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Generation of hybridization probes for diagnostics

Generation of hybridization probes for diagnostics

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Published by ibrukner1107
Method for generating hybridization probes
DNA RNA hybridization diagnostic chip array fast simple cheep fundamental
Method for generating hybridization probes
DNA RNA hybridization diagnostic chip array fast simple cheep fundamental

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Published by: ibrukner1107 on Oct 28, 2008
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02/17/2011

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Ivan Brukner, Guy A.Tremblay, and Bruno Paquin
Université de Montréal,Montréal, Québec, Canada
ABSTRACT
 Here we describe a process for the gen-eration of oligonucleotide libraries repre-sentative of a given nucleic acid. Starting from a random pool of DNA oligonu-cleotides, the technique selects only thosethat hybridize to the nucleic acid template.This selection yields a highly specific li-brary that represents an oligonucleotide im-age of the chosen template. The novel quali-ty of this approach is the generation of amplifiable oligonucleotide probes that areof unique length and are easily subjected todifferential selection. Here we apply thistechnique to produce different genomicoligonucleotide libraries and show that these genomic oligonucleotide libraries donot cross-hybridize. Differential selection of these genomic oligonucleotide libraries produces oligonucleotides that can be used in the identification, characterization, and isolation of nucleic acids.
INTRODUCTION
A number of diagnostic methods thatinvolve nucleic acid hybridization havearisen in recent years. Most of them aredesigned to provide qualitative informa-tion about the presence of a specific se-quence motif in a complex analyticalmixture of nucleic acids and use a de-tection system based on PCR and/orDNA chip hybridization technologies(5,6,12,15,16). For these technologies,diagnostic oligonucleotides constitutean essential part of the detection system.These oligonucleotides are primarilychosen based on the sequence data of the nucleic acids to be detected. In spiteof the power of hybridization to identifycorrectly a complementary strand, itdoes have certain limitations. For exam-ple, the difference in stability between aperfectly matched complement and acomplement mismatched at only onebase can be as little as 0.5°C (7), a factthat constitutes a fundamental limitationto the power of DNA hybridization forspecific identification of a cognatestrand. Therefore, the diagnostic powerof any chosen oligonucleotide must bevalidated on an analytical mixturewhose sequence context is not totallyknown. The problem of adequate probeselection remains time and labor con-suming at a moment when the growingcomplexity of detection systems basedon oligonucleotide technologies re-quires a fast selection of a large numberof suitable 20- to 25-mers.The mismatch-containing oligonu-cleotide probes have better power of discrimination between two sequencemotifs having just one nucleotide dif-ference (7). Also, better mismatch dis-crimination occurred with 9-merprobes containing an additional internalmismatch (9). Additional mismatchedbases located in the 3
-terminal regionsof the specific primers are a way of im-proving the switching characteristics of the primer extension reactions duringSNP detection (2,17). Therefore, differ-ential selection of redundant oligonu-cleotide probes now offers the genera-tion of mismatch-containing probeswith enhanced discrimination poweramong similar sequence motifs.A simple method for generating alarge number of specific probes thatcould potentially improve the signal-to-noise ratio of nucleic acid detectionsystems would prove valuable. Rapidoligonucleotide library manipulationsthat facilitate the duplication, subtrac-tion, and merging of libraries could beused for that purpose. Here we reportthe development of a method called thegenomic oligonucleotide library(GOL). A random pool of DNAoligonucleotides is hybridized to a tem-plate genomic DNA. Thus, the GOL isan oligonucleotide reflection of thetemplate DNA. The GOL is homoge-neous in length and amplifiable byPCR, and as such can be maintainedvirtually forever. Rather than relying ona rational selection of probes based onsequence data, this method depends ona more pragmatic selection based onexperimental signal-to-noise discrimi-nation criteria. This makes the GOLmethod potentially useful for applica-tions that require the rapid generationof a large number of oligonucleotideprobes for nucleic acid detection. Usinga random introduction of mismatches,the selected probes acquired an en-hanced power of detection of small dif-ferences between sequence motifs.
Research Report
2BioTechniquesVol. 33, No. 4 (2002)
DRAFT
Generation of Amplifiable Genome-SpecificOligonucleotide Probes and Libraries
 BioTechniques 33:__-__ (October 2002)
 
MATERIALS AND METHODSDNA/Oligonucleotides
The starting random DNA pool(RAN) was synthesized by Invitrogen(Burlington, Ontario, Canada) (5
-GC-CTGTTGTGAGCCTCCTGTCGAA-N20-TTGAGCGTTTATTCTTGTCT-CCC-3
). The corresponding left andright arms were LEFT (5
-GCCTGT-TGTGAGCCTCCTGTCGAA-3
) andBioRIGHT (5
-BioGGGAGACAAGA-ATAAACGCTCAA-3
). 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
-TTCGACAGGAGGCTCA-CAACAGGC-3
) and RIGHT (5
-GG-GAGACAAGAATAAACGCTCAA-3
).These oligonucleotides are termed“blockers” in the text. The following ge-nomic DNAs were used to produceGOLs: Adenovirus DNA Type 2, (Invit-rogen),
λ 
DNA cI857 ind1 Sam 7 (NewEngland Biolabs, Beverly, MA, USA),pBluescript
®
II SK
+
(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
2
HPO
4
, 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
µ
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
γ 
[
32
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 (
i
) the original genomic DNA fromwhich they were selected (positive con-trol) and (
ii
) 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 
 Bst 
EII-digested
λ 
DNA, 300 ng aden-oviral DNA, and 1
µ
g
 Alu
I-
 Hpa
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
λ 
). The driver GOL was producedfrom the
λ 
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
µ
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 (
λ 
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
λ 
sense strand attached to the mag-
Vol. 33, No. 4 (2002)BioTechniques 3
DRAFT
 
