu-tion frequency for replacing the gene with the deletion was not the expected 50%is unknown.Recovery of the deletion dem-onstrated that the gene was nonessential under these environ-mental conditions and suggested that the apparent lethal effect of the Kn r insertion into hdeA was probably due to an effect of the insertion on elements outside of the cloned segment.Deletion of yjbJ.A similar set of experiments was performed to delete 146bp,or 73%,of yjbJ ,with the rules previously described.Although the deletion product was successfully am-pli?ed,it could not be cloned into pKO3.A PCR assay showed the deletion insert ligated to pKO3,and so we hypothesize that either the protein produced by the deletion mutant was toxic or the insert interfered with plasmid replication in E.coli .An analysis of the genomic region identi?ed a potential promoter 107bp upstream of the yjbJ translational start.We designed a second deletion to remove most of the predicted promoter region while leaving the upstream dinF gene intact.The second deletion extended from 3bp downstream of the dinF stop codon to 36bp upstream of the yjbJ stop codon,deleting 286bp of the region,including 82%of coding region of yjbJ .This second crossover PCR deletion product was successfully cloned into https://www.sodocs.net/doc/3711304753.html,ing the gene replacement protocol,we found that the deletion plasmid had an integration frequency of 7.9?10?5.Similar to hdeA ,only 3%of the

sucrose-resistant

FIG.4.The creation of in-frame deletion constructs.The top line represents a region of the chromosome where genes x ,y ,and z form a polycistronic operon.The second line is an expanded view of gene y showing the two PCRs used to generate fragments (PCR1and PCR2)which will form an in-frame deletion of gene y when fused.The PCR primers B and C are complementary over 21nucleotides (represented by the light gray lines)so that when the two PCR products are mixed,the complementary regions anneal and prime at the 3?overlapping region for a 3?extension of the complementary strand.In the third line,the fused molecule is ampli?ed by PCR with primers A and D.Primers A and D have Bam HI sites incorporated into the 5?ends of both oligonucleotides (represented by the gray lines)so that the fusion product can be restriction digested and cloned into

pKO3.

FIG.5.Constructing and replacing the gene hdeA with a precisely engi-neered deletion.(A)Diagram of the hdeA region.The small arrows marked with capital letters are the PCR primer sites used to construct the deletion and to PCR assay either the wild-type or deletion allele of hdeA .The predicted sizes of the PCR products are shown below the physical map.Primers:A,hdeA-No;B,hdeA-Ni;C,hdeA-Ci;D,hdeA-Co;E,hdeA-Nout2;F,hdeA-Cout2;G,hdeA-Nout3;H,hdeA-Cout3(see Materials and Methods).(B)Analysis of PCR products used to construct precise deletion of hdeA .The left gel shows the two fragments that will form the deletion product and the PCR products made by using primers ?anking hdeA .The sizes of the PCR products are shown.EMG2genomic DNA was used as the template DNA for lanes 1to 7.The DNA primer pairs used for the PCRs are A-D (lane 1),A-B (1:1)(lane 2),A-B (10:1)(lane 3),D-C (1:1)(lane 4),D-C (10:1)(lane 5),G-H (lane 6),and E-F (lane 7)(see panel A).The right gel shows the ampli?ed deletion (?l)product (lane 9).Using the products of lanes 3and 5as templates,the left and right fragments of the deletion were combined,annealed,extended,and PCR ampli?ed by using prim-ers A and D (lane 9).This gel also shows the PCR product made by using the same primer pairs but starting with EMG2genomic DNA as the template (lane 8).The deletion fusion product was cloned into pKO3and used in the gene replacement protocol.The “x”indicates an unknown PCR by-product.The size marker is a 123-bp ladder (lane M).wt,wild type.(C)Veri?cation of the replacement of hdeA with the crossover PCR deletion product.After integration at 43°C,integrates were plated at 30°C on 5%sucrose plates and replica plated to chloramphenicol plates.The chloramphenicol-sensitive,sucrose-resistant colonies were screened by PCR using primers E and F (see panel A).Those containing the precise deletion give a 421-bp product,while those containing the wild-type allele give a 679-bp product.This gel shows a subset of the colonies screened for the deletion (lanes 1to 16).The size marker is a 123-bp ladder (lane M).

6234LINK ET AL.J.B ACTERIOL .

and chloramphenicol-sensitive resolved-integrate colonies had the deletion replacing the wild-type yjbJ sequence.These re-sults prove that the YjbJ protein is nonessential under these environmental conditions and agree with the earlier results obtained by replacing the gene with the Kn r insertion allele. Competition experiments to compare insertional and dele-tion phenotypes.To compare the phenotypes of the various mutant strains,isogenic strains with the insertional yjbJ and hdeA alleles were compared in a growth and survival compe-tition with wild-type E.coli.In a second experiment,the yjbJ insertion and deletion strains were compared(see Materials and Methods).Figure6A shows the survival of the yjbJ and the viable hdeA insertion mutants in competition with the wild-type strain.Under these conditions,the hdeA deletion causes a slight growth defect with respect to wild-type EMG2,while the yjbJ insertion strain outcompetes the wild-type strain.Figure 6B shows the competition results for the strain with the yjbJ insertional allele versus the strain with the deletion yjbJ allele. Surprisingly,two different phenotypes are observed for the different mutant alleles.In this assay,the yjbJ insertion strain outcompetes the yjbJ deletion strain.

