Technological advancement
Double-joint PCR:a PCR-based 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
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 cassettes 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 assem-bly procedure and applied it to delete genes in filamentous fungi.While the principle of this procedure is essentially the same as other recently reported PCR-based tools,our technique has been effectively used to delete 31genes in three fungal species.Moreover,this PCR-based method was used to fuse 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-expressed are presented.In conjunction with the availability of genome sequences,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 $100samples/person/day is also presented.
Ó2004Elsevier Inc.All rights reserved.
Keywords:PCR;Filamentous fungi;Gene replacement;Overexpression;Reporter fusion
1.Introduction
The availability of whole genome sequences for a number of filamentous fungi including the model organ-isms Aspergillus nidulans and Neurospora crassa as well as plant and human pathogens opens new research ave-nues.The first step of understanding gene functions gen-erally involves disruption and/or over-expression of individual genes.Unlike in yeast where only about
50bp of homologous DNA sequences are required for targeted integration,gene disruption by homologous replacement in filamentous fungi usually requires longer DNA sequences (preferentially 500bp or more).Thus,construction of gene-disruption cassettes in filamentous fungi is usually accomplished through several tedious and time-consuming cloning steps.A more efficient functional characterization of genes in filamentous fungi requires a simple and fast procedure to build gene-dis-ruption constructs.
The method of Chaveroche et al.(2000)ensures the presence of very long flanking sequences,but involves a laborious step of in vivo recombination in Escherichia coli .In contrast,PCR-based fusion techniques obviate cloning steps (Davidson et al.,2002;Kolkman and
1087-1845/$-see front matter Ó2004Elsevier Inc.All rights reserved.doi:10.1016/j.fgb.2004.08.001
*
Corresponding authors.Fax:+16082631114(J.-H.Yu),+33169157808(Z.Hamari).
E-mail addresses:jyu1@wisc.edu (J.-H.Yu),zsuzsanna.hamari@igmors.u-psud.fr (Z.Hamari).1
These authors contributed equally.
www.elsevier/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 these studies,we have developed a PCR-assisted tech-nique that can be used 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 composed of the chosen selective maker with0.5–3.0kb upstream and downstreamflanking regions.In this report,we present a detailed protocol to carry out this technique,which was effectively used 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 Research 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 assembling three components using the arg
B gene of A.nidulans as a selective 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(see below and Fig.1
)
远在北京的家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 polymerase
974J.-H.Yu et al./Fungal Genetics and Biology41(2004)973–981
Note .Use 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 used routinely by the Orsay group.This product can be used directly without further purification.
For long constructs,a Long Expand polymerase (Roche,etc.)should be used in preference to the Taq polymerase.
2.2.
3.Third round PCR:amplification of the fused 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
醇醚燃料1l l (usually 0.5l l)of the second round product by using nested primers.
(c)Confirm the size of the product and cut with selected restriction enzymes to verify the PCR product.
(d)Purify the double-jointed final PCR product and
directly use it for transformation.
2.2.4.Conditions for long (>5kb)recombinant DNA The recommended conditions for second round PCR for relatively long (>5kb)recombinant DNA molecules are (with Long Expand polymerase)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 polymerase)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 representation of the construction of a gene replacement cassette.A typical reaction will fuse DNA fragments of a 50flanking sequence,a 30flanking sequence and a marker (M).Primers
2and 3should carry 25–30bases of homologous sequence overlapping with the ends of the selectable marker of choice.The arrows numbered from 1to 10represent primers for the PCRs and primers 2and 3are 45–60bases long chimeric primers.If a polymerase that incorporates an A to the 30end of the PCR product is used,it is strongly suggested that nested primers (7and 8)are designed after a T in the genomic sequence.(A)First round PCR:amplification of the components using the specific and chimeric primers.(B)Second round PCR:the assembly 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 cassette was transformed into RMS011(carrying the D argB ::trpC +allele)selecting 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 cassette (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
sence of an Eco RI site in the gprA ORF,the D gprA amplicon gave rise 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 based on the length of the desiredfinal product.In most cases, 15–30l l of thefinal PCR product can be used for transformation.
双缩脲试剂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 cases)is amplified with a primer pair where the50forward primer car-ries about15–25bases overlapping sequences with the alcA(p),50-tcc aat cct atc acc tcg cct caa a-ATG-target gene ORF sequences-30.
2.3.2.Second round PCR
Both amplicons are purified as described above and mixed in1:1molar ratio.These 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 used for cloning into vectors.
2.3.4.Cloning and sequencing
(a)Clean-up thefinal PCR product,cut with appropri-
ate restriction enzyme(s)or blunt-ended and clone it to a vector of choice, e.g.,pSH96(Wieser and Adams,1995).
(b)Pick three tofive clones,establish the direction of
the insert and sequence the clones to ensure no mutations are introduced by the PCR.
2.4.Fungal genomic DNA isolation(adapted and mod-ified from a commonly used rapid yeast genomic DNA isolation protocol)
These methods enable one person to isolate more than100genomic DNA samples a day and thus improve the efficiency of analysis of genetic alterations.These methods can be used 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).
2008奥运会会徽
(d)Tightly close the cap and insert 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)phase 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
电气石粉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 RNase A(10l g/mlfinal concentration). (k)Use0.5–1l l of genomic DNA solution for20–50l l PCR.Alternatively,5–10l l of DNA solution can be used for a restriction enzyme digestion for Southern blot analyses.
2.5.Total RNA isolation
(a)Place pieces of mycelia(about0.1g and no more
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)Insert 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 phase to a new tube and add
1volume of isopropanol(approximately500l l)to aqueous phase.Mix thoroughly by inverting tube several 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.
后退哥
(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 release 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 used in reverse transcription of the transcriptome.
2.6.Primers and polymerases
PCRs were run on a MJ Research Gradient Cycler PTC-225and PTC-100or an Applied Biosystems Gene-Amp PCR system2700with heated lids.Primers were purchased from Integrated DNA Technologies,or from Sigma Genosys.Amplifications were carried out using Long Expand Polymerase(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(Wieser and Adams,1995)vectors were used.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 sepa-rately amplified by a conventional PCR.In the protocol used by the Yu group(see below for an alternative meth-od)the primers used to amplify the30end of the50flanking sequence and the50end of the30flanking se-quence of the target gene carried25–30bases comple-mentary to50and30of the selective marker, e.g., argB+,respectively.The three,50flanking region,mar-ker and30flanking region,amplicons are mixed in 1:3:1molar ratio and the second round of thermo-cy-cling is carried out.These three DNA fragments will be specifically joined together during the second 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 used for transformation.While the construction of cassettes is an extremely rapid procedure and many independent cassettes can be constructed in a short time,the limiting step is the identification of transformants carrying the desired insertion and/or deleti
on without any extrane-ous events.A useful 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 sequences 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 cassette for the hhoA gene.As mentioned in the text,the chimeric primers(primers5and6)can be used to amplify the selective marker (M),not theflanking sequences.Many genes including hhoA were deleted employing this method.The photograph of the agarose gel electrophoresis shows the resulting products of each PC
R step in constructing a deletion cassette 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 set),and the30flanking(30)regions,which are3.3,2.2,and3.5kb,respectively.The fourth lane shows the resulting products(10l l loaded)of the second round PCR where the50,M,and30components were optimised 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 second round PCR product as the template.Although the assembled product can be9.0kb,the use of the nested primers resulted in afinal product of8.3kb.A1kb DNA ladder(Promega)is shown. Thefinal product was used to transform a riboB2mutant strain.
J.-H.Yu et al./Fungal Genetics and Biology41(2004)973–981977
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