F O C U S 1 9 N U M B E R 1 1

O N F O C U S

Manuscripts describing novel techniques,improvements of common techniques, sim-plified protocols, and trouble-shooting are invited. “Instructions toAuthors” are available upon requestfrom the editor:

Dr. Doreen CupoEditor, FOCUS

Life Technologies, Inc.8451 Helgerman CourtGaithersburg, MD 20884-9980(800) 828-6686(301) 840-8000 (outside the U.S.)E-mail: dcupo@lifetech.com

Editorial Review Board:Holly Anderson, Paul Battista, StephenGorfien, James Hartley, Curtis Henrich,Roger Lasken, Larry Mertz, WilliamWhitford

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© Copyright Life Technologies, Inc. 1996

FOCUS® is published triannually by LifeTechnologies, Inc.POSTMASTER: Send address changes toFOCUS, Life Technologies, Inc.,P.O. Box 6009, Gaithersburg, MD20884-9980.

Requests for subscriptions and address changesshould be directed to the nearest LTI office.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . All referenced trademarks are theproperty of their respective owners.Purchase of Taq DNA Polymerase isaccompanied by a limited license to use it in thePolymerase Chain Reaction (PCR) process forresearch and development in conjunction with athermal cycler whose use in the automatedperformance of the PCR process is covered bythe up-front license fee, either by payment toPerkin-Elmer or as purchased, i.e., anauthorized thermal cycler.

A R T I C L E S

62 CULTURE OF HUMAN KERATINOCYTES IN DEFINED

SERUM FREE MEDIUM

David A. Judd, Paul J. Battista, and Darrin D. Behm

68 A HIGHLY SENSITIVE METHOD FOR ONE-STEP

AMPLIFICATION OF RNA BY POLYMERASE

CHAIN REACTION

Eui Hum Lee, Kalavathy Sitaraman, David Schuster,and Ayoub Rashtchian

T O O L

72 A NEW BACULOVIRUS EXPRESSION VECTOR FOR THE

SIMULTANEOUS EXPRESSION OF TWO HETEROLOGOUS

PROTEINS IN THE SAME INSECT CELL

Ray Harris and Deborah A. Polayes

F R A G M E N T S

75 USING DNA LADDERS AS SIZE STANDARDS FOR

POLYACRYLAMIDE GEL ANALYSIS OF DNAHeather Jordan and Jim Hartley

78 DNA FINGERPRINTING IN COTTON USING AFLPS

Xiang Feng, Sukumar Saha, and Khairy Soliman

C L A S S I C F O C U S A R T I C L E

79 ETHANOL PRECIPITATION: AMMONIUM ACETATE AS AN

ALTERNATIVE TO SODIUM ACETATE

Joseph Crouse and Douglas Amorese

80 Announcements

80 Focus Volume 18 Index

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David A. JuddPaul J. BattistaCell CultureResearch andDevelopmentLife Technologies, Inc.

Darrin D. BehmQuality ControlLife Technologies, Inc.Grand Island, New York 14072

Avariety of systems have beendeveloped to culture humankeratinocytes. Early work usedserum-supplementation with media

such as Medium 199 (1) and NCTC 168 (2).Keratinocyte growth and colony formationwere improved by plating cells on lethallyirradiated 3T3 fibroblasts and adding epidermalgrowth factor (EGF) and hydrocortisone tothe medium (3). One of the first serum-freeformulations developed was based on Medium199 containing a growth factor cocktail thatincluded bovine brain extract (4). Serum-freeculture of human keratinocytes without 3T3fibroblast feeder layers became widely acceptedwith the development of MCDB-153 (5).Serum-free MCDB-153 medium included traceelements, ethanolamine, phosphoethanolamine,hydrocortisone, EGF, and bovine pituitaryextract (BPE). This medium and severalenhanced versions have been used widely forhuman keratinocyte cultivation (6–8).

Serum-free medium containing BPE as theprimary mitogen has several drawbacks. Theundefined composition of BPE complicatesexperimental models and interpretation ofresults. It may stimulate or inhibit humankeratinocyte cultures, depending on theconcentration and the presence of othercomponents (9). In addition, BPE requirestitration in different systems and its stability in

medium is limited to ~4 weeks under normaluse conditions.

Defined Keratinocyte-SFM eliminates BPEby inclusion of defined growth promotingadditives including insulin, EGF, and FGF.The medium is designed for the isolation andinitiation of primary keratinocytes as well asexpansion of keratinocyte cultures. DefinedKeratinocyte-SFM demonstrates superiorprimary cell growth while maintainingmorphology and physiological markers.

METHODSIsolation and culture of human keratinocytes.

Unless otherwise indicated, all media andreagents were GIBCO BRL brand. Neonatalforeskins were placed in serum-free medium(without growth factors) containing 5 µg/mlgentamycin and stored at 4°C. Foreskins canbe stored in this manner for ~5 days withoutsignificant loss of cell viability. Foreskins werebriefly rinsed in 70% isopropanol and thenplaced in Dulbecco’s phosphate buffer saline(DPBS), without Ca++ and Mg++, containing20 µg/ml gentamycin for 60 min. Foreskinswere cut into halves or quarters, depending onthe size of the tissue, and the pieces weretransferred, dermis side down, to a petri dishcontaining 25 units/ml dispase and incubated18 to 24 h at 4°C. Epidermal sheets wereseparated from the full-thickness skin with

2 F O C U S 1 9 N U M B E R 1

A R T I C L E

CULTURE OF HUMAN KERATINOCYTES IN DEFINED

SERUM-FREE MEDIUM

FIGURE 1. Phase contrast microscopy of human keratinocytes. Cells were cultured in Defined Keratinocyte-SFM (panelA) and Keratinocyte-SFM (contains BPE) (panel B) (100X).

A B

forceps, pooled in 60-mm culture dishescontaining 5 to 7 ml of 0.05% trypsin/0.53 mMEDTA, and incubated at 37°C for 15 to 20 minwith gentle pipetting to aid in tissue dissocia-tion. Pooling of tissue specimens is performedto reduce the effects of donor-to-donorgrowth variation. Trypsin activity was ter-minated by addition of soybean trypsin inhibitor(10 mg/ml in DPBS). Any remaining pieces ofepidermal sheets were carefully removed anddiscarded. The cell suspension was transferredto a sterile centrifuge tube and the cells pelletedby centrifugation at 40 × g for 5 min (22°C)and washed once with SFM. The supernatantwas discarded, the cell pellet resuspended inthe appropriate medium, and cell densitiesdetermined using a hemocytometer. Cells wereplated in culture flasks or dishes.

Secondary cultures were established byremoving the spent medium, briefly washingthe cell monolayer with Versene (1:5,000), andadding an appropriate volume of 0.05%trypsin/0.53 mM EDTA. Cells were incubatedat 37°C until they became round (~5 min),trypsin was removed, and the cells wereincubated at 37°C until they detached from theculture surface with gentle tapping (~5 min).Trypsin activity was stopped by addition of10 mg/ml soybean trypsin inhibitor solution;cells were pelleted by centrifugation at 40 × gfor 5 min (22°C), washed once with SFM,and resuspended in the appropriate medium.Trypsinization times are critical to the perfor-mance of any keratinocyte medium. Humankeratinocytes that remain in trypsin too longhave lower plating efficiencies and may beinduced to differentiate. Secondary cell cultureswere also established from primary keratinocytesobtained from Cell Systems Corporation withresults comparable to those found with culturesestablished from neonatal foreskins. Cultureswere incubated at 37°C in a humidifiedatmosphere consisting of 5% CO2/95% air.Stock cultures were maintained at a split ratio of1:2 to 1:3 and subcultured at 70% to 80%confluence. Keratinocytes at passage 0 through4 were used for experimental evaluation.

Morphology and growth assays. Morpho-logical analysis and immunostaining of cellswere performed in 8-chamber glass cultureslides. Keratinocytes were plated at 2 × 104

cells/cm2 in a total volume of 400 µl/0.8-cm2

chamber. Cells were incubated for 24 h, thenfixed with 3.7% formaldehyde, permeabilizedwith 0.5% Triton® X-100 in DPBS, and allowedto react with rabbit anti-cytokeratin 14 antibody(1:200 dilution). Cells labeled with antibodieswere visualized using goat anti-rabbit F(ab´)2FITC conjugate (1:50 dilution).

Human keratinocyte growth assays wereperformed in 24-well culture dishes (2 cm2

growth area) utilizing a seeding density of1 × 104 cells/cm2. Endpoint growth assays wereassessed at 6 days postseeding for primary cellsand 72 h for secondary cells. Growth kineticassays were counted at 24-h intervals over96 h without media replacement. Single-cell cloning assays were performed in 96-welltissue-culture-treated plates by serial dilution ofcell suspensions to 5 cells/ml in the appropriatemedium and plating 100 µl/well. Plates wereincubated for 5 days before observation.

F O C U S 1 9 N U M B E R 1 3

FIGURE 2. Expression of keratin 14. Human ker-atinocytes were cultured in Defined Keratinocyte-SFM(100X).