DISCUSSION

We have presented an improved method for performing gene replacements in E.coli.The method is similar to the pop-in/pop-out method used for Saccharomyces cerevisiae(6, 31,33)and the hit-and-run procedure used for mouse embry-onic stems cells(17).Unlike other methods used for gene replacements in E.coli that use ColE1plasmids in a polA1 background or transformation of linear DNA into recBC,sbcB, or recD strains,this protocol can be performed directly in wild-type strains(15,21,37,44).Since the system is plasmid based,gene replacements are easily performed in any genetic background that is recombination pro?cient and supports the replication of https://www.sodocs.net/doc/3711304753.html,ing this system,we have created another44E.coli strains with in-frame deletions of other ORFs(27a).

Although not attempted in our lab,the pKO3gene replace-ment method can be used for constructing E.coli strains with multiple mutations without the need for multiple drug resis-tance markers or for replacing DNA sequences in the chro-mosome with precise point mutations.Finally,the method can be used for altering large exogenous fragments of DNA cloned into the single-copy P1or BAC vectors which use E.coli as the host cell(38,42).

In contrast to the deletion method,the insertion method creates mutations by inserting a Kn r gene(interposon)into cloned chromosomal DNA segments similar to a previous pro-tocol(29).We designed this method for a gene that is pre-dicted to be essential and uses selection instead of a screen to assess gene replacement.The Kn r gene was chosen as the marker since the gene has no homology to either the gene replacement vector pKO3or the E.coli chromosome.We engineered the Kn r interposons with a different multiplex se-

quencing tag?anking each side of the interposon so that mu-tagenized clones could be sequenced by either cycle or multi-plex sequencing(10,27).

The two distinct phenotypes resulting from the insertional mutagenesis of hdeA highlight the unreliability of insertional mutagenesis.The comparison of the yjbJ insertion and deletion strains in the competition experiment also illustrates the phe-notypic differences that can occur as a result of the particular type of mutation created.The yjbJ insertion strain appears to have an advantage over both the wild type and its respective deletion strain under the selection condition tested.Insertional mutagenesis has the potential for several undesired side ef-fects,including polar termination-induced reduction of down-stream operon expression(3),fusion products(2,20),and misregulation of adjacent genes due to the insertion marker’s promoter(11,22,43).Assigning a phenotype to a mutated gene may be problematic if the phenotype is actually a conse-quence of both the primary mutation and its effects on the surrounding genes.

This system,of replacing targeted ORFs with in-frame de-letions was developed to reduce the inherent problems of in-sertional mutagenesis.Sensitive to the existence of

transcrip-FIG.6.Survival and growth competition between isogenic strains having either insertion or deletion alleles of hdeA and yjbJ.Equivalent numbers of cells from each strain were inoculated into rich medium and grown in competition under aerobic conditions at37°C.At various time points,the cells were plated on rich media with and without kanamycin,and the viable cell density of each strain was assayed.(A)Relative survival of EMG2yjbJ::plekan04and EMG2 hdeA::plekan01insertion strains when competed against wild-type EMG2.(B) Comparative survival of the EMG2yjbJ::plekan04insertion strain and the EMG2?yjbJ deletion strain when cultured together.

V OL.179,1997PRECISE E.COLI GENOME ENGINEERING6235

tional and translational overlap in prokaryotic operons(13), our deletions were designed to retain translational coupling and to minimize the disruption of the regulation of neighbor-ing genes in an operon(13,24).The?rst six codons(18bp)at the5?end of the gene were retained to maintain the gene’s translation start signals.The last12codons(36bp)at the3?end of the gene were retained based on the maximum overlap of coding regions observed in a sequence analysis of E.coli and Salmonella typhimurium operons(30).The largest observed overlap of a gene’s5?coding region into a neighboring gene’s 3?coding region was20bp(5?-cbiF-cbiG-3?in the cob operon of S.typhimurium).The36-bp overlap was chosen to maintain translational coupling in a gene cluster for operons with po-tentially even greater overlapping regions and for ambiguity in downstream translation start site assignment.

The expected frequency of colonies bearing the deletion allele after resolution of the plasmid integrates is50%.As expected,the frequency of colonies bearing the Kn r insertion allele of yjbJ after plasmid resolution was approximately50%. However,the observed resolution frequency of colonies with PCR-generated deletion alleles of both yjbJ and hdeA was only 3to7%.We speculate that this reduction in resolution fre-quency is caused by the reduced length of homologous se-quences combined with possible PCR-generated DNA mis-matches that?ank one side of the duplications,causing the resolution of the integrate to be asymmetric(1,34–36).In E. coli,a Chi site represented by the octanucleotide sequence 5?-GCTGGTGG-3?stimulates recombination,depending on the length of the recombination interval and the location of the Chi site with respect to the interval(40,41).We searched the genomic regions?anking yjbJ and hdeA and did not?nd the octanucleotide sequence in the vicinity of the two genes. This research indicates that in the emerging post-genome sequencing era,when high-throughput evaluation of unchar-acterized ORFs becomes a necessity,insertional mutagenesis by traditional methods will not be suf?ciently reproducible to assign phenotypes based on subtle strain-by-strain variations. Because the engineering of in-frame deletions enables us to avoid many of the phenotypic artifacts mentioned earlier,we should be able to attach signi?cance to a greater number of the phenotypes that we observe.This method will help investiga-tors to systematically assign functions to the vast number of new ORFs revealed by current microbial sequencing projects.

