U.S. patent number 4,302,204 [Application Number 06/054,200] was granted by the patent office on 1981-11-24 for transfer and detection of nucleic acids.
This patent grant is currently assigned to The Board of Trustees of Leland Stanford Junior University. Invention is credited to George R. Stark, Geoffrey M. Wahl.
United States Patent |
4,302,204 |
Wahl , et al. |
November 24, 1981 |
Transfer and detection of nucleic acids
Abstract
Improvements in the transfer and detection of separated nucleic
acids, both RNA and DNA, are provided. For analysis of large DNA,
the molecular weight segregated fractions of DNA are depurinated
and fragmented to provide fractions having less than about 2 kb as
single strands. With both RNA and DNA, the nucleic acid fractions
are transferred after resolution to a chemically treated substrate
and covalently affixed to the substrate. The resulting nucleotides
affixed to the substrate are hybridized with labeled nucleotide
probes and a volume exclusion agent, particularly a water soluble
ionic polymer.
Inventors: |
Wahl; Geoffrey M. (Menlo Park,
CA), Stark; George R. (Ladera, CA) |
Assignee: |
The Board of Trustees of Leland
Stanford Junior University (Stanford, CA)
|
Family
ID: |
21989413 |
Appl.
No.: |
06/054,200 |
Filed: |
July 2, 1979 |
Current U.S.
Class: |
436/501;
435/6.16; 435/805; 422/504 |
Current CPC
Class: |
C12N
15/1034 (20130101); C12Q 1/6834 (20130101); C12Q
1/6832 (20130101); Y10S 435/805 (20130101) |
Current International
Class: |
C12Q
1/68 (20060101); C12N 15/10 (20060101); C12Q
001/68 (); G01N 031/22 (); G01N 033/16 (); G01N
033/48 () |
Field of
Search: |
;435/6,7,172,805
;23/23B,230.3,230.6 ;424/1,2,1.5 ;422/56 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Wetmur, "Acceleration of DNA Renaturation Rates", Biopolymers, vol.
14, (1975), pp. 2517-2524. .
Noyes et al., "Nucleic Acid Hybridization Using DNA Covalently
Coupled to Cellulose", Cell, vol. 5, (1975), pp. 301-310. .
Noyes et al., "Nucleic Acid Hybridization Using DNA Covalently
Coupled to Cellulose", Chem. Absts., vol. 83, No. 9, p. 247 (1975),
Abs No. 160314x. .
Wetmur, "Acceleration of DNA Renaturation Rates", Chem. Absts.,
vol. 84, No. 7, p. 140 (1976), Abs. No. 39846g. .
Reiser et al., "Transfer of Small DNA Fragments From Polyacrylamide
Gels to Diazobenzyloxymethyl-Paper and Detection by Hybridization
With DNA Probes", Biochem. Biophys. Res. Comm., vol. 85, No. 3
(1978), pp. 1104-1112. .
Kohne et al., "Room Temperature Method for Increasing the Rate of
DNA-Association by Many Thousand Fold: The Phenol
Emulsion-Association Technique", Biochem., vol. 16, No. 24 (1977),
pp. 5329-5341..
|
Primary Examiner: Wiseman; Thomas G.
Attorney, Agent or Firm: Rowland; Bertram I.
Claims
What is claimed is:
1. A process for the analysis of polynucleotides of at least ten
bases which comprises:
combining a solid substrate having polynucleotides covalently
affixed thereto with a hybridization solution containing labeled
polynucleotides suspected of being complementary to said affixed
polynucleotides and a charged polysaccharide of at least 10,000
molecular weight present in at least about 2 weight %; and
detecting the presence of labeled polynucleotides annealed to said
affixed polynucleotides.
2. A method according to claim 1, wherein said charged
polysaccharide is present in at least about 5 weight %.
3. A method according to claim 2, wherein said charged
polysaccharide is dextran sulfate.
4. A method according to claim 1, wherein said polynucleotide is
RNA.
5. A method according to claim 1, wherein said polynucleotide is
DNA.
6. A method according to any of claims 4 and 5, wherein said
charged polysaccharide is dextran sulfate and is present in at
least about 5 weight %.
