Technological advancement
Double-joint PCR:a PCR-bad molecular tool for gene manipulations in filamentous fungi
Jae-Hyuk Yu a,*,1,Zsuzsanna Hamari b,*,1,Kap-Hoon Han a,1,Jeong-Ah Seo a,1,
Yazmid Reyes-Domı
´nguez b ,Claudio Scazzocchio b,c a
Department of Food Microbiology and Toxicology,The University of Wisconsin,Madison,WI 53706,USA
b
Institute de Ge
´ne ´tique et Microbiologie,Universite ´Paris-Sud,UMR 8621CNRS,Ba ˆtiment 409,Centre d ÕOrsay,91405Orsay Cedex,France c
Institut Universitaire de France,France
Received 7July 2004;accepted 4August 2004
Abstract
夸美
Gene replacement via homologous double crossover in filamentous fungi requires relatively long (preferentially >0.5kb)flanking regions of the target gene.For this reason,gene replacement casttes are usually constructed through multiple cloning steps.To facilitate gene function studies in filamentous fungi avoiding tedious cloning steps,we have developed a PCR-assisted DNA asm-bly procedure and applied it to delete genes in filamentous fungi.While the principle of this procedure is esntially the same as other recently reported PCR-bad tools,our technique has been effectively ud to delete 31genes in three fungal species.Moreover,this PCR-bad method was ud to fu more than 10genes to a controllable promoter.In this report,a detailed protocol for this easy to follow procedure and examples of genes deleted or over-expresd are prented.In conjunction with the availability of genome quences,the application of this technique should facilitate functional characterization of genes in filamentous fungi.To stream line the analysis of the transformants a relatively simple procedure for genomic DNA or total RNA isolation achieving $100samples/person/day is also prented.
Ó2004Elvier Inc.All rights rerved.
Keywords:PCR;Filamentous fungi;Gene replacement;Overexpression;Reporter fusion
1.Introduction
The availability of whole genome quences for a number of filamentous fungi including the model organ-isms Aspergillus nidulans and Neurospora crassa as well as plant and human pathogens opens new rearch ave-nues.The first step of understanding gene functions gen-erally involves disruption and/or over-expression of individual genes.Unlike in yeast where only about
50bp of homologous DNA quences are required for targeted integration,gene disruption by homologous replacement in filamentous fungi usually requires longer DNA quences (preferentially 500bp or more).Thus,construction of gene-disruption casttes in filamentous fungi is usually accomplished through veral tedious and time-consuming cloning steps.A more efficient functional characterization of genes in filamentous fungi requires a simple and fast procedure to build gene-dis-ruption constructs.
The method of Chaveroche et al.(2000)ensures the prence of very long flanking quences,but involves a laborious step of in vivo recombination in Escherichia coli .In contrast,PCR-bad fusion techniques obviate cloning steps (Davidson et al.,2002;Kolkman and
1087-1845/$-e front matter Ó2004Elvier Inc.All rights rerved.doi:10.1016/j.fgb.2004.08.001
*
Corresponding authors.Fax:+16082631114(J.-H.Yu),+33169157808(Z.Hamari).
E-mail address:jyu1@wisc.edu (J.-H.Yu),zsuzsanna.hamari@igmors.u-psud.fr (Z.Hamari).1
食品卫生手抄报口叫The authors contributed equally.
/locate/yfgbi
Fungal Genetics and Biology 41(2004)
973–981
Stemmer,2001;Shevchuk et al.,2004;Stemmer, 1994a,b)and PCR-assisted gene manipulations were carried out in the pathogenic fungus Cryptococcus neo-formans(Davidson et al.,2002).Independently from the studies,we have developed a PCR-assisted tech-nique that can be ud to construct recombinant DNA molecules combining any two or three DNA fragments. Following this technique,deletion of31genes was car-ried out.Thefinal product of each deletion construct compod of the chon lective maker with0.5–3.0kb upstream and downstreamflanking regions.In this report,we prent a detailed protocol to carry out this technique,which was effectively ud for gene manipulations in threefilamentous fungal species,A. nidulans,Aspergillus fumigatus,and Fusarium graminea-rum.In addition,simple and fast methods to isolatefil-amentous fungal genomic DNA or total RNA are described.