ACKNOWLEDGMENTS

We thank Claire and Doug Berg for plasmid pBS-TS and the Kush-ner lab for plasmids pMAK700and pMAK705.We thank Robert Kolter,Richard Baldarelli,Fritz Roth,and Pete Estep for helpful discussions.We are especially grateful to Elizabeth A.Malone and Martha Bulyk for critical reading of the manuscript.

This work was funded by DOE grant DE-FG02-87ER60565.

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crispr-cas9基因敲除

CRISPR/Cas9 是细菌和古细菌在长期演化过程中形成的一种适应性免疫防御,可用来对抗入侵的病毒及外源DNA。CRISPR/Cas9 系统通过将入侵噬菌体和质粒DNA 的片段整合到CRISPR 中,并利用相应的CRISPR RNAs(crRNAs)来指导同源序列的降解,从而提供免疫性。 原理 此系统的工作原理是crRNA(CRISPR-derived RNA )通过碱基配对与tracrRNA (trans-activating RNA )结合形成tracrRNA/crRNA 复合物,此复合物引导核酸酶Cas9 蛋白在与crRNA 配对的序列靶位点剪切双链DNA。而通过人工设计这两种RNA,可以改造形成具有引导作用的sgRNA (singleguide RNA ),足以引导Cas9 对DNA 的定点切割。 作为一种RNA 导向的dsDNA 结合蛋白,Cas9 效应物核酸酶是已知的第一个统一因子(unifying factor),能够共定位RNA、DNA 和蛋白,从而拥有巨大的改造潜力。将蛋白与无核酸酶的Cas9(Cas9 nuclease-null)融合,并表达适当的sgRNA ,可靶定任何dsDNA 序列,而sgRNA 的末端可连接到目标DNA,不影响Cas9 的结合。因此,Cas9 能在任何dsDNA 序列处带来任何融合蛋白及RNA,这为生物体的研究和改造带来巨大潜力。 应用 基因敲除动物模型一直以来是在活体动物上开展基因功能研究、寻找合适药物作用靶标的重要工具。但是传统的基因敲除方法需要通过复杂的打靶载体构建、ES细胞筛选、嵌合体小鼠选育等一系列步骤,不仅流程繁琐、对技术的要求很高,而且费用大,耗时较长,成功率受到多方面因素的限制。即使对于技术比较成熟的实验室,利用传统技术构建基因敲除大、小鼠一般也需要一年以上。 2013 年1 月份,美国两个实验室在《Science》杂志发表了基于CRISPR-Cas9 技术在细胞系中进行基因敲除的新方法,该技术与以往的技术不同,是利用靶点特异性的RNA 将Cas9 核酸酶带到基因组上的具体靶点,从而对特定基因位点进行切割导致突变。该技术迅速被运用到基因敲除小鼠和大鼠动物模型的构建之中。通过一系列研究,首先证明了通过RNA 注射的方式将CRISPR-Cas 系统导入小鼠受精卵比DNA 注射能更有效的在胚胎中产生定点突变。在此基础上,又发现了该方法没有小鼠遗传品系的限制,能够对大片段的基因组DNA 进行删除,也可以通过同时注射针对不同基因的RNA 序列达到在同一只小鼠或大鼠中产生多个基因突变的效果。此外,还证明了利用CRISPR-Cas 技术构建的基因敲除大鼠模型与传统方法构建的同一基因(肥胖相关G 蛋白偶联受体Mc4R)突变大鼠相比具有一致的表型。该方法构建的基因突变动物具有显著高于传统方法的生殖系转移能力,是一种可靠、高效、快速的构建敲除动物模型的新方法。 CRISPR-Cas 技术是继锌指核酸酶(ZFN)、ES 细胞打靶和TALEN 等技术后可用于定点构建基因敲除大、小鼠动物的第四种方法,且有效率高、速度快、生殖系转移能力强及简单经济的特点,在动物模型构建的应用前景将非常广阔。 技术优缺点 CRISPR (Clustered Regularly Interspersed Short Palindromic Repeats)是细菌用来抵御病毒侵袭/躲避哺乳动物免疫反应的基因系统。科学家们利用RNA引导Cas9核酸酶可在多种细胞(包括iPS)的特定的基因组位点上进行切割,修饰。Rudolf Jaenisch 研究组将Cas9与Te1和Tet2特异的sgRNA共注射到小鼠的受精卵中,成功得到双基因敲除的纯合子小鼠,效率高达80%。他们将Cas9/sgRNA 与带突变序列的引物共注射,能准确在小鼠两个基因引入所要的点突变。在ES细胞中他们更是成功的一次敲除了五个基因。与ZFN/TALEN相比,CRISPR/Cas更易于操作,效率更高,更容易得到纯合子突变体,而且可以在不同的位点同时引入多个突变。但该系统是否有脱靶效应尚需进一步的研究。 传统的转基因和基因打靶技术,由于技术稳定成熟,可以对小鼠和大鼠的基因组序列进行各种修饰,仍将是模式动物的构建的主要技术。核酸酶ZFN/TALEN 尤其是CRISPR/Cas技术如果能解决脱靶效应的话,有可能会广泛应用于小鼠,大鼠及其他模式动物的制备和研究中,成为传统的转基因和基因打靶技术的重要补充。

