Cell, Vol. 5, 301-310,
`
`July
`
`1975, Copyright@1975
`
`by MIT
`
`Using DNA
`Nucleic Acid Hybridization
`Covalently Coupled
`to Cellulose
`
`and
`
`E. Noyes
`Barbara
`R. Stark
`George
`Department
`of Biochemistry
`Stanford
`University
`School
`Stanford,
`California
`94305
`
`of Medicine
`
`Summary
`
`RNA and DNA
`linking
`for
`a method
`We describe
`through
`a di-
`cellulose
`to finely
`divided
`covalently
`primarily
`with
`reacts
`aryl amine,
`which
`azotized
`residues
`of single
`and
`uracil
`(thymine)
`guanine
`of coupling
`and high
`The high efficiency
`strands.
`of the cellulose
`for nucleic
`acid make pos-
`capacity
`sible
`a product
`with as much
`as 67 pg of nucleic
`acid per mg of cellulose.
`The product
`is especially
`suitable
`for hybridization
`experiments
`where
`very
`low backgrounds
`are
`important,
`and
`it is stable
`in
`99%
`formamide
`at 60°C
`so
`that hybridized
`nucleic
`acid
`can
`be
`recovered
`easily.
`Full
`length
`linear
`Simian Virus 40 (SV40) DNA, produced
`by cleavage
`of SV40(1) DNA with Sl nuclease,
`can be coupled
`to diazo
`cellulose
`with an efficiency
`of 60-90%,
`and
`is effective
`in hybridization
`experiments
`with SV40
`DNA,
`complementary
`RNA
`synthesized
`in vitro
`from SV40(1)
`DNA with E. coli RNA polymerase,
`and
`the SV40-specific
`fraction
`of
`total RNA
`from
`SV40-infected
`and
`transformed
`cells.
`In these
`ex-
`periments
`an excess
`of cellulose-bound
`DNA was
`used, and
`the efficiency
`of hybridization
`was about
`90% when
`ribonuclease
`treatment
`of
`the hybrids
`was omitted.
`
`Introduction
`
`in solution
`formed
`hybrids
`or DNA-RNA
`DNA-DNA
`for single
`specific
`can be detected
`with a nuclease
`strands
`or isolated
`by chromatography
`on hydroxy-
`apatite.
`(For
`example,
`with SV40 DNA, see Sam-
`brook,
`Sharp,
`and Keller,
`1972; Hansen,
`Pheiffer,
`and
`Hough,
`1974.)
`DNA-DNA
`reannealing
`competes
`with DNA-RNA
`hybridization
`in solution,
`especially
`if the DNA
`is in excess. Although
`this dif-
`ficulty
`can be eliminated
`by
`immobilizing
`the DNA
`on nitrocellulose
`filters
`(Gillespie
`and Spiegelman,
`1965) or
`in agar
`(Bolton
`and McCarthy,
`1962; Han-
`sen et al., 1974),
`variable
`loss of DNA
`from
`the
`filter
`and high background
`levels
`in agar complicate
`the
`results.
`(For examples
`of the use of filters with SV40
`DNA,
`see Westphal
`and Dulbecco,
`1968;
`Haas,
`Vogt, and Dulbecco,
`1972; Holzel and Sokol,
`1974).
`Loss
`from
`the
`filter
`can be particularly
`significant
`in experiments
`designed
`to quantitate
`low
`levels of
`specific
`RNA within
`a
`larger
`heterogeneous
`pool,
`since any RNA which
`hybridizes
`to DNA
`in solution
`is not detected.
`
`we have devel-
`problems,
`these
`To circumvent
`using DNA co-
`oped a new method
`for hybridization
`valently
`linked
`to cellulose.
`Shih and Martin
`(1974)
`coupled
`SV40 DNA
`to cellulose
`powder
`through
`the
`terminal
`phosphate
`groups
`using
`a water-soluble
`carbodiimide
`according
`to Gilham
`(1971).
`Poonian,
`Schlabach,
`and Weissbach
`(1971)
`and Arndt-Jovin
`et al. (1975)
`have coupled
`DNA
`to agarose
`activated
`with CNBr. These preparations
`have been used suc-
`cessfully
`in affinity
`chromatography,
`but
`their appli-
`cation
`to sensitive
`analytical
`hybridization
`may be
`limited
`because
`of the
`large amount
`of support ma-
`terial
`used.
`Residual
`positive
`charges
`on
`the
`agarose,
`generated
`as a consequence
`of CNBr acti-
`vation, might
`also
`contribute
`to high
`background.
`The coupling
`procedure
`we describe
`is a modifica-
`tion of
`the one developed
`by Gurvich,
`Kuzovleva,
`and Tumanova
`(1961)
`for
`linking
`proteins
`covalently
`to
`finely
`divided
`aminobenzyloxymethyl
`cellulose.
`and
`The high
`capacity
`of the cellulose
`the
`facility
`with which
`the reaction
`can be performed
`are major
`advantages
`of the method.
`The DNA-cellulose
`can
`be used analytically
`to detect
`low
`levels of a specific
`nucleotide
`sequence
`within
`a larger
`heterogeneous
`pool
`as in
`filter hybridization
`techniques,
`or
`it can
`be used preparatively.
`
`Results
`
`to
`
`to Cellulose
`of DNA
`Linkage
`Covalent
`covalently
`DNA
`can be
`linked
`stranded
`Single
`finely
`divided
`m-aminobenzyloxy-
`reprecipitated,
`after
`the primary
`aryl amino
`groups
`methyl
`cellulose
`have
`been
`diazotized
`as described
`by Miles
`and
`Hales
`(1968).