7. A method for analyzing double stranded DNA in a mixture having
DNA molecules having chain lengths greater than 1 kb which
comprises:
distributing said DNA mixture according to molecular weight on a
polysaccharide gel by means of gel electrophoresis;
fragmenting and denaturing said DNA to provide single stranded DNA
of less than about 2 kb;
transferring at least a portion of said fragmented single stranded
DNA to a chemically reactive solid substrate to covalently bond
said transferred DNA to said substrate to provide
DNA-substrate;
combining said DNA-substrate with a hybridizing solution containing
labeled single stranded DNA suspected of being complementary to the
DNA of said DNA substrate; and
determining the presence of labeled DNA bound to said DNA of said
DNA-substrate by means of said label.
8. A method according to claim 7, wherein said chemically reactive
solid substrate is diazosubstituted paper.
9. A method according to claim 7, wherein said fragmenting and
denaturing involves contacting said DNA zone with acid and base
respectively.
10. A method according to any of claims 7 to 9, wherein said
hybridizing solution contains a charged polymeric volume exclusion
agent.
11. A method according to claim 10, wherein said agent is dextran
sulfate of at least about 10,000 molecular weight present in at
least about 10 weight %.
12. A method for analyzing double stranded DNA of chain length
greater than about 1 kb in a mixture of DNA which comprises:
distributing said mixture in a gel according to molecular weight by
means of gel electrophoresis;
fragmenting and denaturing at least a portion of said DNA by
treating with acid and base successively to provide ssDNA of chain
lengths less than about 2 kb;
transferring said ssDNA to diazo substituted paper to covalently
bind said ssDNA to said paper to provide DNA-paper;
hybridizing radioactively labeled single stranded DNA in a
hybridizing solution with said DNA paper, whereby complementary
labeled ssDNA binds to said DNA paper and wherein said hybridizing
solution is an aqueous solution of from about 40 to 60 volume % of
a low molecular weight polar organic solvent and from about 5 to 25
weight % of dextran sulfate of from about 10,000 to 1 M molecular
weight.
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
The ability to probe the chromosome, extra chromosomal genetic
material, messenger, transfer and ribosomal RNA, to synthesize
genetic material, as well as to manipulate genetic material, has
increased the need for means to analyze the composition and base
order of genetic material. It is therefore desirable to provide for
recording various genetic fragments which allow for hybridization
with the complementary fragment, so that mixtures may be analyzed
for the presence or absence of a particular nucleotide sequence. In
the development of a system for analyzing for particular nucleotide
sequences, there are many considerations. The first consideration
is the ability to separate a mixture into its constituent parts,
based on molecular weight and/or electrophoretic mobility. The
second consideration is the ability to accurately determine the
nature of the constituent parts.
One method for determining whether a particular sequence exists is
hybridization. That is, a particular nucleotide sequence is marked
with a detectable label, conveniently a radioactive label, and is
combined with the nucleotide sequence to be analyzed. If the two
sequences hybridize so as to form a strong non-covalent
interaction, it may then be reasonably assumed that the sequences
are substantially identical. Various techniques for accurately
determining whether hybridization has occurred and for
qualitatively or quantitatively determining the amount of the
nucleotide sequence have been developed. There is a continuing
interest and need for improved and more accurate techniques for the
rapid determination of the presence of a particular DNA
sequence.
2. Brief Description of the Prior Art
Southern, J. Mol. Biol. 98, 503 (1975) teaches the transfer of DNA
fragments from electrophoretically resolved DNA in agarose gels as
single strands to strips of nitrocellulose. Noyes and Stark teach
the transfer of DNA and resulting immobilization to
diazobenzyloxymethylcellulose, Cell, 5, 301 (1975). Alwine et al,
PNAS, USA 74, 5350 (1977) teaches the detection of specific RNA's
in agarose gels by transfer to diazobenzyloxymethyl-paper and
hybridization with DNA probes. Reiser et al, Biochem. Biophys. Res.
Comm. 85, 1104 (1978) teaches the transfer of small DNA fragments
from polyacrylamide gels to diazobenzyloxymethyl-paper and
detection with DNA probes. Wetmur, Biopolymers, 14, 2517 (1975)
teaches the use of dextran sulfate for renaturation of DNA. See
also U.S. Pat. No. 4,139,346.