ABO血型分型方法

华科基因ABO血型分型方法 按照Bernstein三复等位基因学说,ABO基因座主要有三个等位基因A、B和O。A、B对O为显性,O为隐性。ABO血型的表型与基因型的关系是:A型为AA或AO;B型为BB 或BO;AB型为AB;O型为OO。因为隐性基因O存在,ABO基因型不能直接用凝集反应来确定,但可通过DNA分型技术直接检测基因型,或通过表型的家系调查来推定。 ABO血型分型方法 一、ABO血型血清学分型方法 ABO血型是根据红细胞与特异性抗体的反应来分型。即用抗A抗体判定A抗原,用抗B抗体判定B抗原,这种检查法称作正定型试验(direct grouping)。同时依据血清中的凝集素与标准型别红细胞膜上的凝集原反应的性质来检验结果。即用标准的A型红细胞判定抗A抗体,用标准的B型红细胞判定抗B抗体,称作反定型试验(reverse grouping)。为使结果准确,应进行正反两个试验来分型,同时也应用抗H抗体判定H抗原。当正反试验的结果与常规的ABO分型原则不符的时候,提示可能是弱亚型或变异型。 二、ABO血型DNA分型方法 分子生物学技术将ABO血型分型由血清学水平深入到基因水平。DNA分型方法都是针对核苷酸顺序差异而设计的。常用的有序列特异性引物PCR技术与PCR-RFLP技术等。序列特异性引物PCR(PCR-sequence-specific primers,PCR-SSP)是根据等位基因的序列,设计具有序列特异性的引物,对样本进行DNA分型。该方法是以引物决定分型特异性,具有特异性强、重复性好、结果易于判定的优点。 ABO血型分型可提供以下样本: 常用样本:血液,血痕,带毛囊的毛发,口腔粘膜细胞(口腔拭子)等。 特殊检材:如精斑、混合斑、肌肉、烟蒂、胎儿的羊水等都可以采用。 采样建议: 1.我们建议您采用血痕或口腔棉签作为检测样本,或采用毛发(带毛囊)作为检测样本。 2.如果您的孩子未满四周岁,请采用血痕、口腔棉签作为样本。 3.如果孩子还未出生,则参考羊水样本抽取方法。

基因敲除技术研究进展

兰州交通大学化学与生物工程学院综合能力训练Ⅰ——文献综述 题目:基因敲除技术研究进展 作者:王振宇 学号:201207744 指导教师:谢放 完成日期:2014-7-16

基因敲除技术研究进展 摘要基因敲除是自20世纪80年代末以来发展起来的一种新型分子生物学技术,是通过一定的途径使机体特定的基因失活或缺失的技术。在总结已有研究成果的基础上,本文对基因敲除技术的概况、原理方法应用以及近年来基因敲除技术的研究进展作一个简单的综述。 关键词基因敲除 RNA i生物模型基因置换基因打靶同源重组1. 基因敲除技术简介 基因敲除(Gene knockout)是指一种遗传工程技术,针对某个序列已知但功能未知的序列,改变生物的遗传基因,令特定的基因功能丧失作用,从而使部分功能被屏障,并可进一步对生物体造成影响,进而推测出该基因的生物学功能。 它克服了随机整合的盲目性和偶然性,是一种理想的修饰、改造生物遗传物质的方法。基因敲除借助分子生物学、细胞生物学和动物胚胎学的方法,通过胚胎干细胞这一特殊的中间环节将小鼠的正常功能基因的编码区破坏,使特定基因失活,以研究该基因的功能;或者通过外源基因来替换宿主基因组中相应部分,以便测定它们是否具有相同的功能,或将正常基因引入宿主基因组中置换突变基因以达到靶向基因治疗的目的。基因敲除是揭示基因功能最直接的手段之一。通常意义上的基因敲除主要是应用DNA同源重组原理,用设计的同源片段替代靶基因片段(即基因打靶),从而达到基因敲除的目的。随着基因敲除技术的发展,除了基因打靶技术外,近年来新的原理和技术也逐渐被应用,比较成功的有RNA干扰技术,同样也可以达到基因敲除的目的。简单的说基因敲除是指将目标基因从基因组中删除。基因敲除技术主要应用于动物模型的建立,而最成熟的实验动物是小鼠,对于大型哺乳动物的基因敲除模型还处于探索阶段。这项技术的诞生可以说是分子生物学技术上继转基因技术后的又一革命。尤其是条件性、诱导性基因打靶系统的建立,使得对基因靶位时间和空间上的操作更加明确、效果更加精确、