DefinedKeratinocyte-

SFM

Cel

ls/

Wel

l (×1

05 )

Keratinocyte-SFM

Supplier A

2.0

2.5

1.5

1.0

0.5

0.0

*

FIGURE 3. Growth of primary human keratinocytes. Growth was determined 6 dayspostseeding. Values represent the mean ± SEM, n = 7. * p < 0.05 versus Keratinocyte-SFMand Supplier A.

Supplier A is a defined medium for humankeratinocytes. Keratinocyte-SFM is a BPE-containing formulation (10).

RESULTS AND DISCUSSIONHuman keratinocytes cultured in Defined

Keratinocyte-SFM exhibited the same contact-inhibited, “crazy paving” pattern morphology(11) as cells grown in the presence of BPE(figure 1). Monolayer cultures had distinctborders and prominent nuclei. All culturesstained positive for keratin 14, a standardmarker for basal human keratinocytes (figure 2).

Primary cultures established in DefinedKeratinocyte-SFM demonstrated significantlybetter growth when compared to other ker-atinocyte media (figure 3). Population doublingtimes (PDTs) were: Defined Keratinocyte-SFM,46.3 ± 5.9 h, Keratinocyte-SFM, 66.6 ± 12.8 h,and Supplier A, 83.5 ± 19.1 h.

Growth of secondary cultures was similarbetween Defined Keratinocyte-SFM andKeratinocyte-SFM, although better cell growthwas achieved in Defined Keratinocyte-SFM thanin Supplier A’s defined medium (figure 4).PDTs for secondary keratinocytes culturedwere: Defined Keratinocyte-SFM, 25.0 ± 1.1 h,Keratinocyte-SFM, 29.0 ± 1.6 h, and Supplier A,35.4 ± 4.1 h. Daily growth kinetic experimentsusing secondary cultures confirmed that cellscultured in defined medium proliferated at arate comparable to BPE-containing medium(figure 5, p > 0.05). Cloning efficienciesof ~40% have been achieved with humankeratinocytes cultured in Defined Keratinocyte-SFM in single-cell cloning experiments and arecomparable to those found for cells cultured inBPE-containing medium. Cultures can bemaintained for at least 6 passages in DefinedKeratinocyte-SFM with split ratios of 1:2performed twice weekly. Fully supplementedmedium had a shelf life of >14 weeks, consider-ably longer than medium containing BPE(figure 6).

Culture systems to propagate humankeratinocytes have evolved to reduce theundefined components and to increase culturelongevity and cell yields. The results presentedhere demonstrate that BPE can be replacedwithout adversely affecting cellular proliferationrates and general physiology of humankeratinocytes. The removal of BPE as a

4 F O C U S 1 9 N U M B E R 1

Per

cent

of

Con

trol

Gro

wth

Time (weeks)

100

50

80

60

70

40

30

90

50 10 15

Cel

ls/

Wel

l (×1

05 )

Time (hours)

3.5

1.0

2.5

1.5

2.0

0.024 48 72 96

0.5

3.0

4.0

FIGURE 5. Growth kinetic analysis of human keratinocytes. Secondary cells werecultured in Defined Keratinocyte-SFM (■) or Keratinocyte-SFM (❏). Values represent themean ± SD, n = 2.

FIGURE 6. Evaluation of media shelf life using primary human keratinocytes. Cellswere cultured in Keratinocyte-SFM (solid line) or Defined Keratinocyte-SFM (dashed line)over a 15-week period. Cells were counted after 6 days in medium stored for given times andcompared to cells cultured in fresh medium.

DefinedKeratinocyte-

SFM

Cel

ls/

Wel

l (×1

05 )

Keratinocyte-SFM

Supplier A

1.4

1.8

0.4

1.0

0.6

0.8

0.0

0.2

*

1.2

1.6

FIGURE 4. Growth of secondary human keratinocytes. Growth was determined 72 hpostseeding. Values represent the mean ± SEM, n = 7. * p < 0.05 versus Supplier A.

F O C U S 1 9 N U M B E R 1 5

medium component while maintaining mediumperformance represents a step forward in humankeratinocyte culture by providing a morestandardized and controlled culture environ-ment (12).

ACKNOWLEDGEMENTWe thank Carl Soderland (Cell Systems Corp.)for providing human keratinoytes.

REFERENCES1. Marcelo, C.L., Kim, Y.G., Kaine, J.L., and

Voorhees, J. (1978) J. Cell Biology 79, 356.2. Price, F.M., Camalier, R.F., Gantt, R., Taylor,

W.G., Smith, G.H., and Sanford, K.K. (1980) InVitro 16, 147.

3. Rheinwald, J.G. and Green, H. (1975) Cell 6,331.

4. Gilcrest, B.A., Calhoun, J.K., and Maciag, T.(1982) J. Cell. Physiol. 112, 197.

5. Boyce, S.T. and Ham, R.G. (1983) J. Invest.Dermatol. 81, 33.

6. Pittelkow, M.R. and Scott, R.E. (1986) MayoClinic Proceedings 61, 771.

7. Pirisi, L., Yasumoto, S., Feller, M., Doniger, J.,and DiPaolo, J. (1987) J. Virol. 61, 1061.

8. Shipley, G.D. and Pittelkow, M.R. (1987) Arch.Dermatol. 123, 1541.

9. Wille, J.J., Pittelkow, M.R., Shipley, G.D., andScott, R.E. (1984) J. Cell. Physiol. 121, 31.

10. Daley, J.P., Epstein, D.A., and Hawley-Nelson,P. (1990) FOCUS 12, 68.

11. Daniels, J.T., Harris, I.R., Kearney, J.N., andIngham, E. (1995) Exp. Dermatol. 4, 183.

12. Watson, C.A., Camera-Benson, L., Palmer-Crocker, R., and Pober, J.S. (1995) Science 268,447.

be carried out either in two-step or one-stepformats. In two-step RT-PCR, cDNA synthesisis first performed with RT in an appropriatebuffer. The RT step is followed by PCR ampli-fication with a thermostable DNA polymerasein another appropriate buffer (5). This two-stepformat requires opening the reaction tubeafter cDNA synthesis to either remove a cDNAaliquot for subsequent PCR or to add PCRreagents. The method is widely used and effec-tive for cDNA cloning and characterization,RACE techniques, and cDNA library construc-tion, as well as gene expression detection.

In the one-step RT-PCR method, reversetranscription and PCR take place sequentially ina single tube under conditions optimized forboth the RT and DNA polymerase withoutopening the tube. This simplifies the procedureand minimizes the potential for cross-samplecontamination. One-step RT-PCR is suitable forroutine and high-throughput screening of geneexpression. When using SUPERSCRIPT II RT,the ONE-STEP RT-PCR System detects RNAmolecules present in low abundance.

METHODSRNAs. The 891-bp CAT mRNA was a

run-off transcript of pTEPA-CAT plasmid DNAby T7 RNA polymerase. The CAT RNA wastreated with DNase I, Amplification Grade(Cat. No. 18068), for removal of DNA tem-plate, followed by phenol extraction and ethanolprecipitation. Total HeLa RNA was isolated byTRIZOL® Reagent or the GLASSMAX® RNAMicroisolation Spin Cartridge System (6).

One-step RT-PCR. One-step RT-PCR wascarried out using the GIBCO BRL SUPERSCRIPT

ONE-STEP RT-PCR System (Cat. No. XXXXX).Reactions (50 µl final volume) were assembledby mixing 25 µl of 2X reaction mix [2X buffer,2.4 mM MgSO4, 400 µM dNTPs each, and 4µg/ml BSA], 1 ml of enzyme mix [SUPERSCRIPT

II RT and recombinant Taq DNA polymerase in20 mM Tris-HCl (pH 7.5 at 25oC), 100 mMNaCl, 0.1 mM EDTA,1 mM DTT, 50% glycerol (v/v), and stabilizer],

6 F O C U S 1 9 N U M B E R 1

ABSTRACTUsing SUPERSCRIPT™ II RT (1) and

Taq DNA polymerase (2), we developed aconvenient and sensitive SUPERSCRIPT

ONE-STEP RT-PCR system. The system usestwo premixed solutions: 1) an optimizedmixture of SUPERSCRIPT II RT and Taq DNApolymerase and 2) a 2X reaction mix con-taining buffers, dNTPs, and MgSO4. Withthis system, 10 copies of an in vitrotranscript RNA and β-actin mRNA from100 fg of total HeLa RNA were detected.The high sensitivity and premixed formatof the ONE-STEP RT-PCR system make it aneasy and convenient tool for rapid androutine screening of RNA expression.

Coupling reverse transcription andpolymerase chain reaction (RT-PCR)is a sensitive and powerful method todetect RNA (3). Using SUPERSCRIPT

II RT for cDNA synthesis improves the efficien-cy and sensitivity of RT-PCR as compared toMMLV RT or AMV RT (4). RT-PCR can

Eui Hum LeeKalavathy SitaramanDavid SchusterAyoub RashtchianMolecular BiologyResearch andDevelopmentLife Technologies, Inc.Gaithersburg,Maryland 20884

A HIGHLY SENSITIVE METHOD FOR ONE-STEP AMPLIFICATION OF

RNA BY POLYMERASE CHAIN REACTION

TABLE 1. Primer sequences.

