Ansorge, W.

Benes, V.

Kilger, C.

Pääbo, S.

Voss, H.

English

MPG.PuRe

Direct primer walking on P1 plasmid DNA

BenchmarksDirect Primer Walking onP1 Plasmid DNABioTechniques 23:98-100 (July 1997)The development of vectors such asP1, P1-derived artificial chromosome(PAC) or bacterial artificial chromo-some (BAC) (2,6,8) has permitted thecloning of large DNA fragments. Thesevectors have become common in large-scale sequencing and mapping projects.Therefore, a reliable primer walkingprotocol for P1 plasmids is desirable.It has been demonstrated previouslythat sequencing of P1 plasmid DNA isgenerally feasible (4,11). However, theprotocols included several laborioussteps such as a host strain change fromNS3145 to DH10B or tedious purifica-tion procedures and were limited to theuse of primers located in the vectorarms. Another development for se-quencing P1 plasmid DNA uses a poly-merase chain reaction (PCR)-basedtechnique termed “thermal asymmetricinterlaced PCR” (5); however, thismethod also relies on numerous experi-mental steps. We present an optimizedsequencing protocol for direct primerwalking on P1 plasmid DNA based oncycle sequencing (7). The effect of the following parame-ters on the success rate of P1 sequenc-ing was evaluated: host cell type, purifi-cation procedure, template amount,primer design, primer amount and cy-cle sequencing conditions. Sequencingreactions were carried out with eitherfluorescein isothiocyanate (FITC)- orCy5-labeled primers and analyzed onthe corresponding sequencing appara-tus (ALF DNA Sequencer and ALF-express; Pharmacia Biotech, Piscat-away, NJ, USA). The read lengthassigned to sequencing results was de-termined by the number of nucleotidescalled after automatic processing by theALF software.P1 plasmid DNA was isolated fromits E. coli host strain NS3145 (librarystrain) and transformed into the newhost strain DH10B by electroporation(3). When performing parallel P1 plas-mid DNA preparations under identicalconditions (2× YT medium, 0.5-L cul-ture volume, 50 µg/mL kanamycin, sin-gle-colony inoculate, two QIAGEN100 columns [Qiagen, Hilden, Ger-many]), the E. coli host strain NS3145consistently yielded between 5 and 8times more P1 plasmid DNA than theDH10B host strain. On average, a 0.5-Lculture (NS3145 strain) yielded 80 µg(±10%) plasmid DNA.P1 plasmid DNA purification wasperformed with a conventional alkalinelysis/phenol:chloroform protocol (1)(Nucleobond; Macheray and Nagel,Düren, Germany) or an alkaline lysisprotocol followed by column purifica-tion (Qiagen). The Nucleobond and theQiagen protocols provided by the sup-pliers were both modified as follows.Lysis time was shortened from 5 to 1min, and volumes of the resuspension,lysis and neutralization buffers were98 BioTechniques Vol. 23, No. 1 (1997)Figure 1. Direct P1 plasmid sequencing using 6 pmol of an FITC-labeled primer (20-mer) and 8 µg of column-purified P1 plasmid DNA.doubled. Elution was done with pre-heated buffer (55°C) and a volume in-creased by one fourth. In both cases,the precipitated P1 plasmid DNA wasrinsed twice with 70% ethanol. Carewas taken not to overdry the pellet afterthe final wash. Multiple sequencing re-actions were performed on plasmidDNA grown in either NS3145 orDH10B (ELECTROMAX; Life Tech-nologies, Berlin, Germany) and isolat-ed by both methods.DNA purified from the host strainNS3145 yielded better sequencing re-sults than identical amounts from thehost strain DH10B. Although plasmidDNA isolated by conventional alkalinelysis/phenol:chloroform purificationyielded usable sequence, column puri-fication reduced the background in thesequencing reactions and producedlonger readouts with fewer ambiguities. Best results were obtained when5–10 µg of plasmid DNA were used inthe reaction. Thus, substantially largeramounts of DNA are necessary for suc-cessful cycle sequencing of P1 clonesthan of smaller multicopy plasmids.Walking primers were selected anddesigned with the aid of the EuropeanMolecular Biology Laboratory (EMBL)integrated software package Gene-Skipper (9). In general, primers weredesigned by choosing a region with thehighest possible melting temperature(calculated melting temperatures wereabove 45°C). This