CRISPR cas9基因敲除原理及其应用

CRISPR/Cas9基因敲除原理及其应用 CRISPR(clustered,regularly interspaced,short palindromic repeats)是一种来自细菌降解入侵的病毒DNA或其他外源DNA的免疫机制。在细菌及古细菌中,CRISPR系统共分成3类,其中Ⅰ类和Ⅲ类需要多种CRISPR相关蛋白(Cas蛋白)共同发挥作用,而Ⅱ类系统只需要一种Cas蛋白即可,这为其能够广泛应用提供了便利条件[1]。 目前,来自Streptococcus pyogenes的CRISPR-Cas9系统应用最为广泛。Cas9蛋白(含有两个核酸酶结构域,可以分别切割DNA两条单链。Cas9首先与crRNA及tracrRNA结合成复合物,然后通过PAM序列结合并侵入DNA,形成RNA-DNA复合结构,进而对目的DNA双链进行切割,使DNA双链断裂。 由于PAM序列结构简单(5’-NGG-3’),几乎可以在所有的基因中找到大量靶点,因此得到广泛的应用。CRISPR-Cas9系统已经成功应用于植物、细菌、酵母、鱼类及哺乳动物细胞,是目前最高效的基因组编辑系统[1]。 通过基因工程手段对crRNA和tracrRNA进行改造,将其连接在一起得到sgRNA(single guide RNA)。融合的RNA具有与野生型RNA类似的活力,但因为结构得到了简化更方便研究者使用。通过将表达sgRNA的原件与表达Cas9的原件相连接,得到可以同时表达两者的质粒,将其转染细胞,便能够对目的基因进行操作[2,3]。

目前常用的CAS9研究方法是通过普通质粒,质粒构建流程如下:Cas9质粒构建 设计2条单链oligo序列; 退火形成双链DNA pGK1.1 将双链DNA连接到载体 中 转化G10competent cell 筛选阳性克隆;测序验证 序列;质粒大提;电转染 靶细胞 在细胞内crRNA识别靶 位点,Cas9对靶位点进行 随机剪切 Cruiser TM酶切细胞池,计 算突变率;Cruiser TM酶切 初筛阳性克隆;将阳性克 隆测序验证;做敲除序列 比对分析。

基因敲除具体步骤

The following protocols take MLCK (myosin light chain kinase) as an example. General steps: 1.BAC extraction (It is necessary for us to identify the BAC by PCR) 2.Transform BAC to EL350 ( Cm+) 3.Retrieving (Cm+ Amp+) 4. Targeting 1st lox P (Amp+ Amp+ and K+) 5. Transform MLCK 1st lox P to EL350 to get purify MLCK 1st lox P ( Amp+ and K+) 6. MLCK 1st lox P pop out (Amp+ and K+ AmP+) 7. Transform MLCK 1st lox P pop out to EL250 (Amp+) 8. Targeting 2nd lox P (Amp+ Amp+ and K+) 9. Transform MLCK 2nd lox P to DH-5α or XL1-Blue ( Amp+ and K+) 10. Linearization 1. BAC extraction Solution I: Tris.Cl 0.025 M EDTA 0.01M Glucose 0.05M pH 8.0 Solution II: SDS 1 % NaOH 0.2M fresh prepared (1Volume 2% SDS + 1Volume 0.4M NaOH) Solution III: (120 ml 5 M KAc + 23 ml HAc + 57 ml H2O) / 200 ml

SNP基因分型的高通量方法

Chapter16 High-Throughput Methods for SNP Genotyping Chunming Ding and Shengnan Jin Abstract Single nucleotide polymorphisms(SNPs)are ideal markers for identifying genes associated with complex diseases for two main reasons.Firstly,SNPs are densely located on the human genome at about one SNP per approximately500–1,000base pairs.Secondly,a large number of commercial platforms are available for semiautomated or fully automated SNP genotyping.These SNP genotyping platforms serve different purposes since they differ in SNP selection,reaction chemistry,signal detection,throughput,cost,and assay flexibility.This chapter aims to give an overview of some of these platforms by explaining the technologies behind each platform and identifying the best application scenarios for each platform through cross-comparison.The readers may delve into more technical details in the following chapters. Key words:Whole genome association,fine mapping,single nucleotide polymorphism,copy number variation,haplotyping. 1.Introduction Single nucleotide polymorphisms(SNPs)are best known as genetic markers in disease-association studies to identify genes associated with complex diseases(1,2).However,SNPs are also used in many other clinically and biologically important applica- tions(3).A large variety of commercial platforms are available for semiautomated or fully automated SNP genotyping analysis.On the basis of the purposes of the study,SNP genotyping can be divided into two domains:whole genome association(WGA)and fine mapping(Fig.16.1).Most of the genotyping platforms can be classified accordingly.This chapter aims to briefly explain the principles behind various platforms which lead to a comparison of these platforms so that the readers will get a quick overview before delving into the technical details of some of these methods in the following chapters. A.A.Komar(ed.),Single Nucleotide Polymorphisms,Methods in Molecular Biology578, DOI10.1007/978-1-60327-411-1_16,aHumana Press,a part of Springer Science+Business Media,LLC2003,2009 245