Gene Primer Product size (bp)

CAT sense CGACCGTTCAGCTGGATATTAC 500antisense TTGTAATTCATTAAGCATTCTGCC

β-actin sense TGAAGTACCCCATCGAGCACG 174antisense CAAACATGATCTGGGTCATCTTCTC

β-actin sense CAGGGCGTGATGGTGGGCA 253antisense CAAACATGATCTGGGTCATCTTCTC

β-actin sense GCTCGTCGTCGACAACGGCTC 353antisense CAAACATGATCTGGGTCATCTTCTC

β-actin sense TGAAGTACCCCATCGAGCACG 755antisense AGTGATCTCCTTCTGCATCCTGT

β-actin sense GCTGGTCGTCGACAACGGCTC 976antisense AGGAGCAATGATCTTGATCTTCATT

β-actin sense ATGGCCACGGCTGCTTCCAGCTCC 1,026antisense ATTCAACTGGTCTCAAGTCAGTGTA

β-actin sense GCTCGTCGTCGACAACGGCTC 1,684antisense ATTCAACTGGTCTCAAGTCAGTGTA

β-actin sense GCCAGCTCACCATGGATGATGAT 1,715antisense ATTCAACTGGTCTCAAGTCAGTGTA

* Primer sequences for the polε, RPA, PP2A, and CBP PCR products are listed in the Internet versionof this article at http://www.lifetech.com/focus/1910xx.pdf.

A R T I C L E

200 nM of each primer (table 1), and the appro-priate amount of sample RNA. Alternatively, forexperiments utilizing the same primer or targetRNA, a master mix of enzyme and buffer withprimer or target RNA was made. The sampleswere incubated at 45oC-55oC for 30 min; then94oC for 2 min followed by amplification of 40cycles of 94oC for 15 s, 50oC-65oC range for 30s, and 68oC-72oC for 1-3 min (1 kb/1 min); fol-lowed by one cycle of 72oC for 5-10 min. PCRproducts (10 µl) were analyzed on0.8%-1.5% (w/v) agarose gels containing0.5 µg/ml ethidium bromide.

RESULTS AND DISCUSSIONSeveral reports have suggested inhibition

of amplification when RT was mixed withTaq DNA polymerase for one-step RT-PCR (7).From our studies (data not shown), the inhibi-tion appears to be related to the amount ofenzyme and buffer conditions. By examiningthe ratio of enzymes in combination with avariety of buffers, a one-step RT-PCR systemwas developed that permits optimal activityfor both SUPERSCRIPT II RT and Taq DNApolymerase. The procedure is shown in figure 1.

The system detected 10 copies of a 500-bpCAT product (figure 2). No PCR products wereobserved from control reactions that omitted

F O C U S 1 9 N U M B E R 1 7

cDNA synthesis and pre-denaturation1 cycle

45oC -55oC for 30 min94oC for 2 min

PCR40 cycles

Denature 94oC for 15 sAnneal 50 oC -65oC for 30 sExtend 68oC-72oC for 1kb/min

Final extension (optional)72oC for 5-10 min

Assemble Reaction

Thermal Cycling

Analyze Products

2X reaction mixture 25 mlSense primer (10 µM) 1 mlAntisense primer (10 µM) 1 mlRNA template X mlEnzyme mixture 1 mlDistilled water X mlFinal Volume 50 µl

�

�

FIGURE 1. The SUPERSCRIPT ONE-STEP RT-PCR protocol.

FIGURE 2. Amplification of CAT mRNA. Reactionswere incubated at 45oC for 30 min; 94oC for 2 min; then 40cycles of 94oC for 15 s, 58oC for 30 s, and 68oC for 90 s;followed by 68oC for 5 min. Lane 1. No RNA template.Lanes 2 to 7 contain 5, 10, 102, 103, 104, and 105 copies ofCAT mRNA, respectively.

FIGURE 3. Amplification of β-actin mRNA. The incu-bations were as in figure 1 except the cDNA synthesis was at50oC and the annealing temperature was 55oC. Lane 1.No RNA template, Lanes 2 to 6 contain 0.1, 1, 10, 102, and103 pg total HeLa RNA.

100

Bp

DN

AL

adde

r

1 2 3 4 5 6 7 100

Bp

DN

AL

adde

r

1 2 3 4 5 6

500–� 353–�

RT with up to 109 copies of CAT mRNA (datanot shown). Application of one-step RT-PCR tosamples containing limited quantities of totalcellular RNA was tested. A 353-bp β-actinfragment was detected from 0.1 pg total HeLaRNA (figure 3).

SUPERSCRIPT II RT improves the versatilityof the SUPERSCRIPT ONE-STEP System. The RTreaction can be performed between 42oC and

55oC (1). This may facilitate amplification ofRNAs with secondary structure. Cosolventssuch as dimethyl sulfoxide and glycerol that mayhelp RT-PCR (8, 9) were excluded from thereaction buffer, since no significant improve-ment was observed for the amplicons tested(data not shown). Since SUPERSCRIPT II RT ishighly efficient in the one-step RT-PCR buffer,incubation times may be decreased for shorttemplates (<300 bp) to 1-2 min at 45oC. A10-min incubation was sufficient for detectionof the 1.68-kb b-actin mRNA target (figure 4).The 30-min RT incubation was chosen to per-mit efficient cDNA synthesis for a wide range ofprimer sets. Decreased yield of specific productand increased nonspecific bands were observedwith some of the primer sets with incubationtimes beyond 30 min (data not shown).

The SUPERSCRIPT ONE-STEP System wasused with RNA targets ranging from 100 bp to3.5 kb (figure 5). The RNA targets includedβ-actin (10), DNA polymerase ε (polε) (11),cap binding protein (CBP) (12), replicationprotein A (RPA) (13), and phosphatase 2A(PP2A) (14), representing genes with differentlevels of abundance. The system detected specif-ic mRNA targets using total RNAs from avariety of sources, including HeLa cells, human

8 F O C U S 1 9 N U M B E R 1

FIGURE 4. Incubation time for cDNA synthesis. The1,684-bp β-actin fragment amplified after RT incubation at45oC for 2, 5, 10, 15, and 20 min, respectively, in duplicate(lanes 1-5).

FIGURE 5. RT-PCR products of different sizes. 4-8 µl of RT-PCR products were loaded on a 1.5% (Panel A) or 1.0%(Panel B) agarose gel containing ethidium bromide. Panel A. Lanes 1 and 2. β-actin, 174 and 253 bp. Lane 3. PP2A, 331bp. Lane 4. CBP, 495 bp. Lane 5. RPA, 514 bp. Lane 6. polε, 606 bp. Lane 7. β-actin, 755 bp. Panel B. Lane 1. β-actin,976 bp. Lanes 2 and 3. polε, 1,081 and 1,475 bp. Lane 4. β-actin, 1,715 bp. Lanes 5-7. pole, 2,036, 2,531, and 3,520 bp,respectively.

A B

1 2 3 4 5

�–1,684

DN

A L

ow M

ass

Lad

der

1 2 3 4 5 6 7 λD

NA

/H

ind

III

Frag

men

ts

1 2 3 4 5 6 7

174–�

755–�

976–�

3,520–�

tissue (submaxillary salivary gland cell), rattissue (liver, brain, and spleen), and tobaccoplant leaves (data not shown). In addition, one-step RT-PCR has a large capacity for RNA, sinceas much as 5 µg total RNA template was used,which can be useful for the detection of veryrare mRNAs.

One important parameter for PCR is themagnesium concentration. Optimal concentra-tion can vary depending on the primer sets.Analysis of >600 RT-PCRs with 40 differentprimer sets designed for 11 different genes(tested at 1 to 2 mM magnesium), showed thatthe 1.2-mM magnesium concentration of theSUPERSCRIPT ONE-STEP System detected thesetargets. Only 4 primer sets showed low yield anda slightly higher magnesium optimum (1.4 to2.0 mM). These reactions were easily optimizedby addition of magnesium ion. (These data areavailable in the Internet version of this article athttp://www.lifetech.com/focus/1910xx.pdf).

The data presented uses a gene-specificprimer for cDNA synthesis. Use of oligo(dT) isnot recommended for the one-step proceduresince this system uses higher temperatures(45oC-55oC) which would give poor yield ofcDNA with oligo(dT). If oligo(dT) is necessary,a two-step system is recommended.

In this paper, we have described theSUPERSCRIPT ONE-STEP RT-PCR System forrapid screening and sensitive amplification ofRNA in a one-step protocol. A total of 40primer sets for 11 separate mRNAs of varyingabundance successfully amplified and detecteddifferent regions ranging between 100 bp and3.5 kb.

ACKNOWLEDGMENTSWe thank Domenica Simms for providing

the RNA and Paul Nisson and Donna Fox forsome of the primers. We are grateful to GaryGerard, Roger Lasken, and Wu Bo Li for help-ful discussions.