`In the
`first experiment
`of Table 1, only
`8% of the
`input SV40 DNA was coupled
`to the diazo
`cellulose
`in borate
`buffer
`under
`conditions
`used
`successfully
`by Miles and Hales
`(1968)
`for
`the cou-
`pling
`of proteins.
`The DNA
`in this experiment
`had
`been
`denatured
`at pH 12
`immediately
`before
`the
`diazo
`cellulose
`was added,
`but was extensively
`re-
`natured
`during
`coupling
`at high DNA concentration
`at 4°C
`(pH 8). However, when
`the DNA
`is kept dena-
`tured by doing
`the coupling
`in 70% or 80% dimethyl-
`sulfoxide
`(DMSO), more
`than 80% of the
`input
`can
`be added
`to
`the cellulose
`routinely.
`No DNA
`re-
`mains
`stably
`bound
`to amino
`cellulose
`which
`has
`not been
`diazotized.
`Experiments
`2-4
`of Table
`1
`show
`that
`the efficiency
`of the
`reaction
`depends
`on
`the concentration
`of diazo cellulose,
`but
`is relatively
`independent
`of the concentration
`of DNA.
`In experi-
`ments using
`from 20-800
`pg of SV40 DNA at con-
`centrations
`from 100-300
`pg/ml,
`about
`90% of the
`DNA can be linked
`covalently,
`provided
`that
`the cel-
`lulose
`concentration
`in
`the
`reaction
`mixture
`is at
`least 8-10 mg
`(dry weight)/ml.
`The percentage
`of
`DNA coupled
`decreases
`to 40-50%
`when
`the cellu-
`
`Page 1 of 10
`
`HOLOGIC EXHIBIT 1007
`Hologic v. Enzo
`
`

`
`Cell
`302
`
`Table
`
`1. Coupling
`
`of DNA
`
`to Diazo Cellulose
`
`Experiment
`
`1
`
`SV40 DNA
`
`Sonicated
`(6-7s)
`
`linears
`Sl
`(16%
`
`linears
`Sl
`(1’3s)
`
`linears
`Si
`(16s)
`
`4
`
`Suffer
`
`Borate
`
`70% DMSO
`
`70% DMSO
`
`70% DMSO
`80% DMSO
`
`80% DMSO
`
`DNA
`(Wml)
`
`245
`
`90
`
`280
`
`100
`
`80% DMSO
`
`200
`
`Diazo Cellulose
`O-w/ml)
`
`% DNA
`Coupled
`
`Temperature
`(6
`
`12
`
`9.5
`
`10
`
`(NH2-cellulose)
`
`a
`11
`
`22
`
`2.2
`
`0.5
`
`2.2
`
`77
`
`a
`a7
`
`0
`
`80
`88
`
`99
`
`43
`
`4
`
`45
`
`5.1
`
`4
`
`4
`
`25
`
`25
`
`4
`
`was suspended
`(16s)
`linear size
`length
`full
`to
`with Sl nuclease
`or digested
`size of 6-7s
`to an average
`sonicated
`SV40(1) DNA,
`32P-labeled
`alone was
`denatured
`at pH 12, placed
`on
`ice,
`DNA
`in borate
`additional
`buffer
`or DMSO.
`in 0.2 M borate
`buffer
`(pH a), and diluted with
`and
`readjusted
`to pH 6 immediately
`before
`addition
`to cellulose.
`DNA
`solutions
`were
`added
`to diazotized
`cellulose
`in small
`tubes,
`and
`the suspensions
`were
`stirred
`continually
`for 46 hr. The percentage
`of DNA coupled
`was determined
`from
`3zP stably
`bound
`to
`the cellulose
`followina
`extensive
`washina
`as described
`in the
`text.
`
`the
`and
`to 2 mg/ml,
`is reduced
`lose concentration
`if the cellulose
`con-
`is very poor
`coupling
`efficiency
`centration
`is reduced
`further. When E. coli DNA or
`salmon
`sperm DNA
`(200 pg/ml)
`are coupled
`to cel-
`lulose
`(10 mg/ml),
`about
`60% of
`the DNA
`remains
`stably
`bound.
`This decrease
`in the percent
`of DNA
`which
`couples
`compared
`to SV40 DNA
`is not under-
`stood. E. coli
`tRNA
`(2 mg/ml)
`coupled
`to diazo
`cel-
`lulose
`(2 mg/ml)
`in 70% DMSO with 47% efficiency.
`At cellulose
`concentrations
`above
`8-10 mg/ml,
`the
`reaction
`is complete
`within
`24 hr. At lower
`con-
`centrations,
`48 hr are
`required
`to achieve maximum
`coupling.
`The
`time course
`and extent
`of
`the cou-
`pling
`reaction
`are about
`the same at 4°C or 25°C.
`The amount
`of DNA stably bound
`to the cellulose
`is determined
`after
`thorough
`washing
`with
`80%
`DMSO and 0.1 x standard
`saline
`citrate
`(SSC, 0.15
`M NaCI-0.015
`M sodium
`citrate). When DNA dena-
`tured before
`addition
`of cellulose
`is coupled
`without
`DMSO,
`as much as 50% of the DNA
`initially
`bound
`can be removed
`by washing with 80% DMSO or 99%
`formamide.
`However,
`less
`than 10% of bound
`DNA
`is lost during
`such washing
`after
`reaction
`in DMSO,
`and most of this material
`is released
`in the
`low salt
`washes.
`The percentage
`of polynucleotide
`found
`linked
`covalently
`to
`the
`cellulose
`was
`same
`the
`whether
`determined
`using
`radioactively
`labeled
`DNA, by spectral
`analysis
`of acid
`hydrolysates
`of
`the DNA-cellulose,
`or by spectral
`analysis
`of
`the
`DNA which
`did not couple.