基因敲除小鼠的制作方法

.. 一、常规基因敲除鼠(Conventional Knockout) 常规基因敲除是通过基因打靶,把需要敲除的基因的几个重要的外显子或者功能区域用Neo Cassette 替换掉。这样的小鼠其全身所有的组织和细胞中都不表达该基因产物。此类基因敲除鼠一般用于研究某个基因在对小鼠全身生理病理的影响,而且这个基因没有胚胎致死性。 二、条件性基因敲除小鼠(Conditional Knockout) 条件性基因敲除小鼠是通过基因打靶,把两个loxP 位点放到目的基因一个或几个重要的外显子的两边。该小鼠和表达Cre酶小鼠杂交之前,其目的基因表达完全正常。当和组织特异性表达Cre酶的小鼠进行杂交后,可以在特定的组织或细胞中敲除该基因,而该基因在其他组织或细胞表达正常。 条件性基因敲除鼠适用范围为:(1)该基因有胚胎致死性;(2)用于研究该基因在特定的组织或细胞中的生理病理功能。 三、基因敲入小鼠(Knockin) 基因敲入小鼠是通过基因打靶,把目的基因序列敲入到小鼠的相应基因位点,使用小鼠的表达调控元件指导目的基因表达。 此类基因敲入鼠一般用于药物的筛选,信号通路的研究等。 获得嵌合体及之后品系纯化详细流程: 基因敲除其他方法: 一、ZFN技术制作基因敲除鼠 ZFN能够识别并结合指定的基因序列位点,并高效精确地切断。随后细胞利用天然的DNA 修复过程来实现DNA的插入、删除和修改,这样研究人员就能够随心所欲地进行基因组编辑。这在过去是无法想象的,传统的基因敲除技术依赖细胞内自然发生的同源重组,其效率只有百万分之一,而ZFN的基因敲除效率能达到10%。利用这些技术进行小鼠基因的定点敲除和敲入,可以把时间从一年缩短到几个月。 这项技术中设计特异性的ZFN是最关键的环节,目前研究者采用计算生物学方法设计高特异性的ZFN,但ZFN的脱靶(off target),也就是把不该切的地方切了的问题仍是一个挑战。也正因为这个原因,利用ZFN技术进行小鼠的基因修饰还无法完全取代传统技术。 二、TALEN技术制作基因敲除鼠 TALEN 技术是一种崭新的分子生物学工具。科学家发现,来自一种植物细菌的TAL蛋白的核酸结合域的氨基酸序列与其靶位点的核酸序列有恒定的对应关系。利用TAL的序列模块,可组装成特异结合任意DNA序列的模块化蛋白,从而达到靶向操作内源性基因的目的,它克服了ZFN方法不能识别任意目标基因序列,以及识别序列经常受上下游序列影响等问题,而具有ZFN相等或更好的灵活性,使基因操作变得更加简单方便。然而同样因为脱靶的问题,利用TALEN技术进行小鼠的基因修饰仍然无法取代传统技术。 ;.

分子生物学综述论文(基因敲除技术)

现代分子生物学 课程论文 题目基因敲除技术 班别生物技术10-2学号 10114040220 姓名陈嘉杰 成绩

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基因敲除技术

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基因敲除技术的原理、方法和应用 2010-01-24 17:03:43 来源:易生物实验浏览次数:6302 网友评论 0 条 1.基因敲除概述 2.实现基因敲除的多种原理和方法: 2.1.利用基因同源重组进行基因敲除 2.2利用随机插入突变进行基因敲 除。 2.3.RNAi引起的基因敲除。 3.基因敲除技术的应用及前景 4.基因敲除技术的缺陷 关键词:基因敲除 1.基因敲除概述: 基因敲除是自80年代末以来发展起来的一种新型分子生物学技术,是通过一定的途径使机体特定的基因失活或缺失的技术。通常意义上的基因敲除主要是应用DNA同源重组原理,用设计的同源片段替代靶基因片段,从而达到基因敲除的目的。随着基因敲除技术的发展,除了同源重组外,新的原理和技术也逐渐被应用,比较成功的有基因的插入突变和iRNA,它们同样可以达到基因敲除的目的。 2.实现基因敲除的多种原理和方法: 2.1.利用基因同源重组进行基因敲除 基因敲除是80年代后半期应用DNA同源重组原理发展起来的。80年代初,胚胎干细胞(ES细胞)分离和体外培养的成功奠定了基因敲除的技术基础。1985 年,首次证实的哺乳动物细胞中同源重组的存在奠定了基因敲除的理论基础。到1987年,Thompsson首次建立了完整的ES细胞基因敲除的小鼠模型 [1]。直到现在,运用基因同源重组进行基因敲除依然是构建基因敲除动物模型中最普遍的使用方法。 2.1.1利用同源重组构建基因敲除动物模型的基本步骤(图1): a.基因载体的构建:把目的基因和与细胞内靶基因特异片段同源的DNA 分子都重组到带有标记基因(如neo 基因,TK 基因等)的载体上,成为重组载体。基因敲除是为了使某一基因失去其生理功能,所以一般设计为替换型载体。