approach makes itpossible to use a high annealing temper-ature during cycle sequencing and thusto reduce nonspecific priming events.Primers either 20 or 27 nucleotides inlength were tested. There was no note-worthy difference in their performance.Walking primers were placed an aver-age of 40 bases away from the end ofthe sequence determined by the previ-ously used primer.The optimal amount of primer wasfound to be 6 pmol with a range of4–10 pmol per reaction giving good re-sults. Too high an amount of primer ledto nonspecific priming, which renderedsequence interpretation difficult. Bestresults were achieved with a two-stepcycling protocol as follows: (i) initialdenaturation at 95°C for 3 min and (ii)40 cycles at 60°C for 30 s and 95°C for30 s. In general, the commerciallyavailable Thermo Sequenase Kit(Amersham International plc, Cam-bridge, England, UK) (10) was used.However, other enzymes performedequally well (data not shown).Applying the above protocol, 44primer walks on various P1 clones havebeen performed, and almost 17 kb ofsequence have been determined. Theaverage reading length per sequencingreaction was 385 bases using 30-cmglass plates. The estimated sizes of theclones (about 95 kb) were well withinthe range of average P1 clones. Theabove protocol has been applied toBAC clones with equal success. Table 1gives an overview of all walks per-formed on P1 plasmid DNA, and Figure1 shows a typical sequencing reaction.In summary, a bacterial host strainchange is superfluous when P1 plasmidDNA for sequencing is prepared, be-cause DNA isolated from the NS3145strain performs well in cycle sequenc-ing. The best results were obtained us-ing 5–10 µg of P1 plasmid DNA and 6pmol of fluorescently labeled primer incombination with a two-step cycle se-quencing protocol with a high anneal-ing temperature (60°C). Column-puri-fied DNA performed better than DNAisolated by alkaline lysis only.Note Added in Proof: We recentlyfound that a cycling protocol consistingof an initial denaturation at 95°C for 3min and 40 cycles at 60°C for 30 s,68°C for 30 s and 95°C for 30 s furtherimproves the average length read.REFERENCES1.Birnboim, H.C. and J. Doly. 1979. A rapidalkaline extraction procedure for screening re-combinant plasmid DNA. Nucleic Acids Res.7:1513-1523.Vol. 23, No. 1 (1997) BioTechniques 99Table 1. Overview of Walks Performed on P1 Plasmid DNABases Read in Number of FailedNumber of Each Sequencing Sequencing Average Length Total BasesClone Number Primers Used Reaction Reactions Read Sequenced3A 11 253, 345, 423, 253, 0 385 bases 4239 bases487, 253, 418, 578, 405, 487, 3376A 10 323, 271, 100, 251, 1 311 bases 3107 bases0, 334, 266, 549, 505, 50812B 23 534, 279, 360, 329, 0 417 bases 9597 bases384, 239, 194, 273,149, 441, 619, 408,476, 413, 348, 633,168, 462, 448, 649,513, 661, 617Total 44 1 385 bases 16 943 basesA total of 44 primers were used on three different P1 clones. A total of 16 943 bases were sequenced with an overall averageof 385 bases read per sequencing reaction. One primer gave no sequence.Benchmarks2.Ioannou, P.A., C.T. Amemiya, J. Garnes,P.M. Kroisel, H. Shizuya, C. Chen, M.A.Batzer and P. de Jong. 1994. A new bacterio-phage P1-derived vector for the propagationof large human DNA fragments. Nat. Genet.6:84-89.3.Kilger, C. and H. Zischler. 1997. Electro-transformation of bacterial cells with P1 plas-mid DNA. Trends Genet. 13:41.4.Kimmerly, W.J., A.L. Kyle, V.M. Lustre,C.H. Martin and M.J. Palazzolo. 1994. Di-rect sequencing of terminal regions of genom-ic P1 clones. Genet. Anal. Tech. Appl. 11:117-128.5.Liu,Y.G. and R.F. Whittier. 1995. Thermalasymmetric interlaced PCR: automatable am-plification and sequencing of insert end frag-ments from P1 and YAC clones for chromo-some walking. Genomics 25:674-681.6.Monaco, A.P. and Z. Larin. 1994. YACs,BACs and MACs: artificial chromosomes asresearch tools. Trends Biotechnol. 12:280-286.7.Murray, V. 1989. Improved double-strandedDNA sequencing using the linear polymerasechain reaction. Nucleic Acids Res. 17:8889.8.Pierce, J.C., B. Sauerand and N. Sternberg.1992. A positive selection vector for cloninghigh molecular weight DNA by the bacterio-phage P1 system: improved cloning efficacy.Proc. Natl. Acad. Sci. USA 89:2056-2060.9.Schwager, C., S. Wiemann and W. Ansorge.1995. GeneSkipper—an integrated softwareenvironment for DNA sequence assembly andanalysis. Genome Digest 2:8-9.10.Tabor, S. and C.C. Richardson. 