REFERENCES1. Gerard, G., Schmidt, B.J., Kotewitz, M.L., andCampbell, J.H. (1992) FOCUS 14, 91.2. Chien, A., Edgar, D.B., and Trela, J. (1976) J.Biol. Chem. 127, 1550.3. Murakawa, G.J., Zaia, J.A., Spallone, P.A.,Stephens, D.A., Kaplan, B.E., Wallace, R.B., andRossi, J.J. (1988) DNA 7, 287.4. Nathan, M., Mertz, L.M., and Fox, D.K. (1995)FOCUS 17, 78.5. Hyone-Myong, E. (1996) Enzymology Primer forRecombinant Technology, Academic Press, 345.6. Farrell, R.E., (ed.) (1993) RNA Methodologies - ALaboratory Guide for Isolation and Characterization.Academic Press.7. Sellner, L.N., Coelen, R.J., and Mackenzie, J.S.(1992) Nucleic Acids Res. 20, 1487.8. Bassel-Duby, R., Spriggs, D.R., Tyler, K.L., andFields, B.N. (1986) J.Virol. 60, 64.9 Sidhu, M.K., Liao, M.J., and Rashidbagi, A. (1996)BioTechniques 21, 44.10. Ponte, P., Mg, S.Y., Engel, J., Gunning, P., andKedes, L. (1984) Nucleic Acids Res. 12, 1687.11. Kesti, T., Frantti, H., and Syvaoja, J.E. (1993) J.Biol. Chem. 268, 10238.12. Rychlik, W., Domier, L.L., Gardner, P.R., andHellmann, G.M. (1987) Proc. Natl. Acad. Sci. USA84, 945.13. Erdile, L.F., Wold, M.S., and Kelly, T. (1990) J.Biol. Chem. 265, 3177.14. Arino, J., Woon, C.W., Brautigan, D.L., andMiller, T.B., Jr. (1988) Proc. Natl. Acad. Sci. USA 85,4252.

F O C U S 1 9 N U M B E R 1 9

Passive PCR?

Chained to the reactionabsorbed into the fundamentalsthe truth cannot escape

Poly Poly Polyit’s my racethe recipe to repeat

Primed more specificto the pur breed ofMore More More

Still chained to the reactionScience isn’t mere scienceanymore…

—LY N N SH O O K S

In this paper, a new vector, pFASTBAC™DUAL, which allows for the cloning andsimultaneous expression of two heterologousproteins, is presented. This vector has two latepromoters, the polyhedrin promoter (polh) andthe p10 promoter. By inserting separate genesin the 2 multiple cloning sites (MCS), it ispossible to generate a recombinant baculovirusthat produces 2 heterologous proteins in thesame insect cell. This is particularly useful in theinvestigation of protein-protein interactions orthe expression of multisubunit proteins.

10 F O C U S 1 9 N U M B E R 1

The BAC-TO-BAC™ BaculovirusExpression System was developed tosimplify the generation of recombi-nant baculoviruses. This system is

based on site-specific transposition of anexpression cassette from the recombinant donorplasmid into a shuttle vector of baculovirusDNA (bacmid) that is propagated in E. coli (1).Recombinant bacmid DNA is rapidly isolatedfrom E. coli cells, transfected into insect cells,and viral stocks (>107 pfu/ml) are harvestedfrom insect cells for protein expression, purifica-tion, and analysis.

T O O L

A NEW BACULOVIRUS EXPRESSION VECTOR FOR THE

SIMULTANEOUS EXPRESSION OF TWO HETEROLOGOUS PROTEINS

IN THE SAME INSECT CELL

Ray HarrisResearch andDevelopmentLife Technologies, Ltd.Paisley, Scotland

Deborah A. PolayesMolecular BiologyResearch andDevelopmentLife Technologies, Inc.Gaithersburg,Maryland 20884

FIGURE 1. Map of pFASTBAC DUAL expression vector. For MCS I, +1 corresponds to the transcriptional start for the polyhedrin (polh) promoter. TheATG site of original polh start was mutated to ATT. The stop codons are shown in bold. For MCS II, +1 corresponds to the transcriptional start for the p10promoter. Digestion at the Bbs I site generates a BamH I compatible overhang.

Kp

n I

Sp

h I

Nsi

IP

vu I

IN

he

IN

co I

Xh

o I

Sm

a I

Bb

s I

Ba

mH

IR

sr I

IB

ssH

II

Eco

R I

Stu

IS

al

IS

st I

Sp

e I

No

t I

Nsp

VX

ba

IP

st I

Hin

d I

II

Tn7R

pFASTBAC™DUAL5237 bp

Apr

Tn7L

f1 intergenicregion

Gmr

ori

MCS IIHSV tk polyA pPolhp10 MCS I SV40 poly A

polyhedrin promoter

pFASTBAC DUAL multiple cloning sites: 4515-4730

MCS I

AAATAAGTAT TTTACTGTTT TCGTAACAGT TTTGTAATAA AAAAACCTAT AAATATTCCG GATTATTCAT ACCGTCCCAC CATCGGGCGC GGATCCCGGT CCGAAGCGCG CGGAATTCAA

4515 BamH I BssH II EcoR IRsr II

AGGCCTACGT CGACGAGCTC ACTAGTCGCG GCCGCTTTCG AATCTAGAGC CTGCAGTCTC GACAAGCTTG TCGAGAAGTA CTAGAGGATC ATAATC – 3�

Stu I Sal I Sst I Spe I Not I Nsp V Xba I Pst I Hind III

→ +1

5�–

5�–

stop codons

stop codons

p10 promoterAA ATAAGAATTA TCAAATCATT TGTATATTAA TTAAAAAATA CTATACTGTA AATTACATTT TATTTACAAT CACTCGACGA AGACTTGATC ACCCGGGATC TCGAGCCATG GTGCTAGCAG CTGATGCATA

4417 Bbs I Sma I Xho I Nco I Nhe I Pvu II Nsi I

GCATGCGGTA CCGGGAGATG GGGGAGGCTA ACTGAAACAC – 3�

Sph I Kpn I

→ +1MCS II

METHODSThe cloning vector pFASTBAC DUAL

(figure 1, Cat. No. 10712) comes with controlDNA that contains the chloramphenicolacetyltransferase (CAT) gene cloned into thepolh MCS of pFASTBAC DUAL at the BamH Iand Pst I sites and the β-glucuronidase (gus)gene cloned into the p10 MCS at the Nco I andNsi I sites. pFASTBAC DUAL control DNA wastransformed into MAX EFFICIENCY DH10BAC™Competent Cells, and the cells were selected asdescribed previously (2). Recombinant bacmidDNA, isolated as described in the BAC-TO-BAC

system manual, was transfected into Spodopterafrugiperda (Sf 9) cells using CELLFECTIN™Reagent, and virus was collected after 72 h. Theexpression of gus was demonstrated in situ (2).

For expression of reporter genes, 1 × 106

cells (Sf 9, Sf21, or BTI-TN-5B1-4) wereseeded into a 35-mm dish. The cells wereallowed to attach for 1 h at 27°C and theninfected with recombinant baculovirus at anMOI of 5. Sf 9 and Sf21 cells were cultured at27°C in Sf-900 II SFM (Cat. No. 10902), andBTI-TN-5B1-4 cells were cultured at 27°Cin EXPRESS FIVE™ SFM (Cat. No. 10486).All media were supplemented with 50 U/mlpenicillin and 50 µg/ml streptomycin. All cellculture media and reagents were GIBCO BRLbrand. At appropriate time points postinfection,cells were collected by centrifugation, washedone time in PBS, and resuspended in 50 µl TEbuffer. Cells were lysed by a rapid freeze/thawat –70°C, then an equal volume of 2X SDSloading buffer [4% SDS, 125 mM Tris-HCl(pH 6.7), 30% glycerol, 0.002% bromophenolblue, 2% 2-mercaptoethanol] was added.Samples were boiled for 5 min and analyzed bySDS-PAGE.

To quantify expression of reporter genes,infections were set up as described above. Atappropriate time points, the dishes were washedone time with PBS and then 1 ml of lysis buffer[0.1 M Tris-HCl (pH 8.0) containing 0.1%Triton® X-100] was added to the dish. Disheswere stored at –70°C for 2 h, thawed at 37°C,and chilled on ice. Cell lysates were clarified bycentrifugation at 12,000 × g and divided intotwo equal-volume samples in fresh tubes. Oneset of tubes was heat treated, then stored at–70°C until they were assayed for CAT activity(3). The other set of lysates was stored at –70°C

F O C U S 1 9 N U M B E R 1 11

FIGURE 2. SDS-PAGE analysis of cell extracts. Sf 9, Sf21, and BTI-TN-5B1-4 cells wereinfected at an MOI of 5. Samples were analyzed by SDS-PAGE on a 12.5% gel. For eachsample, 25 µg of total protein was loaded. Lane 1. Uninfected Sf 9 cells. Lane 2. Sf 9 cellsinfected with both genes (48 hpi). Lane 3. Sf21 cells infected with both genes (48 hpi).Lane 4. BTI-TN-5B1-4 cells infected with both genes (72 hpi). Lane 5. Sf 9 cells infectedwith CAT (48 hpi). Lane 6. Sf 9 cells infected with gus (48 hpi). Lane 7. Sf 9 wild-typeAcNPV infected cells (48 hpi).

without further treatment and assayed for gusactivity (4).

RESULTS AND DISCUSSIONThe simultaneous expression of 2 heterolo-

gous proteins has been achieved using thepFASTBAC DUAL vector and the BAC-TO-BAC

System. For expression from the polh promoter,an in-frame ATG must be provided by thecloned gene. For the p10 promoter, cloninginto the Bbs I, Sma I, or Xho I sites requires anATG sequence for translation initiation. Whencloning into the Nco I site or sites downstream,make sure the reading frame of the gene ofinterest is in-frame relative to the ATG sequenceof the Nco I site.