SUMMARY OF THE INVENTION
Methods for determining the presence of a particular nucleotide
sequence are provided, whereby a nucleotide sequence is transferred
from a separation zone, e.g. electrophoretic gel, to a chemically
reactive substrate, e.g. a diazo substituted paper, to become
affixed to said substrate. Where the nucleotide sequence is DNA,
the DNA is normally treated sequentially with acid, followed by
base to provide for depurination, cleavage and denaturation to
single stranded fragments of moderate molecular weight, which can
be efficiently transferred to the paper and affixed. The nucleotide
sequence which has been affixed can be determined by hybridization
with a nucleotide sequence of known composition, employing a
detectable label bonded to the sequence of known composition, or
the affixed label can be used to determine the presence of a
complementary nucleotide sequence in a composition to be
assayed.
The affixed nucleotide sequences are found to be stable for long
periods of time and capable of repeated hybridization, so that the
paper may be used in assaying a number of different compositions.
Greatly enhanced efficiency in hybridization is achieved by
including in the hybridization medium a sufficient amount of a
volume exclusion agent, particularly an ionic water soluble
polymer.
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
The subject invention involves the preparation of resolved
nucleotide sequences covalently affixed to a stable substrate,
which are then used for hybridization with nucleotide sequences for
determination of the presence of a complementary nucleotide
sequence. In an initial phase, a particular nucleotide sequence is
prepared for transfer from a source of the sequence to a chemically
reactive substrate, e.g. diazo substituted paper, to affix the
nucleotide sequence to the substrate, by covalent bonding of the
chemically reactive functionality to the polynucleotide, to provide
for storage stability. When the nucleotide sequence has been
previously subjected to resolution, particularly a resolution based
on molecular weight and electrophoretic mobility, the position of
the nucleotide sequence on the paper will be related to its
chemical composition and molecular weight. Once the nucleotide
sequence is transferred from the resolving medium to the substrate,
hereafter referred to as paper, and covalently affixed to the
paper, the paper may now be used for probing compositions having
unknown nucleotide sequences to determine the presence of a
sequence complementary to the affixed nucleotide sequence. A
hybridization buffer is employed including a volume exclusion or
renaturing agent, which greatly enhances the rate and efficiency at
which a complementary nucleotide strand hybridizes to the affixed
strand.
Prior to hybridization, DNA is treated differently from RNA.
Depending upon the molecular weight of the DNA, during
electrophoresis, differing mixtures of materials are employed to
enhance resolution. Where relatively low molecular weight DNA is
involved, cross-linked polymers are employed to provide for a hard
polymer, where the cross-links therein are susceptible to cleavage
without adverse affects on the DNA. Where the DNA is of a size in
excess of about 200 bases in length, the DNA is subject to
successive treatments of depurination and degradation and
denaturation, so as to provide for randomly formed single stranded
smaller fragments.
The nucleotide sequences to be assayed by the paper may be labeled,
particularly with a radioactive marker. After hybridization, the
presence of the radioactively marked nucleotide sequences may be
determined by autoradiography. In this manner, the pesence or
absence of a particular sequence can be determined, as well as a
quantitative evaluation of its amount. By employing fragments, the
method is particularly sensitive, since a fragment having a
complementary sequence to the affixed nucleotide sequence may have
an unhybridized tail which can oligomerize with a plurality of
labeled sequences, so as to multiply the number of labels for each
nucleotide sequence which hybridizes with the affixed nucleotide
sequence.
The subject method provides for the separation of small DNA
fragments obtained from restriction enzyme digests on
polyacrylamide-agarose composite gels and transferring the
denatured DNA to diazo activated paper, and detecting the affixed
DNA by hybridization with radioactively labeled DNA probes. This
procedure is useful for high resolution mapping of plasmid and
viral DNAs, for detecting cloned DNA sequences within mixtures of
DNA fragments protected by nucleosomes during digestion of
chromatin with nucleases, and for mapping of binding sites of
non-histone proteins in DNA and chromatin.
In discussing the subject invention, the various steps which are
involved will be described individually followed by generalizations
which cover the overall method.
RESOLUTION
The nucleotide sequences which are to be assayed are treated
differently, depending upon whether RNA or DNA is involved. For
RNA, before resolution by electrophoretic purification and
separation, the RNA samples are normally purified, precipitated
with ethanol and dried. Since large amounts of ribosomal RNA
compete with transfer of mRNA, it is frequently desirable to purify
the composition by selecting poly A.sup.+ RNAs with poly-U
Sepharose or oligo-dT cellulose before electrophoresis, thus
removing ribosomal RNA.