1995. A sin-gle residue in DNA polymerases of the Es-cherichia coli DNA polymerase I family iscritical for distinguishing between deoxy- anddideoxyribonucleotides. Proc. Natl. Acad. Sci.USA 92:6339-6343.11.Wang, Q. and M.T. Keating. 1994. Isolationof P1 insert ends by direct sequencing. Bio-Techniques 17:282-284.The excellent synthesis of fluorescentlylabeled primers by Heiko Drzonek and Wal-ter Schartau as well as the fine technicalsupport by Karin Bauer are gratefully ac-knowledged. Financial support came fromthe DFG. Address correspondence to Chris-tian Kilger, Institute of Zoology, Universityof Munich, P.O. Box 202126, D-80021 Mu-nich, Germany. Internet: kilger@zi.biologie.uni-muenchen.deReceived 7 August 1996; accepted 24January 1997.V. Benes, C. Kilger1, H. Voss,S. Pääbo1 and W. AnsorgeEuropean Molecular Biology LaboratoryHeidelberg1University of MunichMunich, Germany Fabrication of Ion-Selec-tive Microelectrodes by aCentrifugation/SuctionMethodBioTechniques 23:100-102 (July 1997)The preparation of ion-selectivemicroelectrodes can be both time-con-suming and frustrating. Since mosttechniques involve filling the tip withresin before back-filling the rest of theelectrode with electrolyte, the step mostcommonly found annoying is the elimi-nation of air bubbles between the elec-trolyte and the resin. Since the tip is notvisible, trying to draw liquid ion ex-changer (LIX) into a plugged electrodetip is also frustrating. In fabricating cal-cium ion-sensitive microelectrodes foruse as extracellular vibrating probes,Kühtreiber and Jaffe (2) avoided theseproblems by sequential suction-front-filling, first with electrolyte and thenwith resin. Although these steps pre-cluded the problem of air bubbles be-tween the two filling materials, theelectrodes described had open tips of1–10 µm. Intracellular ion-selectiveelectrodes require smaller tip sizes, andfront-filling the electrolyte becomes in-convenient (1) or impossible. Back-fill-ing the tip through the capillary actionof a filling fiber (3,5) is undoubtedlythe most common practice today forpreparing standard potassium chlorideelectrodes, but due to the hydrophobici-ty of the coating, silane-coated mi-cropipets cannot be filled in this man-ner. Yet, if the problem of fully fillingthe electrode tip with electrolyte can beovercome, the advantage of subsequentbubble-free front-filling with LIX canbe achieved. I describe a simple tech-nique by which this may be accom-plished and that nearly always results ina useful ion-selective electrode.First, electrodes are batch-silane-coated. Depending on the method ofsilane coating employed, capillaryglass tubing used may be either with orwithout filling fibers. The technique Iuse is based on procedures describedby Spray and Zavilowitz (4) for double-barreled electrodes, but works wellwith single-barreled electrodes with afilling fiber. One part trimethylchloro-silane (silane) (Sigma Chemical, St.Louis, MO, USA) is mixed with 10parts carbon tetrachloride. A smallamount of silane is introduced into theback of the electrode and allowed tomove into the tip. Because of the fillingfiber and the low surface tension of thesolution, the silane travels easily to thetip. Excess solution is then withdrawn,and the electrodes are placed horizon-tally in a carrier made from an alu-minum block and baked in an oven at100 BioTechniques Vol. 23, No. 1 (1997)Figure 1. Holder for electrolyte filling ofmicropipet tips by centrifugation.Figure 2. A smooth interface free from airbubbles results when LIX is drawn into a mi-croelectrode previously filled with electrolyte.

Kilger, Christian

Pääbo, Svante

Ansorge, Wilhelm

Max Planck Society eDoc Server

Direct Primer Walking on P1 Plasmid DNA

https://pure.mpg.de/pubman/item/item_2105148_8/component/file_2110134/Benes_Direct_Biotechniques_1998.pdf

Direct primer walking on P1 plasmid DNA

Abstract

Similar works

Full text

Available Versions

MPG.PuRe

MPG.PuRe

Max Planck Society eDoc Server