乙型肝炎病毒基因分型方法简述

乙型肝炎病毒基因分型方法简述 邵 玲 张 男 【摘要】乙型肝炎病毒是一种嗜肝脱氧核糖核酸病毒,属于一种复合体DNA病毒。乙型肝炎病毒可按两种方法分型:血清型和基因型。随着分子生物学的发展以及对乙型肝炎病毒研究的深入,乙型肝炎病毒血清分型法已不能适应对该病毒感染研究的需要,而出现的基因分型法则引起广泛的重视。 【关键词】乙型肝炎病毒;基因分型方法 H epatitis B virus gene minute method summ ary S HA O L in Z HA N G N an 【Abstract】The hepatitis B virus is one kind is addicted to the liver deoxyribonucleic acid virus,belongs to one kind of complex DNA virus.The hepatitis B virus may according to two method minutes:Blood serum and genotype.Along with molecular biology’s development as well as to hepatitis B virus research’s thorough,a hepatitis B virus blood serum minute law has not been able to adapt to this virus infection research need,but appears a gene minute principle brings to the widespread attention. 【K ey w ords】Hepatitis B virus;Gene minute method 乙型肝炎病毒是一种嗜肝脱氧核糖核酸病毒,属于一种复合体DNA病毒。乙型肝炎病毒可按两种方法分型:血清型和基因型。随着分子生物学的发展以及对乙型肝炎病毒研究的深入,乙型肝炎病毒血清分型法已不能适应对该病毒感染研究的需要,而出现的基因分型法则引起广泛的重视。1988年Ok2 mamoto[1]对18株不同亚型的HBV基因序列两两进行比较后,根据核苷酸序列异源性>8%的原则,将18株HBV DNA序列分为A~D4个基因型,提出了HBV基因型的概念。1992年Norder[2]发现ayw4和adw4q-两旧亚型之间及基因型A~D 之间S基因差异>4%,提出了两种新的基因型E,F,1994年Norder通过全基因序列P3测定加以证实。2000年Stuyver[3],在研究来自法国和美国的慢性乙肝病人血清样本时,发现有13株病毒无法归入A~F型,命名为G型。随后,日本和德国也相继发现了G基因型。2002年Arauz~Ruiz[4]对10株HBV进行基因型研究,发现其中3株虽与F型相近,但与F型又有明显的不同,进而命名为H型。截止现今,HBV基因型可分为A~H八型。 目前,国内外对HBV进行基因分型主要有“基因序列测定法、聚合酶链反应———限制性片段长度多态性分析法、基因型特异性表位单克隆抗体的EL ISA、基因型特异性线形探针检测法、基因型特异性引物PCR法和基因芯片技术”。 1 基因分型原理 1.1 全基因序列测定。全基因序列测定是根据HBV所有病毒核苷酸异源性>8%进行分型的。Okamoto对从日本及印度尼西亚adw2慢性携带者中分离出的3株HBV进行全序列测序及比较,其核苷酸的异质性为3.9%~5.6%,而与美国2株相同血清亚型HBV序列比较,异质性达8.3%~9.3%,达到甚至超过不同血清亚型HBV的异质性,从而说明血清学分型不能真正反映HBV基因变异。再经对18株HBV DNA进行两两比较分析,根据同源性<92%、异质性>8%,将其分为A, B,C及D4个基因型,初步建立了基因分型体系。12年后Stuyver使用该方法,发现了一种新的3248bp的HBV基因型G 。 1.2 S基因序列测定。由于乙型肝 炎病毒基因可分为p基因、前s基因、编 码HBs4的s基因、C基因及X基因(如 图),可分别对它们进行研究,从而找出各 个基因型在各个基因之间的差异。Nor2 der[5]对32例HBV患者s基因测序结果 进行分析,并建立进化树,基因型间异质 性>4%。除证实了Okamoto的A~D分型外,还发现了2个新的基因型E和F,使HBV基因型达到6个(A~F)。在其后对28例HBV全基因组、p基因、前s基因、编码HBs4的s基因、C 基因及X基因分别比较并建立进化树,进一步证实根据s基因序列分型最接近全基因组,从而证明了单独使用S基因进行分型的可靠性。目前,此法尚在使用,主要有SSP和SSO[6],即基因型特异性引物PCR法和基因型特异性线形探针检测法。 2 基因分型方法 2.1 序列测定法。即直接测定核苷酸序列,根据差异分型。自Okamoto据HBV基因型之间的全序列异质性8%进行分型以来,测序由于方法直接、可靠而成为主要鉴定HBV基因型的方法。同全序列进化树图比较,发现S基因的序列变化同全基因序列的变化一致,可用S基因序列代替全基因序列进行分型,界限为核昔酸序列的异质性4.0%。该法虽较为可靠但操作繁琐、费用昂贵,不适于临床大量标本检测。 2.2 聚合酶链反应———限制性片段长度多态性分析法(PCR~RFL P)。目前常用的基因分型方法,通过PCR扩增出目标基因片段(通常为S基因或Pres/s基因),用特定的限制性内切酶进行酶切,根据酶切图谱进行基因分型。Mizokami[7]通过分子进化方法对已知基因型的68例HBV患者全基因、106例HBV患者s基因序列进行分析,发现并确认基因型特异性酶切位点区域。Lindh[8]对不同基因型S基因的特异酶切位点进行分析,设计使用限制性内切酶Trp509I和Hinf I使S基因PCR产物产生不同长度的酶切片段,成功地将166/180例患者HBV实现A-F基因分型。RFL P敏感性高,但酶切位点易受基因变异影响,且遇混合感染或酶切不完全,会出现复杂条带,影响分型结果判断。 2.3 基因型特异性表位单克隆抗体的酶联免疫吸附法(EL ISA)PreS2多肽有多组抗原表位。基因型不同抗原表位也不同,从而可以鉴定不同基因型。Usuda[9]等用此法制备前S2区域基因型特异性表位的单克隆抗体,并用辣根过氧化酶进行标记,对68例HBV阳性患者血清检测,分型结果与S基因测序分型完全一致。在后期实验中发现,适用于大规模的流行病学调查,使较大范围的HBV的研究成为可能。 2.4 基因型特异性线形探针检测法。该方法是设计型特异的探针,检测HBV扩增产物,以产物的不同长度或与探针的反应性来区分不同型别。Kato[10]利用G基因型的病毒在核心区有36个核苷酸的插入,设计引物用PCR的方法可以对G基因型进行特异的筛查。早在1983年Wu用酶切的方法研究血清型的酶切图谱,来区分不同的血清型。王虹[11]等采用PCR2核酸杂交/EL ISA检测,主要是联合利用PCR、核酸杂交和酶联免疫技术,设计前C和C区的探针,可以快速准确的区分HBV的基因型。另外Van G eyt[12]根据A~F基因型的保守序列设计了18种型特异性探针与HBV S (下转12页)