`
`Table 2. Coupling
`
`of Nucleotide
`
`Hamopolymers
`
`to Diazo Cellulose
`
`Polymer
`(A,A,,/ml)
`
`Concentration
`
`% Couoled
`
`SV40 DNA
`
`POlYW
`
`Poly(dT)
`
`POlYP-2
`
`PoIYG)
`
`5.65
`
`6.15
`
`6.00
`
`6.25
`
`5.65
`
`a7
`
`66
`
`20
`
`<5
`
`41
`
`<5
`
`6.65
`
`PoIYW
`in 60%
`resuspended
`and
`with ethanol
`precipitated
`were
`Polymers
`weights
`of
`buffer
`(pH 6). The molecular
`in 0.2 M borate
`DMSO
`which was
`than 1 x 105, except
`poly(C)
`all polymers
`were greater
`of
`the polymers
`were
`determined
`before
`3 x 104. Concentrations
`Coupling
`was
`carried
`out at 4°C
`for 46
`the DMSO was
`added.
`hr
`in a total
`volume
`of 0.2 ml at a cellulose
`concentration
`of
`IO
`mg/ml.
`
`the diazo
`acids and
`nucleic
`between
`linkage
`The
`nucleotide
`using
`cellulose
`was
`investigated
`homopolymers.
`The data of Table
`2 suggest
`that
`coupling
`occurs
`best
`through
`guanine
`and
`uracil
`residues.
`Poly(dT)
`couples
`less well
`than
`poly(G)
`and
`poly(U),
`whereas
`poly(A)
`and poly(C)
`do not
`react with
`the diazo
`cellulose
`appreciably
`under
`the
`conditions
`tested. Although
`a low
`level of reaction
`with poly(A)
`and poly(C)
`cannot
`be excluded,
`the
`results do indicate
`that guanine
`and uracil
`(thymine)
`residues
`are probably
`the major
`sites of reaction.
`
`Page 2 of 10
`
`

`
`Hybridization
`303
`
`with
`
`Immobilized
`
`Nucleic
`
`Acids
`
`of DNA-Cellulose
`Micrographs
`1. Electron
`Figure
`in 50%
`formamide
`were
`spread
`Cellulose
`samples
`(a) SV40 DNA-cellulose,
`22 pg DNA/mg.
`(b) E. coli DNA-cellulose,
`23 pg DNA/mg.
`(c) Control
`cellulose
`diazotized
`and washed
`
`as described
`
`by Davis
`
`et al., (1971).
`
`in the absence
`
`of DNA.
`
`DNA
`of Coupled
`for Hybridization
`Accessibility
`for molec-
`to cellulose
`of DNA
`linked
`The availability
`in several
`examined
`has been
`ular
`hybridization
`of SV40
`ways. Figure
`1 shows electron micrographs
`DNA-cellulose,
`E. coli DNA-cellulose,
`and control
`cellulose
`(subjected
`to the diazotization
`and wash-
`ing procedures,
`but never
`exposed
`to DNA). Even
`though
`at
`least 8 equivalents
`of
`full
`length
`SV40
`DNA are coupled
`to the cellulose
`particle
`in Figure
`la,
`there
`appears
`to be minimal
`self-annealing
`of
`the DNA as judged
`by comparison
`with
`the E. coli
`DNA-cellulose
`in Figure
`1 b, where
`the DNA must
`
`that
`par-
`
`It is probable
`stranded.
`single
`be predominantly
`the cellulose
`more DNA
`is actually
`present
`on
`titles
`than
`is visible
`in
`the electron
`micrographs.
`DNA-cellulose
`was tested
`for
`its ability
`to function
`in both DNA-DNA
`and DNA-RNA
`hybridizations.
`50%
`formamide
`buffer
`at 37°C
`for 24 hr, 96-99%
`of sonicated
`SV40 DNA (6-7s)
`hybridized
`selective-
`ly to SV40 DNA-cellulose
`at weight
`ratios of
`immo-
`bilized
`DNA:sonicated
`DNA of 24:l
`or 48:l.
`RNA
`complementary
`to SV40 DNA
`(cRNA) was synthe-
`sized
`in vitro using E. coli RNA polymerase.
`As illus-
`trated
`in Table 3, more
`than 70% of the cRNA hybrid-
`
`In
`
`Page 3 of 10
`
`

`
`Cell
`304
`
`in-
`DNA-cellulose,
`SV40
`with
`selectively
`ized
`per mg
`of DNA coupled
`the amount
`dependent
`of
`with
`range.
`Treatment
`an 11
`fold
`cellulose
`over
`RNAase A caused
`a 15% decrease
`in
`the percent-
`age of cRNA
`hybridized.
`This
`result
`is expected
`since
`sedimentation
`of the cRNA
`in 99%
`formamide
`in-
`and 0.1% sodium
`dodecyl
`sulfate
`(SDS)
`at 35°C
`dicated
`that about
`12% of the preparation
`is larger
`than
`full
`length
`linear
`SV40 DNA. The
`relatively
`small effect of RNAase
`on
`the percentage
`of cRNA
`hybridized
`indicates
`that
`long
`regions
`of single
`stranded
`DNA must be available
`for hybridization.
`An alternative
`method
`of examining
`the accessi-
`bility of the DNA
`is to hybridize
`increasing
`amounts
`of cRNA
`to a
`fixed
`amount
`of DNA-cellulose,
`as
`shown
`in Figure 2. At least 2.5 ng of cRNA
`(probably
`more at saturation)
`can be bound
`to 50 ng of DNA.