The expression of CAT and gus was mea-sured from virus expressing 1 or both proteinsto verify that the expression pattern and thetotal activity were not affected by the expressionof two proteins. The appropriate protein bandsat ~73 kDa for gus (4) and ~26 kDa for CATwere observed in the 3 commonly used insectcell lines (figure 2). Neither of these bands wasobserved in uninfected or wild-type infected cells.

kDa

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Prot

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Lad

der

1 2 3 4 5 6 7

The activity of CAT was quantitated forvirus expressing CAT or CAT and gus over a96-h time course. Maximal activity was at 72 h(figure 3). The CAT gene is under the controlof the polh promoter in the same context in bothrecombinant viruses. Comparable total CATactivity was observed for the single protein CATvirus (CAT) and the dual CAT/gus construct inSf 9 and Sf21 cells, indicating no detrimentaleffect of expressing two proteins simultaneous-ly. For BTI-TN-5B1-4 cells, there appearedto be more activity for the single-gene constructthan the DUAL construct. This may be a

12 F O C U S 1 9 N U M B E R 1

function of the high level of protein expressionin these cells. Similar results were seen with thegus expression for virus expressing gus or CATand gus (figure 4). The gus gene is 13 bp closerto the p10 promoter in the single construct thanit is in the DUAL recombinant virus.

Differences in total activity were observedfor the various cell lines. The Sf 9 cells had thelowest CAT activity (figure 3). The level of CATactivity observed from infections with theDUAL construct was 4.5 times higher in Sf21cells and 7 times higher in BTI-TN-5B1-4 cellscompared to Sf 9 cells. The specific activity forCAT from Sf 9 cells was 36 U/µg. The specificactivity was 2 times higher for Sf21 cells and 3times higher for BTI-TN-5B1-4 cells. For gusactivity, Sf 9 cells also had the lowest activity(figure 4). However, the difference between Sf 9and Sf21 cells was not as great.

These data demonstrated that the pFASTBAC

DUAL Expression Vector used with theBAC-TO-BAC System produced large quantitiesof 2 heterologous proteins in the same cell.Under the control of the same promoter,comparable levels of gene expression can beobtained when the recombinant virus directedthe expression of 2 heterologous proteins ascompared to when one protein was expressed.The expression of proteins in different cell linesand at different time points indicated the impor-tance of fully characterizing these parametersto optimize the levels of protein obtained.

REFERENCES

1. Luckow, V.A., Lee, S.C., Barry, G.F., and Olins,P.O. (1993) J. Virol. 67, 4566.

2. Anderson, D., Harris, R., Polayes, D., Ciccarone,V., Donahue, R., Gerard G., Jessee, J., andLuckow, V. (1995) FOCUS 17, 53.

3. Ciccarone, V., Hawley-Nelson, P., and Jessee, J.(1993) FOCUS 15, 81.

4. Jefferson, R.A., Burgess, S.M., and Hirsh, D.(1986) Proc. Natl. Acad. Sci. USA 83, 8447.

FIGURE 3. Total CAT activity for gus/CAT- and CAT-infected cells. Sf 9, Sf21, andBTI-TN-5B1-4 cells were infected at an MOI of 5. CAT transcription is controlled by thepolyhedrin promoter.

µU g

us/

µl e

xtra

ct

0

2,000

4,000

6,000

10,000

8,000

12,000

14,000

Sf9 Sf21

Hours Post Infection

BTI-TN-5B1-4

24 48 72 96

DUAL-gus DUAL-gus

24 48 72 96

DUAL-gus

24 48 72 96 24 48 72 96 24 48 72 9624 48 72 96

gus gusgus

mU

CA

T/

5µl E

xtra

ct

0

250,000

Sf9 Sf21

Hours Post Infection

BTI-TN-5B1-4

24 48 72 96

DUAL-CAT DUAL-CAT

24 48 72 96

DUAL-CAT

24 48 72 96 24 48 72 96 24 48 72 9624 48 72 96

CAT CATCAT

50,000

100,000

150,000

200,000

300,000

350,000

400,000

450,000

500,000

FIGURE 4. Total gus activity for gus/CAT- and gus-infected cells. Sf 9, Sf21, and BTI-TN-5B1-4 cells were infected at an MOI of 5.

DNA ladders were designed to sizeDNA on agarose gels. DNAladders have more bands, balancedband intensities, and orientation

bands when compared with DNA standardsderived from restriction digests. There are appli-cations where polyacrylamide gel electrophore-sis (PAGE) may be advantageous for resolutionof small fragments. Since migration of DNAfragments on polyacrylamide is influencedby sequence as well as size (1–3), we haveexamined several DNA ladders to determinetheir usefulness in sizing DNA in PAGE gels. ADNA ladder and a restriction endonucleasedigest of a known DNA were used to calculatethe sizes of DNA fragments separated on anative PAGE gel.

METHODSDNA size standards evaluated on a 6%

nondenaturing polyacrylamide gel includedΦX174 DNA digested with Hae III; pBR322DNA digested with Msp I; 25 bp DNA Ladder(Cat. No. 10597); 50 bp DNA Ladder (Cat.No. 10416); 100 bp DNA Ladder (Cat. No.15628); and 123 bp DNA Ladder (Cat. No.15613). Each standard was diluted in loadingbuffer [final concentration 1 mM Tris-HCl (pH7.5), 1 mM EDTA, 6.5% sucrose, 0.03%bromphenol blue], and 300 ng were loadedonto the gel (well width, 2.5 mm; gel thickness,1 mm).

A restriction digest with a DNA ladder onone side, and the other restriction digest on theother side was electrophoresed at 6 V/cm for90 min in 1X TBE (100 mM Tris, 90 mM boricacid, 1 mM EDTA) and stained in 1X TBE con-taining 1 µg/ml ethidium bromide for 10 minat room temperature. The migration distance ofeach band was determined by measuring thedistance from the bottom of the well to themiddle of the DNA band. Standard curves wereconstructed (figure 1) with the two flankinglanes and used to calculate the apparent sizes ofthe digest in the middle lane. The semilogarith-mic plots of the 25 bp DNA Ladder, 50 bp

F O C U S 1 9 N U M B E R 1 13

DNA Ladder, and pBR322/Msp I were used tocalculate the apparent molecular weight of frag-ments of FX174/Hae III. The semilogarithmicplots of the 100 bp DNA Ladder, 123 bp DNALadder, and ΦX174/Hae III were used tocalculate the apparent molecular weight offragments of pBR322/Msp I. Finally, the valuescalculated using the DNA ladders or the restric-tion digests and were plotted against the knownfragment sizes for either ΦX174/Hae III orpBR322/Msp I (figure 2).

RESULTS AND DISCUSSIONSeveral DNA ladders were examined to

determine their usefulness in sizing DNA onnative PAGE. Figure 2 shows photographs ofthe gels, and the data derived from them. Theline represents perfect correspondence betweenthe calculated and actual sizes of the fragmentsin the center lane of each photograph. Thepoints represent the fragment size determinedusing the DNA ladder or the restriction digestas the standard. The point closest to the linerepresents the most accurate estimate of the sizeof that fragment. For the lower range (25 to250 bp) the 25 bp DNA Ladder was most accu-rate, and for the higher range (250 to 800 bp)

F R A G M E N T S

USING DNA LADDERS AS SIZE STANDARDS FOR

POLYACRYLAMIDE GEL ANALYSIS OF DNA

Heather JordanJim HartleyMolecular and CellBiologyResearch andDevelopmentLife Technologies, Inc.Gaithersburg,Maryland 20884

1 3 5 7

2000

1000

100

80

Act

ual F

ragm

ent

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(bp

)

FIGURE 1. A representative standard curve for the 100 Bp DNA Ladder (+) andΦX174/Hae III fragments (∆).

1500–

700–

600

300–

100–

14 F O C U S 1 9 N U M B E R 1

C

A

B

D

FIGURE 2. Comparing estimated DNA fragment sizedetermined with a DNA ladder and a restriction digest.Panel A. 25 bp DNA Ladder (lane 1,+) and pBR322/Msp I(lane 3, ∆) used to calculate sizes of ΦX174/Hae III frag-ments (lane 2). Panel B. 50 bp DNA Ladder (lane 1, +) andpBR322/Msp I (lane 3, ∆) used to calculate sizes ofΦX174/Hae III fragments (lane 2). Panel C. 100 bp DNALadder (lane 1, +) and ΦX174/Hae III (lane 3, ∆) used tocalculate sizes of pBR322/Msp I fragments (lane 2). PanelD. 123 bp DNA Ladder (lane 1, +) and ΦX174/Hae III(lane 3, ∆) used to calculate sizes of pBR322/Msp I frag-ments (lane 2).

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–622–527

–307

–160147

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bp

–622–527

–307

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F O C U S 1 9 N U M B E R 1 15

the 50 and 100 bp DNA Ladders were mostaccurate. The 100 bp DNA Ladder was betterthan the 123 bp DNA Ladder in closeness tothe actual value, especially in the low range. TheDNA ladders were as accurate as the restrictiondigests in determining the size of an unknownfragment. The increased number of bands in theDNA ladders allows for more accurate sizedetermination in the appropriate range.