In performing the electrophoresis, agarose gel is normally
employed. Desirably, the secondary structure of the RNA is
disrupted, either by pretreatment with glyoxal or by performing the
electrophoresis in the presence of methylmercuric hydroxide.
The DNA is electrophoretically resolved with agarose gels,
frequently having a small portion of acrylamide, usually not
exeeding about 12%. Depending upon the size of the DNA to be
resolved, the hardness of the gel may be enhanced by cross-linking
of the acrylamide. In order to enhance the transfer of small DNA
nucleotide sequences, for example fewer than 50 base pairs, the
cross-linking agent should be capable of cleavage by a reagent
which does not adversely affect the chemical structure of the DNA.
For example, the linking group may have a glycol functionality,
which is readily cleaved by periodic acid. The amount of acrylamide
generally ranges from about 5 to 12% for resolving fragments in the
range of about 2,000 to 10 base pairs.
After the nucleotide sequences have been resolved by
electrophoresis, the gel is then prepared for transfer to the
paper. Because of the short lived nature of the diazo group, the
two processes are normally performed concomitantly.
GEL PREPARATION AND TRANSFER
The RNA gel is treated differently, depending upon its history.
Where the RNA was pretreated with glyoxal, the gel is treated with
aqueous base, generally from about 10 to 100 mM under mild
conditions for a sufficient time to substantially remove all the
glyoxal from the RNA; and to cleave the RNA for efficient transfer.
Where the mecuric compound has been employed, the mecuric compound
is removed by reaction with a sulphur compound, for example
mercaptoethanol. In each case, the gel is then washed with an
appropriate buffer, while with the mecuric compound, an additive is
included to react with the excess mercapto compound, e.g.
iodoacetic acid. The buffer employed provides a mildly acidic pH
generally under 5, preferably from about 3 to 5, more preferably
about 4.
For DNA, particuarly in cases of small DNA fragments (.about.10-100
bases) the acrylamide is cross-linked, and the cross-links are
cleaved to enhance the efficiency of transfer of the small DNA
fragments. In preparing the gel for transfer, the gel is treated
with the cleaving reagent under conditions which do not adversely
affect the DNA fragments. In contrast, where large DNA fragments
are involved, cross-linked acrylamide is not required, and the gel
is treated with mild acid to provide for degradation of the large
DNA to randomly sized smaller fragments. The DNA is then treated
with a denaturing agent, conveniently mild base, generally from
about 0.2 to 1 M hydroxide, preferably about 0.5 M, to cleave and
provide single strands. After sufficient time to denature the DNA,
the gel is neutralized to a mildly acid pH, not lower than about 3,
preferably about 4, for transfer.
The diazo substituted paper is prepared in substantially the same
manner as has been described in Alwine et al, supra. Conveniently,
1-[(m-nitrobenzyloxy)methyl] pyridinium chloride (NBPC) is added to
the paper, preferably Whatman 540 paper, in an aqueous medium and
the paper subsequently dried. After washing with a nonpolar
solvent, the paper is dried and the nitro groups reduced by a
convenient reducing agent, e.g. dithionite. After washing to remove
the reductant and any hydrogen sulfide, the paper may be stored
until required for use.
When the paper is to be used, the amino groups are diazotized,
employing nitrous acid under mild conditions, so as to stabilize
the diazo groups. The concentration of diazo groups should be
sufficient to affix at least 5 .mu.gm of single stranded nucleic
acid per cm.sup.2 of surface area, preferably at least 10 .mu.gm
per cm.sup.2 of surface area.
TRANSFER
The transfer from the gel to the paper is substantially the same
for both RNA and single stranded DNA. The diazotized paper is
placed on top of the gel under a light weight in an appropriate
buffer and the composite structure allowed to stand for a
sufficient time under mild conditions (0.degree. to 25.degree. C.)
to allow for the efficient transfer of the nucleotide sequences to
the paper. The diazo groups form covalent bonds with the nucleic
acid, particularly guanosine and uridine bases, and any unreacted
diazo groups decompose to phenolic groups, which do not adversely
affect the nucleic acids bound to the paper.