基因敲除技术样本

基因敲除技术 点击次数: 2605 发布日期: -5-25 来源: 本站仅供参考, 谢 绝转载, 否则责任自负 1.概述: 基因敲除是自80年代末以来发展起来的一种新型分子生物学技术, 是经过一定的途径使机体特定的基因失活或缺失的技术。一般意义上的基因敲除主要是应用DNA同源重组原理, 用设计的同源片段替代靶基因片段, 从而达到基因敲除的 目的。随着基因敲除技术的发展, 除了同源重组外, 新的原理和技术也逐渐被应用, 比较成功的有基因的插入突变和iRNA, 它们同样能够达到基因敲除的目的。2.实现基因敲除的多种原理和方法: 2.1.利用基因同源重组进行基因敲除 基因敲除是80年代后半期应用DNA同源重组原理发展起来的。80年代初, 胚胎干细胞( ES细胞) 分离和体外培养的成功奠定了基因敲除的技术基础。1985年, 首次证实的哺乳动物细胞中同源重组的存在奠定了基因敲除的理论基础。到 1987年, Thompsson首次建立了完整的ES细胞基因敲除的小鼠模型[1]。直到现在, 运用基因同源重组进行基因敲除依然是构建基因敲除动物模型中最普遍的 使用方法。 2.1.1利用同源重组构建基因敲除动物模型的基本步骤(图1):

图1.基因同源重组法敲除靶基因的基本步骤 a.基因载体的构建: 把目的基因和与细胞内靶基因特异片段同源的DNA 分子 都重组到带有标记基因(如neo 基因, TK 基因等)的载体上, 成为重组载体。基因敲除是为了使某一基因失去其生理功能, 因此一般设计为替换型载体。 b.ES 细胞的获得: 现在基因敲除一般采用是胚胎干细胞, 最常见的是鼠, 而兔, 猪, 鸡等的胚胎干细胞也有使用。常见的鼠的种系是129及其杂合体, 因为这类小鼠具有自发突变形成畸胎瘤和畸胎肉瘤的倾向, 是基因敲除的理想实验动物。而其它遗传背景的胚胎干细胞系也逐渐被发展应用。[2, 3] c.同源重组: 将重组载体经过一定的方式(电穿孔法或显微注射)导入同源的胚胎干细胞(ES cell)中, 使外源DNA与胚胎干细胞基因组中相应部分发生同源重组, 将重组载体中的DNA序列整合到内源基因组中, 从而得以表示。一般地, 显微注射命中率较高, 但技术难度较大, 电穿孔命中率比显微注射低, 但便于使用。[4,5] d.选择筛选已击中的细胞: 由于基因转移的同源重组自然发生率极低, 动物的重组概率为10-2~10-5, 植物的概率为10-4~10-5。因此如何从众多细胞中筛出真正发生了同源重组的胚胎干细胞非常重要。当前常见的方法是正负筛选法( PNS法) , 标记基因的特异位点表示法以及PCR法。其中应用最多的是PNS法。[6]

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