With the 100 bp DNA Ladder (panel C)the migration of the highlight band wasreduced. This fragment migrated very close tothe 700-bp band, when its actual size is 600 bp(4). Somewhat reduced migration of thehighlight bands of the other DNA ladders onpolyacrylamide has also been observed (datanot shown), but it was not obvious on the gelphotographs here. The highlight bands of eachladder are composed of sequences that are not

related to the repeated fragments of the rest ofthe ladder. Their behavior in polyacrylamidegels demonstrates the potential influence of par-ticular DNA sequences upon electrophoreticmobility (3). It has also been reported thatelectrophoresis at lower temperatures (5oC) canenhance anomalous migration in PAGE (2).From the data presented, we conclude thatDNA ladders and restriction digests gave similarresults when used to estimate the sizes of DNAfragments in native PAGE gels.

REFERENCES1. Stellwagen, N.C. (1983) Biochemistry 22, 6186.2. Hsieh, C.H., Wu, M., and Yang, J.M. (1991) MolGen Genet 225, 25.3. Diekmann, S. (1989) Electrophoresis 10, 354.4. Starr, S., Hartley, J., Russell, L., and Longo, M.

(1991) FOCUS 13, 101.

• • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • •

DNA FINGERPRINTING IN COTTON USING AFLPS

Amplified fragment length polymor-phism (AFLP) is based on theselective amplification of restrictionfragments from total genomic DNA

with different primer pairs (1). Fine variationamong samples can be distinguished by AFLPon a DNA sequencing gel. The AFLP methodhas been used for producing high-densitygenetic maps in many crops (2). In this paper,we apply AFLP to the cotton genome.

Genomic DNA from young leaves ofGossypium barbadense (Pima 3-79) was extract-ed following the modified method of Wagner(3). AFLP was performed using the GIBCO BRLAFLP System I (Cat. No. 10544) following themanufacturer’s protocol (2) with minor modifi-cations. The use of 800 ng of the genomic DNAof cotton in the initial reaction provided thebest results in comparison to the 250 ng ofDNA suggested in the instructions. A highamount of DNA might be necessary because thehigh levels of polyphenolic and secondaryproducts in cotton make it difficult to get pureDNA for selective amplification.

Cotton DNA and control tomato DNAwere digested with 3 µl and 2 µl of EcoR I/Mse I(1.25 units/µl each), respectively, at 37°C for 3h, and the fragments were ligated with the EcoRI and Mse I adapters at 18°C for 3 h. Eachligation reaction was diluted 1:10 with TEbuffer. After selective amplification, 4 µl of eachreaction mixture was electrophoresed on a 6%DNA sequencing gel and at 1,800 V until thexylene cyanole dye migrated two-thirds of theway down the gel. The gel was dried andexposed to x-ray film (Biomax-MR) for 16 h.

On average, the primer pairs used in AFLPprovided about 60 bands, ranging from 30 bpto 600 bp (table 1). The results, using the samesample, demonstrated that AFLP bands vary

Xiang FengSukumar SahaKhairy SolimanDepartment of Plantand Soil ScienceAlabama A&MUniversityNormal, AL 35762

TABLE 1. Number of bands seen with different primer combinations in AFLP

analysis of the cotton genome.

M-CAA M-CAC M-CAG M-CAT M-CTA M-CTC M-CTG M-CTT

E-AAC 87 63 64 79 27 67 26 None

E-ACA 13 65 60 73 48 38 43 20

E-ACG 25 33 60 45 - - - -

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

16 F O C U S 1 9 N U M B E R 1

FIGURE 1. Screening of AFLP primer pairs from Pima-3 79 cotton. Lane 1. Controltomato DNA. Lanes 2–5. E-AAC primer paired with M-CAA, M-CAC, M-CAG, and M-CAT, respectively.

1 2 3 4 5

among the different primer pairs, indicating thateach primer pair amplified different segments ofthe same genome (figure 1). Also, each lanecontained some major bands and some minorbands. The major bands indicated the presenceof more copies of similar sequence in the cottongenome in comparison to that of the minorbands.

In a comparison of the AFLP to RFLP andRAPD methods (4), AFLP identified morepolymorphic bands than RAPD in soybean.Also, our previous genetic analysis (5) in cottonshowed that AFLP provides more bandscompared to the RFLP or RAPD technique.Our results demonstrate that AFLP is a strongtool for genetic dissection of the cotton genome.

REFERENCES1. Vos, P., Hogers, R., Bleeker, M., Reijans, M., Lee,

Vande T., Hornes, M., Fritjers, A., Pot, J.,Peleman, J., Kuiper, M., and Zabeau, M. (1995)Nucl. Acids Res. 23, 4407.

2. Lin, J.J. and Kuo, J. (1995) Focus 17, 66.3. Wagner, D.B., Furnier, G.R., Saghai-Maroof,

M.A., William, S.M., and Dancik, B.P. (1987)Proc. Natl. Acad. Sci. USA. 84, 2097.

4. Lin, J.J., Kuo, J., Ma, J., Saunders, J.A., Beard,H.S., MacDonald, M.H., Kenworthy, W., Ude,G.N., and Matthews, B.F. (1996) Plant Molec.Biol. Report 14:2, 156.

5. Feng, X., Saha, S., Soliman, K.M., Jaggernauth,M., and McMillian, C.E. (1995) InternationalPlant Genome Conference III at San Diego, p.264.

ACKNOWLEDGEMENTSWe acknowledge Drs. Allan Zipf and Peter

Gay for reviewing the manuscript. This work hasbeen supported by Cotton Incorporated andthe Capacity Building Program ofUSDA/CSRS.

Ethanol precipitation is frequently used toconcentrate DNA following enzymatic reactions.In addition, ethanol precipitation is used toremove salts or reaction products and oftenfollows phenol and chloroform extractions. Theuse of sodium salts for precipitating DNA iscommon in most laboratories, and the precipita-tion characteristics of DNA in sodium acetatewere recently re-examined (1). Ammoniumacetate, at a final concentration of 2.5 M, alsohas been used for ethanol precipitation of DNA.There are two instances when ammoniumacetate is used frequently: the removal ofunincorporated nucleotides following a DNAlabeling reaction (2) and the removal of proteinfrom DNA in mini-plasmid preparation protocols(3). The effectiveness of ammonium acetate forprecipitating DNA or removing proteins ornucleotides has not been previously reported.

A study was undertaken to determine the effectsof incubation time, incubation temperature, cen-trifugation time, and centrifugation temperatureon ethanol precipitation of DNA using ammoni-um acetate in place of sodium acetate. Thisstudy also quantitatively examined the efficiencyof removal of proteins and free nucleotides fromDNA by ethanol precipitation in the presence ofammonium acetate.

MethodsPreparation of DNA. Supercoiled pUC19 DNAwas digested with EcoR I and the 3′ recessedtermini were filled-in with dTTP, dGTP, dCTP,and [α-32P]dATP using the large fragment ofDNA polymerase I. Herring sperm DNA wassonicated to give an average size of 200–400 bp.

Ethanol Precipitations. All precipitations wereperformed in a 200-µl volume. Each tubecontained 1 µl of labeled DNA (1 ng),10 µl of herring sperm DNA (1 ng/µl, 10 ng/µl, or100 ng/µl), and 190 µl 10 mM Tris-HCl(pH 7.6), 1 mM Na2EDTA (TE).To precipitate theDNA, 100 µl of 7.5 M ammonium acetate (0.5volumes) and 750 µl of 95% ethanol(2.5 volumes) were added to the tubes. Thetubes were inverted 10 times to mix thecontents and incubated for the specified

period of time. The temperature of the ethanoladded to the vial was the same as theincubation temperature. The –70oC incubationtook place in a dry ice/ethanol bath, the –20oC incubation was in a –20oC ethanol bath,the 0oC incubation was performed on wet ice,and the 22oC incubation was at room tempera-ture. After the appropriate incubation time, thesolutions were centrifuged at 16,000 × g in afixed angle microcentrifuge at 4oC or roomtemperature. The supernate was removed andthe pellets were rinsed with 200 µl of 95%ethanol. The amount of radiation in the pelletswas determined by Cerenkov counting in ascintillation counter. Data points represent theaverages of at least two samples.

Removal of Free Nucleotide. The efficiency ofremoving free nucleotides was monitored byprecipitating nick-translated pUC19 inthe presence of unincorporated nucleotides.Supercoiled pUC19 was labeled using theNick Translation System with 65 µCi[α-32P]dATP. Acid precipitable counts and totalcounts were determined before and after twosequential ethanol precipitations.

Removal of Protein. The efficiency of removingprotein from DNA containing solutions wasmonitored using the 14C-labeled ProteinMolecular Weight Standards. Ammoniumacetate was added to a final concentration of 2.5M to solutions containing 50 µg/ml or 1,000µg/ml of BSA and 37.5 µg/ml 14C-labeledproteins. These protein solutions contained 1 µgof DNA in a 50 µl volume (20 µg/ml) and wereincubated for 0 or 30 min at 0oC or 22oC prior tocentrifugation at 16,000 × g for 15 min at roomtemperature. After the resulting supernate wastransferred to a fresh tube, ethanol was addedto a concentration of 70%, and the solutioncentrifuged at 16,000 × g for 15 min at roomtemperature. After each centrifugation, 2 µl ofthe supernate was removed and counted in 10ml of a scintillation cocktail.