`Since most of the cRNA
`is complementary
`to only
`one of
`the
`two strands
`of SV40 DNA, at least one
`DNA strand
`in
`ten
`is accessible
`for hybridization
`with a long
`sequence
`of cRNA.
`
`of SV40
`
`of Hybridization
`Efficiency
`DNA-Cellulose
`with cRNA
`and
`DNA-cellulose
`The
`reaction
`between
`SV40
`thoroughly
`to deter-
`cRNA was
`investigated
`more
`mine optimal
`standard
`conditions
`for assays of cell
`extracts
`containing
`SV40-specific
`RNA. At DNA
`concentrations
`between
`1 and 120 pg/ml,
`70-80%
`of the
`input
`cRNA hybridized
`reproducibly
`with
`the
`SV40 DNA-cellulose
`within
`24 hr. Figure
`3 shows
`kinetic
`data
`for a DNA concentration
`of 2.5
`,ug/ml
`and a cRNA concentration
`of 12 rig/ml.
`To evaluate
`the efficiency
`of hybridization
`reactions
`with cRNA
`
`Table
`
`3. Hybridization
`
`of SV40
`
`cRNA with DNA Celluloses
`
`% of Input
`Hvbridized
`
`cRNA
`
`DNA-Cellulose
`
`ua DNA/ma
`
`Cellulose
`
`-RNAase
`
`+ RNAase
`
`sv40
`
`6
`
`15
`
`48
`
`67
`
`Salmon
`
`Sperm
`
`IO
`
`74.0
`
`76.7
`
`59.9
`
`70.4
`
`75.7
`
`74.7
`
`71.5
`
`70.8
`
`0.3
`
`0.5
`
`60.0
`
`60.3
`
`49.3
`
`49.9
`
`63.9
`
`66.9
`
`55.9
`
`55.7
`
`0.2
`
`0.3
`
`with SV40
`incubated
`fig of DNA was
`0.6
`containing
`DNA-cellulose
`in 50%
`formamide
`for 18 hr under
`standard
`condi-
`cRNA
`(3.6 ng)
`tions. The
`total amount
`of cellulose
`in each
`reaction
`mixture
`was
`normalized
`to 0.1 mg by adding
`carrier
`cellulose.
`Treatment
`with
`RNAase
`(20
`pg/ml)
`was
`for
`1 hr at
`room
`temperature
`in
`2 x ssc.
`
`A
`
`the
`characterize
`to
`necessary
`it was
`further,
`units of cRNA,
`used. Of 100 arbitrary
`preparation
`incubated
`with
`17 were RNAase
`resistant. When
`conditions
`for
`SV40 DNA-cellulose
`under
`standard
`24 hr, 75 units
`hybridized
`and 25 did not. Only 4
`
`0
`
`1.0
`Log cRNA Input (ng)
`
`2.0
`
`of SV40 DNA-Cellulose
`
`with
`
`Increasing
`
`2. Hybridization
`Figure
`of cRNA
`Amounts
`was
`(50 ng DNA, 22 pg DNA/mg)
`to cellulose
`SV40 DNA coupled
`hybridized
`with
`increasing
`amounts
`of cRNA
`in 50%
`formamide
`buffer
`for 48 hr at 37°C.
`The samples
`were washed,
`treated
`with
`RNAase
`(20
`kg/ml,
`1 hr,
`room
`temperature)
`in 2 x SSC
`and
`washed
`again. The cRNA
`hybridized
`was determined
`after
`elution
`with 99%
`formamide
`and 0.1% SDS as described
`in the
`text. Each
`point
`represents
`the average
`of duplicate
`determinations.
`Similar
`results
`were
`obtained
`when
`the RNAase
`treatment
`was omitted.
`
`80)
`
`60 I 40
`
`20
`
`0
`
`20
`
`60
`
`80
`
`40
`Hours
`with cRNA
`of SV40 DNA-Cellulose
`of Hybridization
`Kinetics
`Figure3.
`(500 ng DNA, 22 pg DNA/mg)
`was
`to cellulose
`SV40 DNA coupled
`in 50%
`formamide
`buffer
`at 37°C.
`incubated
`with 2.5 ng cRNA
`the percentage
`of cRNA
`hybridized
`The samples
`were washed,
`and
`of
`the
`cRNA
`with 99%
`formamide
`was
`determined
`after
`elution
`in the
`text. Each
`point
`represents
`the
`and 0.1% SDS as described
`average
`of duplicate
`determinations.
`Similar
`results
`were obtained
`with samples
`treated
`with RNAase.
`
`Page 4 of 10
`
`

`
`Hybridization
`305
`
`with
`
`Immobilized
`
`Nucleic
`
`Acids
`
`the
`in
`to hybridize
`failed
`the cRNA which
`units of
`DNA-
`fresh
`SV40
`to
`first
`experiment
`hybridized
`Therefore,
`21 units
`cellulose
`in a second
`attempt.
`to hybridize
`in two
`of the original
`preparation
`failed
`attempts.
`This material
`was
`completely
`RNAase
`sensitive,
`indicating
`that
`it was not double
`stranded;
`it probably
`represents
`transcripts
`of small amounts
`of
`cellular
`DNA
`contaminating
`the SV40
`DNA
`preparation
`used as template.
`In addition
`to the 21
`units of material
`not complementary
`to SV40 DNA,
`the cRNA
`contains
`62 units of single
`stranded
`and
`17 units of double
`stranded
`SV40 RNA. Since
`75
`units did hybridize
`in the
`first experiment,
`we con-
`clude
`that
`the
`efficiency
`of
`hybridization
`was
`greater
`than 90%, and
`that
`in this case most of the
`double-stranded
`RNA did hybridize.