2.Materials and methods:protocols
2.1.Strains,culture conditions,genetics,and transformation
All A.nidulans strains derived from the standard lab-oratory Glasgow strain or Fungal Genetics Stock Center and carried auxotrophic markers appropriate for each transformation experiment.Transformat
ion recipient strains include RMS011(pabaA1,yA2;
D argB::trpC+;trpC801),PW1(biA1;argB2;methG1), FGSC237(pabaA1,yA2;trpC801),CS2902(biA1, pyrG89;pyroA4;riboB2),CS2903(biA1;wA3;pyroA4; riboB2),CS2904(pabaA1,pyrG89;pantoB100),CS2905 (yA2,pantoB100,riboB2),CS2906(pyrG89,yA2;argB2; pantoB100,riboB2).Genetic markers as in Clutterbuck (1994).The A.fumigatus strain AF293.1(pyrGÀ)was from Greg May at the University of Texas,MD.Ander-son Cancer aminearum(R-5317,equal to MRC1781and NRRL5908)strains were from the Fusa-rium Rearch Center at the Penn State University.A. nidulans media are described in Martinelli and Kinghorn (1994).Transformation of A.nidulans and A.fumigatus was carried out as described by Tilburn et al.(1983)or Han et al.(2004a).Transformation aminearum was performed as previously described(Lee et al.,2002).
2.2.Double joint PCR(DJ-PCR)to build a gene replacement construct
A typical reaction asmbling three components using the arg
B gene of A.nidulans as a lective marker is described as an example.In general,sufficient amount of maker DNA is pre-amplified,cleaned,and stored as stocks.Template DNA for marker gene amplific
ation can be genomic or plasmid DNA.The argB gene of A.nidulans is amplified with the following primers:argB-For:50-gac cag ttt aga ggc ctc-30,argB-Rev:50-gtg tta ggc ctg gat cta-30.
2.2.1.First round PCR:amplification of50-and30-flanking regions of the gene(s)of interest
(a)Design the following six primers for the1st round
PCR:50-For,50-Rev+marker tail,30-For+marker tail,30-Rev,50-nest and30-nest(e below and Fig.1
)
PCR mixture(final25l l)PCR
1l l of purified50-flanking amplicon94°C2min
1l l of purified30-flanking amplicon94°C30s—> 3l l of purified argB amplicon58°C10min·
10–15cycles
2l l of dNTP(2.5mM each)72°C5min—^ 2.5l l of10·PCR buffer72°C10min 15.25l l of sterile distilled water
0.25l l of Taq polymera
974J.-H.Yu et al./Fungal Genetics and Biology41(2004)973–981
Note .U 1:3:1molar ratio for 50-flanking:argB :30-flanking amplicons.The total DNA amount of the three components should be between 100and 1000ng.The annealing time was routinely 10min in the procedure carried out at the Yu lab.However,the experience of the Orsay laboratory showed that this may be reduced to 2min.The latter was ud routinely by the Orsay group.This product can be ud directly without further purification.
For long constructs,a Long Expand polymera (Roche,etc.)should be ud in preference to the Taq polymera.
2.2.
3.Third round PCR:amplification of the fud knockout construct
(a)Design a nested primer pair preferentially starting
just after a ÔT Õas explained in Section 2.2.1.
(b)Perform a conventional PCR in 100l l with less than
1l l (usually 0.5l l)of the cond round product by using nested primers.
(c)Confirm the size of the product and cut with lected restriction enzymes to verify the PCR product.
(d)Purify the double-jointed final PCR product and
directly u it for transformation.
