MATERIALS AND METHODSDNA/Oligonucleotides
The starting random DNA pool(RAN) was synthesized by Invitrogen(Burlington, Ontario, Canada) (5
). The corresponding left andright arms were LEFT (5
) andBioRIGHT (5
). The 5
-endbiotinylated oligonucleotides were usedto bind complementary strands, usingBioMag magnetic particles (PerSeptiveBiosystems, Framingham, MA, USA).During preparative hybridization, theleft and right arms were blocked bycLEFT (5
) and RIGHT (5
).These oligonucleotides are termed“blockers” in the text. The following ge-nomic DNAs were used to produceGOLs: Adenovirus DNA Type 2, (Invit-rogen),
DNA cI857 ind1 Sam 7 (NewEngland Biolabs, Beverly, MA, USA),pBluescript
(Stratagene, SanDiego, CA, USA). The human HeLaDNA we used in one of our controls isfrom BD Biosciences Clontech (PaloAlto, CA, USA).
Blotting Genomic DNA
The genomic DNA was denatured2–3 min at 95°C and cooled on ice. De-natured genomic DNA (100 ng) wasblotted on the nylon membrane (Hy-bond-N
; Amersham Biosciences, Pis-cataway, NJ, USA), dried for 2 min ona hot plate, and exposed to UV light for8 min. The prehybridization was donefor a minimum of 30 min in the hy-bridization buffer (7% SDS, 0.25 MNa
, pH 7.4, 1 mM EDTA, and1% BSA).
Hybridization and Washing of theStarting Random Pool
The preparative hybridization be-tween random core (20N) and targetedDNA was done with 10 pmol startingrandom pool (RAN). The random poolwas pre-mixed with 100 pmol (10 timesmore than RAN) of LEFT and RIGHTblockers to exclude cross-hybridizationof left and right arms with genomicDNA. The oligonucleotide mixture washeated at 95°C, cooled down to roomtemperature, and added to the hybridiza-tion buffer. The hybridization was doneovernight at 50°C. The membranes werewashed several times, first in 6
SSCand then in 2
SSC, at the same temper-ature as the hybridization was done.
Generating OligonucleotideLibraries by PCR
The dot containing the genomicDNA and bound probes was cut out of the nylon membrane (radius of 2–4mm), soaked in 100
L water, and heat-ed to 95°C for 1–2 min. The solutioncontaining the denatured probe (origi-nally RAN) was then collected andpassed through a Sephadex
G-50 col-umn to eliminate salts and SDS. ThePCR was prepared under standard con-ditions, typical for SELEX-like ampli-fication of DNA (4,13). The RIGHT 5
-end biotinylated primer of the sensestrand (the one that did not hybridizewith genomic DNA) and LEFT primerof the antisense strand were used in thereactions. The temperature cycles were53°C, 72°C, and 95°C, each for 30 s,repeated 20 times.
Probe Labeling and Hybridization
Before labeling, the PCR mixtureswere passed through Sephadex G-50columns. PCR product (100–200 ng)was labeled with 50 pmol
P]ATP(6000 Ci/mmol; I.C.N. Pharmaceuti-cals, Irvine, CA, USA). The totalamount of probe radioactivity was300000 cpm. The probe was added to0.5 mL hybridization buffer. The blot-ting of genomic DNA was done as de-scribed above. Hybridizations weredone overnight at 50°C. The nylonmembranes were washed as describedabove and exposed onto Kodak
X-OMAT film (Eastman Kodak,Rochester, NY, USA).
GOL Labeling and AnalyticalHybridization
The generated GOLs are tested us-ing (
) the original genomic DNA fromwhich they were selected (positive con-trol) and (
) using the unrelated ge-nomic DNA (negative control). TheGOL labeling, hybridization, and probewashing were done as described, ex-cept that the hybridization time wasshorter (60 min).
Southern Blot Hybridization
Electrophoresis was performed in a1% agarose gel with TBE buffer (80mM Tris adjusted to pH 8.0 with boricacid, 2 mM EDTA) and stained withethidium bromide. One microgram of
DNA, 300 ng aden-oviral DNA, and 1
I-di-gested human HeLa DNA were run onthe gel according to specifications (allrestriction enzymes used in this work were purchased from New England Bi-olabs). For Southern hybridization,DNA was transferred to nylon mem-branes by capillary blot procedurefollowing manufacturer’s recommen-dations (Amersham Biosciences). Hy-bridization was performed as describedabove with adenoviral GOL. Autoradi-ographic exposure (using X-OMATfilm) was done at room temperature fora few hours. Stripping was achieved bypouring a boiling 1% SDS solutionover the nylon membrane.
Subtractive Enrichment of GOL
The tester GOL (mixed GOL) wasprepared from equimolar mixtures of the two genomes (adenovirus type 2and
). The driver GOL was producedfrom the
genome only. The produc-tion of sense strand (the one that did nothybridize with genomic DNA) wasdone using 5
-end biotinylated primerin PCR. After denaturing the PCRproduct, the biotinylated sense strandwas bound to streptavidin magneticparticles (200
g, binding capacitygreater than 200 pmol biotinylatedoligonucleotides; Biomag magneticparticles) and bound using a magnet.The complementary antisense strandwas discarded with the liquid phase.The mixed antisense tester GOL (
andadenovirus DNA) was produced in thesame way. This time, the supernatantwith the antisense, non-biotinylatedstrand was hybridized overnight at50°C with 10 times molar excess of dri-ver
sense strand attached to the mag-
Vol. 33, No. 4 (2002)BioTechniques 3