`
`and Transformed
`
`RNA
`
`in
`
`Infected
`
`SV40-Specific
`Cells
`for detecting
`technique
`the utility of the
`To explore
`the total RNA
`RNA within
`low
`levels of SV40-specific
`pool
`of eucaryotic
`cells, RNA was prepared
`from
`monkey
`cells
`(MA-134)
`productively
`infected
`by
`SV40 and
`from a hamster
`line
`transformed
`by SV40
`(C13/SV28).
`MA-134
`cells were
`harvested
`72 hr
`after high multiplicity
`infection
`with SV40 and after
`labeling
`with 3H-uridine
`for 20 min
`(71 s-72
`hr) or
`24 hr (48-72
`hr). RNA was extracted
`from
`the super-
`natant
`solution
`following
`lysis of the cells with SDS
`
`Table
`
`4. SV40-Specific
`
`RNA
`
`in Infected
`
`Monkey
`
`Cells
`
`Experiment
`
`1
`
`2
`
`3
`
`4
`
`Labeling
`Period
`
`20 min
`
`(71 2/3-72
`
`hr)
`
`24 hr
`
`(48-72
`
`hr)
`
`of precipitated
`removal
`of NaCl and
`in the presence
`(Hirt,
`1967).
`Although
`material
`by centrifugation
`RNA prepared
`in this way probably
`represents
`only
`about
`50% of
`the
`total
`cellular
`RNA
`(Aloni,
`1972),
`this procedure
`was chosen
`in order
`to compare
`our
`results with
`those of others.
`As
`indicated
`in Table
`4, 10% of the RNA
`from
`cells pulse-labeled
`for 20
`min with 3H-uridine
`hybridizes
`specifically
`to SV40
`DNA-cellulose.
`This agrees well with values
`of lo-
`20% and 10%
`reported
`by Aloni
`(1972) and Acheson
`et al. (1971)
`for viral-specific
`RNA prepared
`in the
`same way
`from SV40
`and
`polyoma-infected
`cells
`and assayed
`using
`filter
`hybridization
`techniques.
`Table
`4 also shows
`that about
`0.7%
`of
`the RNA
`prepared
`from
`lytically
`infected
`cells
`labeled
`for 24
`hr hybridizes
`with SV40 DNA-cellulose.
`This
`is with-
`in the
`range 0.1-f % reported
`by Khoury
`and Martin
`(1972)
`for
`the percentage
`of SV40-specific
`RNA
`in
`infected
`AGMK
`cells as measured
`by reassociation
`kinetics.
`la-
`after
`cells was extracted
`from Cl 3/SV28
`RNA
`in Table
`for 24 hr. As shown
`beling with 3H-uridine
`total
`labeled
`RNA hybridizes
`5, about
`0.02%
`of the
`specifically
`to
`the SV40 DNA-cellulose.
`This value
`is 10 fold
`larger
`than
`the one
`reported
`by Sambrook
`et al. (1972)
`for SV40-specific
`RNA
`in SV3T3
`cells
`(assayed
`by hydroxyapatite
`chromatography
`after
`hybridization
`in solution),
`but our
`result
`is within
`the
`range
`the values O.Ol-0.025%
`reported
`by
`
`of
`
`DNA
`
`s
`IO
`
`IO
`
`IO
`
`24
`
`24
`
`IO
`
`10
`
`15
`
`15
`
`20
`
`kg RNA
`2.3
`
`1.9
`
`1.7
`
`1.9
`
`0.95
`
`68
`
`143
`
`88
`
`177
`
`34
`
`% RNA Hybridized
`
`8.0,
`
`9.8,
`
`11.1
`
`9.3
`
`12.5
`
`8.9
`
`13.2
`10.4 zk 1.8
`
`0.58,
`
`0.74,
`
`0.76,
`
`0.77,
`
`0.91,
`
`0.67
`
`0.65
`
`0.83
`
`0.73
`
`0.85
`
`The
`
`15
`
`88
`
`0.73
`0.70,
`i 0.09
`0.74
`hr) after
`labeling
`(48-72
`hr) or 24 min
`20 hr (71 2/3-72
`with SV40 and either
`infection
`cells 72 hr after
`from MA-134
`RNA was extracted
`labeled
`for 24 hr,
`cells
`6.4
`x 103 cpmlpg,
`and
`from
`labeled
`for 20 min was
`cells
`specific
`activity
`of RNA
`from
`with XH-uridine.
`at 37OC
`for 40
`was
`carried
`out
`in 50%
`formamide
`and E. coli DNA-cellulose
`Hybridization
`with SV40 DNA-cellulose
`2.6 x 104 cpmlpg.
`Tl RNAase)
`was
`for 1 hr at room
`temperature
`in 0.2
`RNAase
`A and 1 pg/ml
`the
`text. RNAase
`treatment
`(20 pg/ml
`hr as described
`in
`in specific
`activity,
`the conditions
`in Experiments
`I-4
`which
`reflect
`differences
`ml of 2 x SSC. Except
`for different
`RNA
`concentrations,
`were
`the same
`as
`those
`in Experiments
`5-8.
`The percentage
`input RNA hybridized
`was
`corrected
`for a background
`determined
`from
`of
`of
`hybridization
`with E. coli DNA-cellulose.
`For RNA
`labeled
`for 20 min,
`this background
`was
`0.4%
`the
`input,
`and
`for RNA
`labeled
`for
`24 hr
`it was 0.009%.
`In Experiment
`8, unlabeled
`total
`cellular
`RNA
`from
`uninfected
`MA-134
`cells was
`used
`in place
`of yeast
`RNA
`as
`cold
`carrier
`in the hybridization
`mixture.