2.2.4.Conditions for long (>5kb)recombinant DNA The recommended conditions for cond round PCR for relatively long (>5kb)recombinant DNA molecules are (with Long Expand polymera)2min at 94°C;15cycles of (45s at 93°C,2min at 62°C and 12min at 68°C)and 15min post-polymerization.The third round PCR conditions are (using Long Expand polymera)2min at 94°C;35cycles of (45s at 93°C,45s at 62°C,and 12min at 68°C)and 15min
post-polymerization.
Fig.1.Schematic reprentation of the construction of a gene replacement castte.A typical reaction will fu DNA fragments of a 50flanking quence,a 30flanking quence and a marker (M).Primers 左耳朵疼
技艺精湛
2and 3should carry 25–30bas of homologous quence overlapping with the ends of the lectable marker of choice.The arrows numbered from 1to 10reprent primers for the PCRs and primers 2and 3are 45–60bas long chimeric primers.If a polymera that incorporates an A to the 30end of the PCR product is ud,it is strongly suggested that nested primers (7and 8)are designed after a T in the genomic quence.(A)First round PCR:amplification of the components using the specific and chimeric primers.(B)Second round PCR:the asmbly reaction is carried out without using any specific primers,as the overhanging chimeric extensions act as primers.The first two cycles are shown in detail.(C)Third round PCR:amplification of the final product using nested primers.(D)Confirmation of gene replacement:an example of deletion confirmation of the gprA gene encoding a putative G protein coupled receptor (Seo et al.,2004)is shown.The gprA deletion castte was transformed into RMS011(carrying the D argB ::trpC +allele)lecting for arginine prototrophy.Transformants were randomly picked and examined for double crossover-mediated gene replacement pattern by PCR amplification of the gprA locus using a primer pair beyond the flanking regions included in the castte (primers 9and 10).As shown,amplicons of wild type (3.2kb)and deletion (3.8kb)alleles of gprA differ in size (uncut).In addition,restriction enzyme digestion patterns of the individual amplicons further distinguish the wild type and null alleles.When digested with Eco RI,while the wild type amplicon is cut into three fragments (note the $300bp faint band)due to the pre
nce of an Eco RI site in the gprA ORF,the D gprA amplicon gave ri to two bands.Similarly,the different positions of the Eco RV sites in the gprA and argB ORFs resulted in varying digestion patterns.Restriction enzyme abbreviations are for Bam HI (B),Eco RI (E),Eco RV (V),Hin dIII (H),and Pst I (P).
J.-H.Yu et al./Fungal Genetics and Biology 41(2004)973–981975
The annealing temperature depends on the actual primers.The elongation time is determined bad on the length of the desiredfinal product.In most cas, 15–30l l of thefinal PCR product can be ud for transformation.
2.3.Single-joint PCR(SJ-PCR):construction of the alcA(p)::ORF or fusion of any two DNA fragments
2.3.1.First round PCR
(a)Amplification of the alcA promoter is carried out
with the primer pair50For primer-50ccg ttc tgc tta ggg ta30and30Rev primer-50ttt gag gcg agg tga tag gat tgg a30using wild type genomic DNA as a template.
(b)The target gene ORF with the(probable)poly(A)
binding site(AATAAA in most cas)is amplified with a primer pair where the50forward primer car-ries about15–25bas overlapping quences with the alcA(p),50-tcc aat cct atc acc tcg cct caa a-ATG-target gene ORF quences-30.
2.3.2.Second round PCR
Both amplicons are purified as described above and mixed in1:1molar ratio.The fragments are joined by PCR with no primers.Reaction mixture is the same as above.The thermo-cycling conditions are94°C for3min,10cycles of(94°C for30s, 58°C for10min,72°C for5min)and72°C for 10min.
2.3.3.Third round PCR
In this third round PCR,the joined product will be amplified with nested primers carrying restriction en-zyme sites(if necessary)to be ud for cloning into vectors.