RESULTSIncubation Temperature. The effect ofincubation temperature on the efficiency ofethanol precipitation of DNA in the presence of

Joseph CrouseDouglas AmoreseTechnical ServicesBethesda ResearchLaboratories

F O C U S 1 9 N U M B E R 1 17

ETHANOL PRECIPITATION: AMMONIUM ACETATE AS

AN ALTERNATIVE TO SODIUM ACETATE

C L A S S I C F O C U S A R T I C L E

ammonium acetate was determined by incubat-ing DNA solutions ranging from 0.005 µg/ml to 5µg/ml at –70oC, –20oC, 0oC, and 22oC for 0,10, and 30 min and overnight. In general, thetemperature of incubation (and ethanol) did nothave a dramatic effect on the recovery of DNAby ethanol precipitation for incubation timesranging from 0 to 30 min (table 1). The yield ofDNA incubated at -70oC was slightly reduced, inagreement with previous studies (1). The mostdramatic effect of temperature was seen whenthe ethanol precipitations were allowed toincubate overnight (figure 1 and table 1).Although incubation temperature had little effecton more concentrated DNA (5 µg/ml), DNAconcentrations ≤ 0.5 µg/ml showed a markedimprovement in percentage recovery at 0oC and22oC incubation temperatures.

Incubation Time. The effect of incubation timeon the efficiency of DNA precipitation was deter-mined at all four of the incubation temperaturesdescribed above. The same general trend wasobserved for all of the incubation times (table 1).For DNA concentrations of 5 µg/ml, the extend-ed incubation did not increase yields. Althoughthere appears to be little effect of incubationtime from 0 to 30 min, extended incubation didincrease the percentage of DNA precipitated inthe presence of 2.5 M ammonium acetate and70% ethanol for DNA concentrations of≤ 0.5 µg/ml.

Centrifugation Time and Temperature.Centrifugation of ethanol precipitates followingincubation is commonly performed at 4oC. Todetermine the effect of centrifugation timeand temperature, a 0.05 µg/ml DNA solutionwas incubated at 0oC for 10 min, andcentrifuged for 15 or 30 min at 4oC or roomtemperature. The recovery of DNA increasedwith the extended centrifugation time from37% to 57% for centrifugation at 22oC andfrom 22% to 39% for centrifugation at 4oC.In addition to achieving higher recoveries with30 min centrifugations, it is noteworthy thatrecoveries were improved by centrifugation atroom temperature.

Volume. The effect of volume on the recovery ofDNA precipitated with ammonium acetate andethanol was determined with DNAconcentrations of 0.05 µg/ml and 0.5 µg/mlin volumes of 20, 100, and 200 µl (figure 2). Byreducing the volume, the yield of precipitatedDNA at a given concentration was improved. To

18 F O C U S 1 9 N U M B E R 1

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temperature (°C)

–10 –20 0 20

Figure 1. Effect of incubation temperature on ethanol precipitation with ammoni-um acetate. All solutions were incubated overnight at the designated temperature andcentrifuged for 15 min at 22oC. DNA concentrations were 5 µg/ml (×), 0.5 µg/ml (�),0.05 µg/ml (�), and 0.005 µg/ml (�).

20

30

10

40

50

60

70

80

90

100

per

cen

t re

cove

ry

volume of solution (µl)

20 100 200

Figure 2.The effect of volume on the recovery of DNA by ethanol precipitation withammonium acetate. DNA at two concentrations, 0.5 mg/ml (�) and 0.05 mg/ml(�), was precipitated by the addition of 0.5 volumes of 7.5 M ammonium acetate and2.5 volumes of ethanol (at 0oC). Samples were centrifuged for 15 min at 22oC.

control for the ability to remove the supernatereproducibly, each pellet was monitored beforeand after a 70% ethanol rinse. No changes inthe amount of radioactivity associated with thepellet were noted upon washing.

Removal of Free Nucleotides. Two typesof experiments were performed to monitorthe removal of free nucleotides by ethanolprecipitation. In the first set of experiments,a labeled nucleotide (80,000 cpm of[α-32P]dATP in the presence of 20 µM colddNTPs) was added to varying concentrations ofDNA. Room temperature ethanol was addedto the samples, and they were immediatelycentrifuged for 15 min at room temperature.In the presence of 2.5 M ammonium acetateand 70% ethanol, approximately 7% of thefree nucleotides precipitated out of solutionscontaining 100 ng, 1 µg, or 5 µg of DNA in a50 or 200-µl volume (data not shown). Theamount of nucleotide precipitated was indepen-dent of the DNA concentration, and underthese conditions, greater than 90% of the DNAwas precipitated.

In the second set of experiments, pUC19(1 µg) was labeled by nick translation. The acidprecipitable and total counts were determinedafter nick translation and after the first and sec-ond ethanol precipitations in the presence ofammonium acetate or sodium acetate. The ratioof precipitable counts to total counts increasedfrom 68% to 87% after the first precipitation withsodium acetate and from 61% to 90% after thefirst precipitation with ammonium acetate (datanot shown). After two precipitations with eithersalt, the precipitable counts equaled the totalcounts, indicating that unincorporatednucleotides were removed efficiently in bothcases.

Removal of Proteins. Some rapid plasmidpreparation protocols use 2.5 M ammonium

acetate followed by centrifugation to removeprotein from the solution. To test the efficiency ofprotein removal from a DNA-containing solution,14C-labeled protein and BSA were mixed with a20 µg/ml DNA solution. This concentration is thesame as a standard nick translation or restric-tion digestion (1 µg of DNA in 50 µl). Ammoniumacetate was added, and the solution was mixedand incubated for 0 or 30 min at 0oC or 22oCprior to centrifugation at 22oC. In all cases,approximately 90% of the labeled proteinprecipitated out of solution (data not shown).The addition of ethanol to the supernateprecipitated the DNA but failed to precipitate the14C-labeled proteins that had remained in thefirst supernate. The same experiment was per-formed with labeled DNA and unlabeled protein.Again, a protein pellet was observed followingammonium acetate addition and centrifugation,but no labeled DNA was associated with thismaterial. Once again, following the addition ofethanol, greater than 90% of the DNA wasrecovered (data not shown).

DISCUSSIONWhen using ammonium acetate as describedhere for ethanol precipitation of DNA, theincubation temperature and length of incubationtime do not have an effect when DNA concen-trations are ≥5 µg/ml. However, at lower DNAconcentrations, incubation at 0oC and 22oCresulted in higher yields of DNA, especially asthe length of incubation increased to overnight.

Other factors that affected the recovery ofprecipitated DNA were the centrifugation speedand temperature, the length of centrifugation,and the volume of the solution. The recovery ofDNA was improved when solutions were cen-trifuged for 15 min at maximum speed in a fixedangle microcentrifuge at 16,000 × g comparedto a horizontal microcentrifuge at 8,800 × g(data not shown). All results reported here wereobtained using a fixed angle microcentrifuge. A

F O C U S 1 9 N U M B E R 1 19

Table 1. Effect of time and temperature on ethanol precipitation with ammonium acetate

Percent DNA recovered

DNA - 70oC - 20oC 0oC 22oC

Concentration 0 10 30 over- 0 10 30 over- 0 10 30 over- 0 10 30 over-min min min night min min min night min min min night min min min night

5 mg/ml 85 80 89 91 87 78 91 96 88 94 94 96 88 97 93 1000.5 mg/ml 62 46 52 50 57 52 65 83 60 58 63 98 62 64 65 920.05 mg/ml 28 29 30 32 35 33 49 69 36 33 38 92 47 40 36 870.005 mg/ml 25 27 38 33 41 38 49 72 37 33 39 86 40 35 38 85

Note: Data shown in bold type had ∆80% recovery.

greater percentage of DNA was also recoveredwhen samples were centrifuged at 22oC ratherthan 4oC and for 30 min in comparison to 15min. The volume of the solution also had aneffect on recovery, with much better recoveriesbeing observed for small volumes.

Recovery of DNA by ethanol precipitation canbe thought of as taking place in two steps:precipitation and collection of the precipitate.The precipitation appears to take place equallywell at temperatures ranging from –70oCto 22oC, and decreased temperature doesnot substitute for incubation time (table 1). Thecollection of the precipitate requirescentrifugation of the DNA through the 70%ethanol solution. At reduced temperature(4oC versus 22oC) this solution will be moreviscous, making it more difficult for precipitatesto reach the bottom of the tube. Longer centrifu-gation time improves the efficiency of recoverybecause it allows precipitates to reach thebottom of the tube. Likewise, smaller volumesdecrease the time required for the precipitate toreach the bottom of the tube and can improvethe efficiency of recovery.

For the removal of unincorporated nucleotidesby ethanol precipitation, ammonium acetate isslightly more efficient than sodium acetate. Ininstances where there is a substantial amountof unincorporated nucleotides (i.e., kinasereactions), the difference in the absolute amountof radio-activity can be considerable. However,when two successive precipitations are done,the difference in the efficiency between saltsis negligible. Dilution of the DNA solutions priorto precipitation did not reduce the amount ofunincorporated label that precipitated.