`
`Page 5 of 10
`
`

`
`Cell
`306
`
`Table
`
`5. SV40-Specific
`
`RNA
`
`in Transformed
`
`Cells
`
`Experiment
`
`1
`
`RNA
`Fraction
`
`I
`
`DNA
`
`sv40
`
`E. coli
`
`sv40
`
`E. coli
`
`sv40
`
`E. coli
`
`sv40
`
`E. coli
`
`Pcl
`DNA
`a
`
`a
`
`IO
`
`IO
`
`a
`
`a
`
`IO
`
`10
`
`P&l
`RNA
`
`206
`
`206
`
`105
`
`105
`
`346
`
`346
`
`263
`
`263
`
`wm
`Hybridized
`
`Average
`% of Total Labeled
`Material
`Hybridized
`
`3454
`
`4254
`
`634
`
`730
`I 878
`2069
`
`239
`
`297
`
`2316
`
`2648
`
`427
`
`628
`
`1463
`
`3592
`
`322
`
`276
`
`0.018
`
`0.019
`
`0.0021
`
`0.0027
`
`for extraction
`into 3 fractions
`and divided
`homogenizer
`with a Dounce
`disrupted
`were
`for 24 hr with 3H-uridine
`labeled
`cells
`C13/SV26
`supernatant)
`contained
`72% and 23% of
`the
`total
`II (postmitochondrial
`pellet)
`and
`of RNA as described
`in the
`text.
`Fractions
`I (nuclear
`at 17,000
`x g
`for 15 min)
`contained
`5% of
`the
`total
`labeled
`RNA and
`centrifugation
`labeled
`RNA,
`respectively.
`Fraction
`Ill (pellet
`from
`in 50%
`formamide
`at 37°C
`for 40 hr, and
`samples
`were
`processed
`as described
`in
`was
`not assayed.
`Hybridizations
`were
`performed
`A and 1 pg/ml
`Tl RNAase)
`was
`for 1 hr at
`room
`temperature
`in 0.2 ml of 2 x SSC.
`the
`text. RNAase
`treatment
`(20 pg/ml
`RNAase
`The
`specific
`activity
`of
`the RNA was 6.2
`x 104 cpm/pg.
`The
`percent
`of
`the
`total
`labeled
`RNA
`hybridized
`is
`the average
`of duplicate
`determinations,
`corrected
`for
`the average
`background
`values
`obtained
`with E. coli DNA-cellulose.
`
`cells
`for SV3T3
`(1968)
`and Sachs
`Aloni, Winocour,
`labeled
`with 3H-uridine
`for 22 hr (assayed
`by filter
`hybridization).
`
`Discussion
`
`the nucleo-
`reduces
`and nucleosides)
`nucleotides
`reaction
`occurs
`instead
`philicity
`of carbon
`8 so that
`with
`the primary
`amino
`substituent,
`or a ring nitro-
`gen, or both
`(Robins,
`1967; Shapiro,
`1968). Pyrimi-
`dine
`bases
`probably
`react
`with
`diazotized
`aryl
`amines
`through
`electrophilic
`substitution
`at carbon
`5 (Acheson,
`1967; Robins,
`1967). The methyl group
`at this position
`in thymine may account
`for
`the
`re-
`duced
`reactivity
`of
`this base. Barry
`and O’Carra
`(1973)
`reported
`the coupling
`of NAD+
`to sepharose
`through
`a diazotized
`aryl amine. Although
`the site
`of attachment
`to
`the NAD+
`is not known,
`the au-
`thors postulate
`linkage
`through
`the adenine moiety.
`In
`these
`experiments,
`1% or
`less
`of
`the NAD+
`in
`present
`the
`reaction mixture
`actually
`coupled
`to
`the support.
`Such
`low
`levels of reaction with poly(A)
`or poly(C) would
`not have been detected
`in the ex-
`periment
`presented
`in Table
`2.
`much
`react
`Single-stranded
`polynucleotides
`species.
`stranded
`more
`readily
`the
`double
`than
`to prevent
`With denatured
`SV40 DNA,
`it is difficult
`because
`renaturation
`in buffer
`at
`low
`temperature
`the concentration
`of DNA
`is high. For more complex
`is
`DNAs,
`renaturation
`at similar
`high
`concentration
`not as rapid. For example,
`denatured
`salmon
`sperm
`DNA can be coupled
`in borate
`buffer
`at pH 8, al-
`
`Acids
`
`to
`
`Attachment
`
`of Nucleic
`
`Covalent
`Cellulose
`them
`by trapping
`DNA and RNA can be immobilized
`in agar or cellulose
`or on nitrocellulose
`filters,
`or
`by
`linking
`them
`covalently
`to a solid
`support
`through
`the
`single
`terminal
`phosphate
`groups
`or
`through
`multiple
`points
`of
`internal
`attachment
`as
`to agarose
`after activation
`with CNBr
`(see
`review
`by Giiham,
`1974). The method we describe
`results
`in covalent
`attachment
`of single
`stranded
`RNA or
`DNA at multiple
`points
`to very
`finely
`divided
`cellu-
`lose. Based
`on
`the data of Table
`2,
`the
`linkage
`is
`primarily
`through
`guanine
`and uracil
`(thymine)
`resi-
`dues,
`as expected
`from
`the work
`of Cavalieri
`and
`Bendich
`(1950)
`and Robins
`(1958), who
`found
`that
`the disubstituted
`purine
`bases
`guanine
`and
`xan-
`thine did couple with diazotized
`aromatic
`amines
`in
`dilute
`alkali at carbon
`8, whereas
`the monosubsti-
`tuted
`bases
`adenine
`and
`hypoxanthine
`did
`not
`react. Substitution
`of guanine
`at position
`9 (as
`in
`
`Page 6 of 10
`
`

`
`Hybridization
`307
`
`with
`
`Immobilized
`
`Nucleic
`
`Acids
`
`result
`This
`15-20%.