2.3.4.Cloning and quencing
(a)Clean-up thefinal PCR product,cut with appropri-
朝韩战争ate restriction enzyme(s)or blunt-ended and clone it to a vector of choice, e.g.,pSH96(Wier and Adams,1995).
(b)Pick three tofive clones,establish the direction of
the inrt and quence the clones to ensure no mutations are introduced by the PCR.六国论翻译
2.4.Fungal genomic DNA isolation(adapted and mod-ified from a commonly ud rapid yeast genomic DNA isolation protocol)
The methods enable one person to isolate more than100genomic DNA samples a day and thus improve the efficiency of analysis of genetic alterations.The methods can be ud to monitor large number of trans-formants for correct integration of the construction and expression of the relevant mRNA.
(a)Inoculate2ml of liquid minimal medium+5g/L
yeast extract+supplements in an8ml test tube with $105spores and grow$16h(no longer than20h)at 37°C(stationary culture).
(b)Collect the mycelial mat,squeeze excessive medium
using paper towel and transfer the squeeze-dried sample into a2ml screw cup tube with an O-ring cap.
(c)Add500l l of breaking buffer(2%Triton X-100,1%
SDS,100mM NaCl,10mM Tris–HCl,pH8.0,and 1mM EDTA,pH8.0),500l l of phenol:chloro-phorm:isoamyl alcohol(25:24:1)and300l l of
0.5mm zirconia/silica beads(BioSpec).
(d)Tightly clo the cap and inrt up to eight tubes in
Mini-BeadBeater-8(BioSpec)in a cold room and homogenize at the highest speed for2min.
(e)Take out the tubes and centrifuge at16,000g for
10min at room temperature or4°C.
(f)Gently transfer the aqueous(upper)pha into a
new1.5ml microcentrifuge tube.
(g)Add two volumes of ice cold absolute ethanol(kept
atÀ20°C),mix well and centrifuge at16,000g for 10min to pellet DNA.
(h)Decant the supernatant andfill the tubes with the
DNA pellets with70%ethanol.
(i)Decant70%ethanol as much as possible and dry the
DNA pellets.
(j)Resuspend isolated genomic DNA in50l l of TE(pH
8.0)with RNa A(10l g/mlfinal concentration). (k)U0.5–1l l of genomic DNA solution for20–50l l PCR.Alternatively,5–10l l of DNA solution can be ud for a restriction enzyme digestion for Southern blot analys.
2.5.Total RNA isolation
(a)Place pieces of mycelia(about0.1g and no more类似lol的游戏
than0.2g)into a pre-labelled2ml screw cup tube with an O-ring cap.Quick-freeze in liquid nitrogen and store atÀ80°C until all samples are ready.
(b)Remove up to eight tubes with mycelia fromÀ80°C,
keep on ice,and add1ml Trizol(Invitrogen)and add400l l0.5mm zirconia/silica beads(BioSpec) to each sample.
(c)Inrt up to eight tubes into a Mini-BeadBeater-8(in
a cold room)and homogenize for2.5min at max
speed(homogenize).
(d)Let tubes sit at RT for5min and add200l l of chlo-
roform.Shake tubes vigorously by hand for15s.
Incubate at RT for3min.
(e)Microcentrifuge at12,000g for15min at4°C or
RT.
976J.-H.Yu et al./Fungal Genetics and Biology41(2004)973–981
(f)Remove upper aqueous pha to a new tube and add
1volume of isopropanol(approximately500l l)to aqueous pha.Mix thoroughly by inverting tube veral times.
(g)Let sit at RT for10min and centrifuge at9200g at
RT for10min.
(h)Remove all supernatant carefully,gently add1.5ml
of70%ethanol(made with DEPC-treated water) and wait for1min.
(i)Remove nearly all of supernatant by leaving the
tubes upside down.
