Functional Analysis Cassettes with Long Flanking Homology Regions Gene - PDF document

Functional Analysis Cassettes with Long Flanking Homology Regions Gene Disruptions Angewundte Mikrobiologie, Biozentrum, Universitat Busel, Klingelbergstr. Table 1. in this study. Restriction site(s) PmeI half site Underlined sequences in oligonucleotides in plasmid the target locus. logous recombination with small homology regions used. Elongation the homology region flanking the several hundred would solve this problem molecules would and high-throughput this paper long flanking reported together selector module and formation and strain XLI-blue as plasmid growth, the bacteria grown either YT) containing (Fluka AG, (YPD) (Treco, media contained, Laboratories). G418 resistant plates containing (geneticin, Gibco BRL, Gaithersburg, MD). performed according to standard purchased from Finnzymes Thermococcus litoralis obtained from from Boehringer, Mannheim, Germany). selector module was amplified as tem- PCR product treated with phenollchloroform, tated, and with BglII also cut with This procedure created Two-step PCR-synthesis of cassettes with long flanking homology regions. (A) of the gene. Only relevant restriction enzyme sites detailed description of sequence of the kunMXmodule elsewhere (Wach two-step PCR-synthesis bind either the target of its (P5.L), immediately the target the first PCR the region directly (downstream) of the coding region yielding fragment bases extensions homologous gene (open boxes in primer symbols) as template. and D) Ethidium bromide-stained agarose HindIIIIHindIII, EcoRI fragments pUC19, Suu3AI fragments as marker. aliquots out synthesized in with genomic following primer Ps,L; lane together with Fragments obtained fragments produced first reaction (lane primers; lane template (lane 0.1 pg; lane 4: in which the former immediately downstream the selector module Fig. 1A). with short kb PCR was gen- as template and two primers (Table nucleotide homology the multiple sequences in Table 1). These primers which were the region immediately upstream or to the last (A. Diisterhoft, personal communication). PCR product cloning, either site (oligonucleotide 4) were 5’-ends of the oligo- volume with as described (Wach quantified as outlined below. ing regions PCR a polymerases with exonuclease activity was used when per was used gether with polymerase exhib- exonuclease activity from the 720bp or start codon (P3, primer), sequence immedi- ately upstream the second the last schematical depiction primer binding First PCR: Approx. either two separate one primer pair each reaction two primer pairs per reaction each primer supplemented with and dTTP. The was overlayed with light mineral oil executed under following conditions: bp fragment (5’-region) with primer combi- (5’-region) using as primers, and used in together with bp fragment the PCR-mix Here variable pg) were used as template and together with 3’-primers (P3,) light mineral 120 sec, mix were added (hot-start). The 50”C, and mix was for 4 Purijication, concentration, chloroform extracted the presence washed with 10 min deionized water. The cells were resuspended in cells were centrifugation, resuspended deionized water PCR product digested with nucleotide kinase cells were performed analogous the method as described (Wach MARKER CASSETTES a thermo- polymerase with unspecific single nucleotide 3'-overhangs PCR prod- ucts. Suitable in the which being in agree- ment with the expected length joined 5'-fragment As seen in the second Obviously, competition the complemen- tary strands PCR product from the first the complementary region overcome only the different also helped, the two outside primers (P5, P3,, Fig. included in the such conditions those compatible with the also amplified. transformation with PCR-made disruption cassettes carrying flanking homology regions (SFH-PCR prod- cassettes with long flanking homology this purpose, the same target locus (ORF456) using this comparison An approximately be mentioned nine base pairs introduced the primers tion cassette the target locus, because they carried restriction sites convenient cloning the targeted reduce the efficiency of homologous recombination the target locus. the protocol the procedure originally described using G418-containing results with the a PCR primers per reaction (Table slightly modified and Methods details). This eight-base restriction vectors allowing convenient excision of modules in enlarged homology regions flanking the two fragments of yeast the marker module used in this reaction (Table The PCR prod- either produced two separate the primer con- for the plasmid containing EcoRI fragment used. The the gener- fragments observed trophoresis (Fig. 1C) good agreement the theoretical the fragments predicted from the primer binding the chromosomal primer pair) primer pair) ORF456 and bp (P3rL7 primer pair) its 3'-region. marker due overlapping regions PCR products from the from the first these fragments PCR product PCR product for transformation bp at 'Three independent made with independently generated PCR products. Control transfor- a CEN-ARS plasmid carrying the complete efficiencies were LiAc treated 1989) were then plating pre-incubation period in the appearance immediately plated type control. the non-integrated pre-incubation. However, positive growth. Transformed can also then, after growth, replica-plated Hegemann, personal communi- increase in efficiency by introduced in the homology flanking the the choice the primer binding the target locus. templates (plasmid insert, genomic have been and it as template produce these homology regions simultaneously in the second kanMX6 was as template. cassettes in using genomic kanMX6 template, reaction, should but a the genomic locus primers, must Any selectable functions in the PCR-method described above. Although not tested here, should be possible with this marker from locus (Vidal can also complete marker removal linking the two genomic in the fragment containing homology regions, polylinker in this in which for a marker from could be their ability eight-base restriction enzyme recog- resulting in previously constructed vectors with reporter- double-modules (Wach into a centromeric vector using the fragment containing the together with the flanking homol- ogy regions transformation, and repair cloning linear plasmid fragment from marker but not the flanking homology regions have Presently many investigate the systematic sequencing disruptions with PCR-made markers flanked regions, the technique presented here, should allow successful and high-throughput probably any industrial importance). longer homology regions flanking the a disruption cassette might avoid mis-integrations with small flanking homology regions more versatile compared conventional cloning techniques, cassettes with long flanking homology regions and PCR-made disruption this technique most promising many different Molecular Genetics D. and Dunham, Antibiotic Resistance Elements in 869-87 1. Nucl. Acids P., Weber, Micro-homology mediated PCR-targeting in and Takanami, M. Nucleotide Sequence the Kanamycin Resistance and Maniatis, Molecular Cloning. Cold Spring Press. Cold Spring nucleic acids as a Curr. Genet. Expression studies in the filamentous the regulatory thesis, University Philippsen, P. highly expressed filamentous fungus (1989). Basic Current Protocols R., Kingston, Seidman, J. and Struhl, Greene Publishing and Gaber, replacement in Poehlmann, R. and Philippsen, heterologous modules of Convenient experimental work a grant Peter Philippsen Science (BBW conjunction with the Yeast Genome Peter Philippsen with high from lambda bacteriophage templates. Acad. Sci. Cullin, C. deletion in Escherichia coli Galactosidase Selection. Bio Techniques transformation with lithium acetate