`is only
`the efficiency
`though
`that DMSO may enhance
`the efficiency
`of
`suggests
`not only by maintaining
`the DNA
`in dena-
`coupling
`tured
`form,
`but also by
`increasing
`the nucleophil-
`icity of the purine
`and pyrimidine
`rings. E. coli
`tRNA
`also coupled
`to diazo
`cellulose
`in borate
`buffer
`at
`pH 8, but again
`the efficiency
`of coupling
`(5-7%)
`is less
`than
`for polymers
`in DMSO. Reaction
`in this
`case
`is probably
`through
`the guanine
`and uracil
`residues
`of single
`stranded
`regions.
`Duplex mole-
`cules with single
`stranded
`tails could
`probably
`be
`coupled
`selectively
`through
`these
`tails, and
`it may
`be possible
`to develop
`conditions
`for
`selectively
`coupling
`partially
`denatured
`(AT-
`or AU-rich)
`re-
`gions
`of a fully duplex
`structure.
`for coupling,
`In designing
`a particular
`procedure
`use of
`the
`it is important
`to consider
`the
`intended
`immobilized
`nucleic
`acid.
`If
`little
`nucleic
`acid
`is
`available,
`it may be preferable
`to couple
`at
`low
`DNA:cellulose
`ratios
`to increase
`the amount
`of DNA
`bound.
`Large
`amounts
`of support,
`however,
`may
`give somewhat
`higher
`backgrounds
`in hybridization
`experiments.
`Alternatively,
`one
`can
`couple
`large
`amounts
`of DNA
`to very small amounts
`of cellulose
`in order
`to
`reduce
`nonspecific
`binding.
`In the ex-
`periments
`presented
`here,
`from
`6-67
`pg of DNA
`were coupled
`per mg cellulose
`without
`any notice-
`able
`effect
`the efficiency
`of hybridization
`with
`cRNA.
`
`on
`
`Acids
`Nucleic
`Immobilized
`with
`Hybridization
`in 0.6 M
`assays
`can
`be performed
`Hybridization
`or
`in 50%
`formamide
`at 37°C with
`NaCl at 65°C
`equivalent
`results. Because
`backgrounds
`appeared
`it
`to be slightly
`lower
`in
`formamide
`and
`because
`is more convenient
`to work at 37”C,
`the
`lower
`tem-
`perature
`was chosen
`for
`routine
`assays. The sensi-
`tivity of the procedure
`depends
`upon
`reducing
`the
`background
`to a low
`level.
`In addition
`to
`including
`cold,
`carrier
`RNA
`in
`the hybridization
`mixture,
`it is
`important
`to wash
`the hybrid
`cellulose
`extensively.
`No differences
`in background
`levels were observed
`when
`the
`cellulose
`was washed
`with
`0.1, 2, or
`4 x SSC,
`and
`6 washes
`with
`ice cold
`2 x SSC
`were
`sufficient
`to
`reduce
`backgrounds
`to accept-
`able
`levels when
`the
`input was
`less
`than 105 cpm
`of cRNA.
`In experiments
`with high
`levels
`of RNA
`extracted
`from
`cells
`(3 x 107 cpm),
`the cellulose
`was washed
`with
`hybridization
`buffer
`containing
`25%
`formamide
`following
`RNAase
`treatment,
`which
`reduced
`the
`background
`without
`disrupting
`the
`RNA-DNA
`hybrids.
`The washing
`procedure
`most
`suitable
`for a given
`application
`depends
`upon
`the
`nature
`of
`the hybrid
`formed.
`For example,
`when
`d(r4C-pC)200(pT)200
`was
`hybridized
`with
`poly(G)-
`cellulose
`in 25%
`formamide
`at room
`temperature
`for
`5 hr in the absence
`of cold carrier,
`93% of the
`input
`
`and 8%
`to poly(G)-cellulose,
`bound
`radioactivity
`bound
`to control
`cellulose. Washing
`with 99%
`form-
`amide
`and 0.1% SDS at 40°C
`removed
`all of
`the
`radioactivity
`bound
`to the control
`cellulose,
`but only
`10% of that bound
`to the poly(G)-cellulose.
`Material
`which
`had hybridized
`selectively
`to
`the poly(G)-
`cellulose
`was
`then eluted with 99%
`formamide,
`0.1%
`SDS at 85°C.
`labeled
`RNA was
`presented,
`In the experiments
`RNA was eluted
`hybridized
`with
`JH-uridine,
`and
`in the presence
`of
`from
`the cellulose,
`precipitated
`filters
`for counting.
`HCI, and collected
`on glass
`fiber
`where
`quenching
`When
`using
`32P-labeled
`material
`due
`to
`the cellulose
`is not significant,
`it is conve-
`nient
`to collect
`the hybrid
`cellulose
`on glass
`fiber
`filters which may be dried and counted
`directly
`after
`extensive
`washing.
`filter hybridization
`between
`The major difference
`is
`is that
`the DNA
`and
`the procedure
`we describe
`in
`the
`latter. Be-
`covalently
`attached
`to cellulose
`has a high capac-
`cause
`the
`finely divided
`cellulose
`ity for nucleic
`acid, very
`low backgrounds,
`equiva-
`lent
`to
`those obtained
`in filter hybridization
`assays,
`can
`be
`obtained.
`In
`addition,
`the
`coupling
`procedure
`is technically
`simple
`and avoids
`harsh
`treatment
`of the DNA. Both
`the hybridized
`and non-
`hybridized
`fractions
`of a sample
`can be
`recovered
`easily
`for
`further
`use or analysis.