netic beads. The hybridization bufferwas the same as described above butwithout SDS. After removing the frac-tion bound to the magnetic beads, therest of the mixture was used in the ana-lytical hybridization step.
RESULTSInitial Complexity of GOL Probes
The starting random pool of oligonucleotides contains 4
20
(i.e.,10
12
) different 20-mers. Such a mixturecontains a pool of 10
11
extra sequencemotifs, which are not present in the hu-man genome (10
12
–10
9
> 10
11
). Thispool of oligonucleotides is used to gen-erate mismatch-containing probes withthe better specificity/discriminationpower over classical probes. Oneshould note that if the length of the ran-dom sequence motif (N) were longer,then a higher number of mismatch-con-taining motifs would exist in a GOL.Increasing the length of the random se-quence motif from 20N to 25N wouldproduce 10
15
extra sequence motifs,which could not find their full-matchcomplementary sequences, even in themost complex genomes. However, theincrease in the length of N would allowthe generation of additional mis-match/match combinations. That leadsto better discrimination between twosequence motifs that differ by only onenucleotide. Therefore, the discrimina-tion power of detection (specificity)will augment with the length of randommotif of GOL oligonucleotides. A pro-cedure for generating GOLs is de-scribed in detail in the Materials andMethods section.We used blockers to avoid hy-bridization of the flanking arms to thetargeted genome, and this step wasfound to be critical to achieve specifici-ty (WO/0043538; Université de Mon-tréal). The stringency of hybridizationconditions eliminates unbound 20-mers, leaving the specific oligonu-cleotides bound to the membrane viahybridization of the random core to thegenome. This ensemble of selectedoligonucleotides constitutes the GOL.One should note that the startingrandom pool of oligonucleotides(10–20 pmol) contained about eightcopies of each sequence motif duringthe first hybridization step. The numberof copies of each particular 20-mer pre-sent in the random mixture was smallerthan the number of genome copies usedin this procedure.
Specificity and Distribution of GOLProbes
Figure 1 shows that GOLs are ableto discriminate genomes with complex-ities of 10
3
–10
4
. The starting randompool of probes binds to all threegenomes equally (Figure 1, row 1). Af-ter one round of selection, the GOL canhybridize specifically to a single target-ed genome (Figure 1, rows 2, 3, and 4).The GOL can also be selected against amixture of two genomes, and the speci-ficity is conserved for both genomes(Figure 1, row 5).A Southern blot was performed todocument the distribution of aden-ovirus GOL probes along the genome(Figure 2). There was no apparentcross-hybridization of adenovirus GOLto either HeLa or
λ 
DNA (Figure 2b,lanes 1, 4, 5, and 6). The intensity of ra-dioactive signal over adenoviralgenome generated by adenovirus-spe-cific GOL was linearly increasing withthe DNA fragments’ length (Figure 3).Therefore, one could deduce a uniformdistribution of GOL throughout the ge-nomic DNA.We then selected only the subset of 
Research Report
4BioTechniquesVol. 33, No. 4 (2002)
DRAFT
Figure 1. Dot blot hybridization of GOL tar-geted against different genomes.
The first rowrepresents the dot blot hybridization of randomprobes with the specified genomic DNA (aden-ovirus, pBluescript, and
λ 
). The other rows areanalytical dot blot hybridizations of selectedGOLs with the corresponding genomes. The lastrow shows dot blot hybridization of mixed aden-ovirus and
λ 
-selected GOLs. The procedures of preparative and analytical hybridization are de-scribed in the Materials and Methods section.
Figure 2. Specificity and probe distribution of GOL generated from the adenoviral genome.
(A)The corresponding genome and adenoviral DNA were run on a 1% agarose gel stained with ethidiumbromide. The type of restriction enzyme and DNA are indicated on the top of each gel lane: 1,
λ 
 Bst 
EII;2, adenovirus; 3, adenovirus
Kpn
I; 4, HeLa DNA
 Alu
I +
 Hpa
I; 5, adenovirus and HeLa DNA
 Alu
I +
 Hpa
I; 6, adenovirus
Kpn
I and HeLa DNA
 Alu
I +
 Hpa
I. (B) Southern hybridization of the same gel usingadenovirus GOL as a hybridization probe (see Figure 1, row 2). Note that under those conditions, therewas no cross-hybridization with either
λ 
or human DNA. (C) The same membrane was stripped and re-hybridized with a GOL directed against a 3648-bp restriction fragment. This subset of adenovirus GOLwas prepared by cutting the membrane corresponding to the 3648-bp band from a similar Southern blotand re-amplified by PCR as described in the Materials and Methods section. Only those members of ade-noviral GOL that were selected from the particular adenoviral restriction fragment (3645 bp) hybridizedback to the same fragment and only to the fragment from which they were selected. Obviously, theseGOL probes are not promiscuously hybridizing to other fragments of the adenoviral genome. Thus, weshow that GOL specificity could be additionally increased by controlling the choice of targeted DNAfragments in the next round of selection.

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