When ammonium acetate is added to aconcentration of 2.5 M, proteins can beefficiently removed by centrifugation of thesample prior to the addition of the ethanol.Reduced temperature and/or increasedincubation times did not have an effect on theprecipitation of the 14C-labeled proteins.Experiments with labeled DNA indicated thatthe DNA was not precipitated or trappedduring the protein removal. The DNA canthen be recovered from the supernate byethanol precipitation.

In general, ethanol precipitations with ammoni-um acetate can be performed by makingthe DNA-containing solution 2.5 M inammonium acetate, adding 2.5 volumes ofroom temperature ethanol, and centrifugingimmediately at 16,000 × g for 15 min at roomtemperature. Since DNA is recovered moreefficiently in reduced volumes and contaminantssuch as unincorporated nucleotides areremoved just as efficiently at high or low DNAconcentrations, there is no need to dilutesamples to greater than 50 µl prior to theaddition of salt and ethanol. A 70% ethanolwash is recommended after precipitations toremove residual salt and to dilute the smallamount of liquid that is difficult to remove fromthe pellet.

References:

1. Zeugin, J.A. and Hartley, J.L. (1985) FOCUS 7:4, 1.2. Maxam, A.M. and Gilbert, W. (1980) Methods

Enzymol. 65, 499.3. FOCUS (1982) 4:3, 12.

20 F O C U S 1 9 N U M B E R 1

F O C U S 1 9 N U M B E R 1 21

22 F O C U S 1 9 N U M B E R 1

VOLUME 18 INDEX

AUTHOR INDEX

Ally, Abdul H., 19Anderson, Dina, 6, 10Biddle, William C., 62Blakesley, Robert, 19, 73Boger, Hinrich, 57Bowen, Heather, 27Boycott, Kym M., 74Brow, Mary Ann D., 2Burn, T.C., 31Caligiuri, Michael A., 62Castillo, Theresa, 59Chaplin, David D., 25Ciccarone, Valentina, 6, 10,

43, 45Connors, T.D., 31Crouse, Joe, 17Curran, M.E., 31Dadey, Barbara M., 62Dahlberg, James, 2Daley, John P., 62Darfler, Marlene M., 15, 70Dougherty, Catherine, 15El-Badry, Osama M., 70Evans, Krista, 40Fors, Lance, 2Fox, Donna K., 33Furth, Priscilla A., 57Garcia-Assad, Nacyra, 47Goldsborough, Mindy D.,

15Grotelueschen, Jeff, 2Gruss, Peter, 57Harris, Ray, 10Hartley, James L., 27Hawley-Nelson, Pamela, 40,

43Heisler, Laura, 2Henrich, Curtis J., 13Hughes, A. John, Jr., 33Jessee, Joel, 6, 40Keating, M.T., 31Kozyavkin, Sergei, 2Krishnan, B. Rajendra, 25Kuo, Jonathan, 47, 68Landes, G.M., 31Lin, Jhy-Jhu, 47, 68Longo, Mary, 17Ludwig, Christian, 38Lundstrom, Kenneth, 53

Lyamichev, Natasha, 2Lyamichev, Victor, 2Ma, Jin, 47, 68Macdonald, Ann S., 6Mertz, Lawrence M., 22,

75Millholland, J.M., 31Nathan, Margret, 33Oldenburg, Mary, 2Olive, D. Michael, 2Polayes, Deborah, 10, 50Rashtchian, Ayoub, 33Sadava, David, 59Schifferli, Kevin P., 6, 13,

40, 45Schraml, Peter, 38Schuster, David M., 33Shen, J., 31Shipman, Rob, 38Sitaraman, Kalavathy, 22Smith, Lloyd, 2Solus, Joseph, 19Splawski, I., 31St. Onge, Luc, 57Van Raay, T.J., 31Wang, Q., 31Westfall, Barry, 33Whitford, William G., 75Wysocki, Michelle G., 62Xu, Lisha, 73Young, Alice C., 25

SUBJECT INDEX

Cell Adhesion

anti-integrin Abs for charac-terization of cell-ECMadhesion, 13

Cytogenetics

in situ hybridization formouse chromosomes, 15

nonradioactive in situhybridization, 70

cDNA

isolation of specific genesfrom cDNA libraries(GENETRAPPER System), 31

isolation of 5« ends with 5«RACE, 33

isolation of 5« ends withPCR, 38

DNA

mutation detection with theCFLP System, 2

precipitation

with polyethylene glycol(PEG) of small DNA frag-ments, 27

purification

DNAZOL Reagent, forgenomic DNA

from gram-positive bacteri-um, Bacillus, 73

from whole blood, 19, 22

GLASSMAX DNA IsolationSystem, for genomic DNA,22

Electrophoresis

resolution of high molecularweight DNA in agarose, 17

SEPARIDE Gel Matrix for smallDNA fragments, 74

Hybridization

fluorescent, in situ (FISH) ofmouse chromosomes, 15

in situ, nonradioactive, 70

probe

biotinylated oligonucleotide,70

Mapping

exon trapping, 31

fingerprinting plant DNAusing AFLP, 68

genotyping using humangenomic DNA, 19

Media

serum-free

AIM-V for human SCLC orH69 cells, 59

Keratinocyte SFM, 43

STEMPRO-34 SFM for humanhematopoietic progenitorcells, 62

Modifying Enzymes

ELONGASE Enzyme Mix forPCR, 19, 22, 33

TEV protease, 10, 50

PCR

AFLP for plant fingerprinting,68

5« RACE System Version 2.0,33

isolation of the 5« end ofcDNA, 38

long DNA templates(ELONGASE System), 19,22, 33

SEPARIDE Gel Matrix for elec-trophoresis, 74

Plant Biotechnology

DNA isolation fromArabidopsis, 22

hygromycin B, antibiotic, 47

plant DNA fingerprinting(AFLP), 68

Protein Expression Systems

BAC-TO-BAC BaculovirusSystem, 10

multiplicity of infection val-ues, 75

Cre expression vectors, 57

PROEX HT System forprokaryotic expression, 50

effect of E. coli strain on pro-tein yield, 50

Semliki Forest Virus (SFV)System for mammalian pro-tein expression, 53

Tet-regulated expression, 57

Proteins

green fluorescent protein(GFP), 40

purification using Ni-NTAresin, 10, 50

TEV protease for removal ofaffinity tags, 10, 50

RNA

biotinylated probe for in situRNA detection, 70

transfection using cationiclipids, 6

Sequencing

T7-end primer for DELETIONFACTORY vectors, 25

Transfection

cationic lipid reagents

human keratinocytes usingLIPOFECTAMINE Reagent,43

monitor in living or fixed cellsby green fluorescent protein(GFP), 40

suspension cell lines usingDMRIE-C Reagent, 45

Transfection ReagentOptimization System forlipid selection, 6

hygromycin B for selection,47

Presentations Will Include:■ Baculovirus expression■ Mammalian viral expression■ Non-viral expression such as

electroporation and liposomebased transfection

Symposia: “GibberllinControl in Plants:

And the Winners Are

Congratulations to the 1996 FBS Sweepstakes winners!The Grand Prize, a Trek 850 SHX off-road bike, was awarded to: Dr. Nancy Freitag of the Public Health Research Institute in New York City.Five first prizes, Braun espresso machines, were awarded to: Ms. Monika Rydinski of Abbott Laboratories, Dr. Kiao-Kum Zhang of La Jolla CancerResearch Foundation, Ms. Wendy K. Alperin-Lea of Tulane . Medical School, Mr. Christopher K. Yunker of Henry Ford Hospital, and Dr. Sukadev Lavuof the FDA.Ten second prizes, computer screen savers, were awarded to: Dr. Raymond Chan of Salk Institute, Dr. Richard L. Garber of PathoGenesis Corp., Ms.Laura C.Tan of Ohio State U., Dr. Victor Stollar of U. of Medicine and Dentistry of NJ, Dr. Christine O’Day of Oridigm Corp., Ms. Betty E. Caywoodof U. of Kentucky, Dr. Sally Short of Alaska State Fish and Game, Dr. Joseph W. Basler of Washington U./Jewish Hospital, Dr. Ed Ambruzs of U. ofSouth Carolina, and Mr. David Ammar of U. of Michigan.

F O C U S 1 9 N U M B E R 1 23

A N N O U C E M E N T S

Registration and general information:

Hogg-Robinson,c/o Glaxo Wellcome,Stockley Park WestUxbridge, MiddlesexUB11 1BT, England.TEL: 0044 0181 990 2413,FAX: 0044 0181 990 4342Web site: http://www.glaxowellcome.co.uk/

PLANT GROWTH REGULATION

SOCIETY OF AMERICA CONFERENCE

August 8–12 1997

CENTENNIAL OLYMPIC CITY OF ATLANTA, GA.

For information, contact:

Dr Joyce Latimer, Program Chair,Department of Horticulture, GeorgiaStation, Griffin GA 30223-1797,Telephone 770-228-7398,e-mail: jlatime@gaes.griffin.peachnet.edu.

Mlolecular Biology to CommercialApplication” and “BrassinosteroidsPart II”

T R A N S I E N T G E N EE X P R E S S I O N I N A N I M A L CELLS

Jersey, UKMay 11–14, 1997