`The method
`can
`be applied
`analytically
`to determine
`low
`levels
`of
`a specific
`nucleotide
`sequence
`within
`a larger
`het-
`erogeneous
`pool, or it can be applied
`preparatively.
`For example,
`by coupling
`SV40
`cRNA
`to the cellu-
`lose,
`it should
`be possible
`to effectively
`separate
`the
`two strands
`of SV40 DNA. The cellulose
`is not
`suitable
`for column
`chromatography
`because
`it
`is
`so
`finely
`divided,
`but
`it sediments
`rapidly
`to
`form
`a firm pellet
`and can be used very conveniently
`in
`a batch-wise
`fashion.
`The DNA cellulose
`can also
`be reused
`after
`thorough
`washing.
`For example,
`in
`experiments
`with cRNA,
`the efficiency
`of hybridiza-
`tion with
`recycled
`cellulose
`was
`the same as with
`freshly
`prepared
`cellulose.
`In addition
`to RNA-DNA
`hybridization,
`the method
`can be adapted
`for DNA-
`DNA hybridization,
`or hybridization
`using
`nucleo-
`tide homopolymers.
`It may be useful
`in gene
`selec-
`tion, and possibly
`for
`isolating
`DNA or RNA binding
`proteins
`and studying
`their
`interaction
`with nucleic
`acids.
`large
`that
`found
`have
`(1975)
`and Horowitz
`Burrell
`to Se-
`periodate-oxidized
`RNAs
`(16S, 23s)
`couple
`less
`pharose-dihydrazide
`through
`their
`3’
`termini
`smaller
`efficiently
`than
`oxidized
`RNAs
`(5s). Proba-
`bly
`the
`larger molecules
`cannot
`penetrate
`the Se-
`pharose
`beads well.
`It may be possible
`to avoid
`such
`steric
`effects
`by coupling
`periodate
`oxidized
`RNAs
`to
`finely-divided
`cellulose
`rather
`than
`Sepharose.
`
`Page 7 of 10
`
`

`
`Cell
`308
`
`Experinental
`
`Procedures
`
`and Reagents
`Enzymes
`pur-
`A were
`and RNAase
`Tl,
`I, RNAase
`DNAase
`RNAase-free
`A (1 mg/ml)
`Corp. RNAase
`Biochemical
`chased
`from Worthington
`any DNAase.
`to
`inactivate
`for
`IO min
`was
`heat-treated
`at 90°C
`and Sl nuclease
`from EM Laboratories,
`Proteinase
`K was obtained
`was
`prepared
`by Elizabeth
`Swyryd
`from
`from Aspergillus
`oryzae
`Takadiastase
`powder
`(Sankyo
`Co.,
`Ltd.,
`Japan)
`as described
`by
`Sutton
`(1971).
`Preparations
`of E. coli RNA polymerase
`prepared
`from E. coli B according
`to Burgess
`(1969) were
`generously
`pro-
`vided
`by William Wickner
`and
`James
`Alwine.
`DMSO
`(Matheson
`Coleman
`and Bell) was
`redistilled
`under
`vacuum,
`and:he
`fraction
`collected
`at 83°C
`at 17 mm Hg was
`placed
`over
`4A molecular
`sieves,
`flushed
`with
`nitrogen,
`and
`stored
`at
`-20°C.
`Formamide
`(Matheson
`Coleman
`and Bell) was washed
`twice with equal
`vol-
`umes of ether
`and stored
`under
`nitrogen
`at -20°C.
`Cu
`(OH)2 was
`from K and K Labs. The potassium
`salts of poly(A),
`poly
`(U), and
`poly
`(C) were
`from Calbiochem,
`and poly
`(dT)
`and poly
`(G) were
`obtained
`from Miles Laboratories.
`!+H-uridine
`(27.8 Ci/mM),
`5,6-
`‘H-uridine-5’triphosphate
`(3684
`Ci/mM),
`and H$P04
`(carrier
`free) were
`from New England
`Nuclear
`Co.
`
`and Virus
`Cell Lines
`cells
`kidney
`line of Green Monkey
`MA-134
`cells,
`an established
`dishes
`grown
`on 100 mm plastic
`were
`obtained
`from
`J. Pagano,
`supple-
`Eagle’s medium
`(Gibco)
`(Nunc)
`in Dulbecco’s
`modified
`(Microbiological
`Associates),
`mented
`with
`10%
`calf
`serum
`100 pg/ml
`streptomycin
`sulfate
`(Pfizer),
`and 500 units/ml
`penicillin
`G (Squibb)
`in a CO2
`incubator
`at 37°C. C13/SV28
`cells,
`an SV40
`transformed
`hamster
`cell
`line, were
`obtained
`from C. N. Wiblin
`(Wiblin
`and MacPherson,
`1972)
`and maintained
`in suspension
`cul-
`ture at 37°C
`in the above medium.
`The virus
`used was SV40 wild
`type 830
`(M. Herzberg,
`J. E. Mertz,
`P. Berg,
`J. R. Cameron,
`and
`R. W. Davis, manuscript
`in preparation)
`derived
`from
`strain
`SVS
`(Takamoto,
`Kirchstein,
`and Habel,
`1966).
`
`of DNA
`Preparation
`at a multi-
`infected
`cells
`from MA-134
`isolated
`SV40
`(I) DNA was
`of
`the cells
`75-80%
`When
`unit/cell.
`forming
`plicity
`of 0.1 plaque
`was added,
`(100 pCi/plate)
`(10-I
`1 days),
`3*P04
`appeared
`ro