(j)Air dry the pellet at room temperature for5–10min. (k)Resuspend the total RNA samples in50–100l l of DEPC-treated sterile H2O or other appropriate buf-fer.Vortex andflick tube withfingers to relea the pellet from the wall of the tube.Pipette up and down gently and briefly heat to65°C and repeat pip
etting until the entire RNA pellet is dissolved.Spin briefly to collect sample at the bottom of the tube.From this point on keep tube on ice or frozen atÀ80°C.
Quality of total RNA isolated by this procedure is high enough to be ud in rever transcription of the transcriptome.
2.6.Primers and polymeras
PCRs were run on a MJ Rearch Gradient Cycler PTC-225and PTC-100or an Applied Biosystems Gene-Amp PCR system2700with heated lids.Primers were purchad from Integrated DNA Technologies,or from Sigma Genosys.Amplifications were carried out using Long Expand Polymera(Roche)or Triplemaster (Brinkmann).PCR products were purified by High Pure PCR Product Purification Kit(Roche)or Qiagen PCR purification kit.
2.7.Cloning vectors and Escherichia coli strains
The pGEM-Teasy(Promega)and pSH96(Wier and Adams,1995)vectors were ud.Plasmids were pre-pared li DH10B or DH5a.
3.Results and discussion
Fig.1summarizes the whole procedure of our PCR-assisted technique.The individual components are pa-rately amplified by a conventional PCR.In the protocol ud by the Yu group(e below for an alternative meth-od)the primers ud to amplify the30end of the50flanking quence and the50end of the30flanking -quence of the target gene carried25–30bas comple-mentary to50and30of the lective marker, e.g., argB+,respectively.The three,50flanking region,mar-ker and30flanking region,amplicons are mixed in 1:3:1molar ratio and the cond round of thermo-cy-cling is carried out.The three DNA fragments will be specifically joined together during the cond round PCR.The elongation time is according to thefinal size of the desired construct($1min/kb).After each round of PCR,the amplified components are purified using a commercially available PCR clean-up kit.In the third round of PCR,the double-joint product is amplified with a nested primer pair(primers7and8in Fig.1). We found that using nested primers gave almost100% success(single product with no artefacts)whereas using thefirst round primer pairs(primers1and4)generated extremely low success levels with high chance of getting PCR artefacts.
Thefinal amplicon is cleaned and directly ud for transformation.While the construction of casttes is an extremely rapid procedure and many independent casttes can be constructed in a short time,the limiting step is the identification of transformants carrying the desired inrtion and/or deleti
on without any extrane-ous events.A uful way to identify the desired homol-ogous recombination event in a primary screening is to amplify the DNA of relevant transformants with a pri-mer pair complementary to quences outside the dele-tion construct(for example,primers9and10in Fig.
1).Fig.1D shows how the deletion mutant of
gprA Fig.2.An alternative primer design and construction of a deletion castte for the hhoA gene.As mentioned in the text,the chimeric primers(primers5and6)can be ud to amplify the lective marker (M),not theflanking quences.Many genes including hhoA were deleted employing this method.The photograph of the agaro gel electrophoresis shows the resulting products of each PC
R step in constructing a deletion castte of the hhoA.Thefirst three lanes show the purified PCR products of the50flanking(50),the chimeric riboB gene(M amplified with an alternative chimeric primer t),and the30flanking(30)regions,which are3.3,2.2,and3.5kb,respectively.The fourth lane shows the resulting products(10l l loaded)of the cond round PCR where the50,M,and30components were optimid to the 1:3:1molar ,$200,$450,and$210ng,respectively.Thefinal 8.3kb product was then amplified with a nested primer pair using 0.5l l of the cond round PCR product as the template.Although the asmbled product can be9.0kb,the u of the nested primers resulted in afinal product of8.3kb.A1kb DNA ladder(Promega)is shown. Thefinal product was ud to transform a riboB2mutant strain.
J.-H.Yu et al./Fungal Genetics and Biology41(2004)973–981977