DETECTION OF NUCLEOTIDES
The paper to which the nucleic acids have been affixed can now be
used in a number of ways. First, the nucleic acid composition
affixed to the paper can be assayed by employing probes for known
composition and hybridizing the nucleic acids bound to the paper
with labeled, conveniently radioactively labeled, nucleic acids of
known composition. Alternatively, where the nucleic acids bound to
the paper are known, a nucleic acid of unknown composition could be
probed by labeling the unknown composition with a marker,
conveniently a radioactive marker, e.g. .sup.32 P, and after
hybridizing, determining whether hybridization has occurred by
autoradiography. (Labeling with .sup.32 P by nick-translation with
DNA polymerase 1 is described in Rigby et al, J.Mol.Biol. 113, 237
(1977)). Normally, the labeled ssDNA will be a mixture of
complementary ssDNA capable of annealing and renaturation to dsDNA.
The fragments are usually randomly sheared by a DNase, U.V. light,
mechanical shearing or the like to provide the oligomers for
hybridization.
Hybridization is carried out from an appropriate hybridization
buffer solution. The aqueous solution will have from about 40 to
60, usually about 50 volume percent of another polar solvent,
usually a low molecular weight organic solvent (<100 m.w.) e.g.
formamide. In addition, there will be a number of additives for a
variety of purposes to enhance the hybridization. Usually there
will be from about 0.1 to 1.5 M saline and about 0.1 to 1.5 mM
citrate; about 0.005 to 0.05 wt %/vol each of albumin, particularly
serum albumin, a high molecular weight inert polysaccharide and a
polar polymer e.g. polyvinylpyrrolidone, about 0.5 to 5 mg/ml of
sonicated denatured DNA e.g. calf thymus or salmon sperm; and
optionally from about 0.5 to 2% wt/vol glycine.
Also included in the medium is a sufficient amount of a volume
exclusion additive as an annealing accelerating agent. The additive
may achieve the result by antichaotropic effects, that is, ordering
of the solvent, desolvating the medium with a strong solvent shell
or other effects which are not known. As the additive, a polar
water swellable or soluble polymer, particularly a charged
saccharidic polymer, more particularly anionic saccharidic polymer,
e.g., dextran sulfate, is employed. The polymer will generally be
at least of about 10,000 molecular weight and not more than about 2
million molecular weight, usually being from about 100,000 to 1
million molecular weight, and preferably about 400,000 to 600,000
molecular weight. The amount of the additive will generally be at
least about 2 weight percent of the hybridization buffer, more
usually at least about 5 weight percent, and generally not more
than about 25 weight percent, preferably about 8 to 15 weight
percent, more usually about 10 percent.
In cleaving the DNA, it is desirable that for DNA of greater than
about 2 kb, usually 1 kb, the DNA is treated with mineral acid.
e.g. HCl, of from about 0.2 to 0.5 M, particularly about 0.2 to 0.3
M, to provide DNA fragments under about 2 kb, usually approximately
0.5-2 kb.
By employing the cleavage as described previously in combination
with the annealing accelerating agent, efficient transfer of DNA to
the paper is achieved so that enhanced signals can be obtained. In
addition, because labeled fragments are used during hybridization,
the labeled fragments are oligomerized, so as to have a plurality
of labels e.g. radioactive atoms, for each hybridization event.
With radioactive labels, this permits more rapid autoradiography
with less background, so as to provide for sharply defined bands in
the autoradiograph.
The following examples are offered by way of illustration and not
by way of limitation.
EXAMPLES
(All temperatures not otherwise indicated are centigrade. All parts
and percents not otherwise indicated are by weight, except for
mixtures of liquids, which are by volume.)
The following abbreviations are employed: DBM,
diazobenzyloxymethyl; kb, kilobases; PALA,
N-(phosphonacetyl)-L-aspartate; CAD, a multifunctional protein
which comprises carbamyl-P synthetase, aspartate transcarbamylase,
and dihydroorotase, the first 3 enzymes of UMP biosynthesis; SSC:
0.15 M NaCl and 0.015 M trisodium citrate; Denhardt's reagent: 0.2%
(w/v) each of bovine serum albumin, polyvinyl pyrrolidone, and
ficoll (MW 400,000); SDS, sodium dodecyl sulfonate.
EXAMPLE I
Preparation of NBM-paper
Cut a sheet of Whatman 540 paper to fit into the bottom of a
rectangular enamel, stainless steel, or glass pan. The size of the
paper can be much larger than the size of each gel. Float the pan
on a water bath at about 60.degree.. For each cm.sup.2 of paper,
prepare a solution of 2.3 mg of 1-[(m-nitrobenzoyloxy)methyl]
pyridinium chloride (NBPC) (8.14 .mu.moles, M.W. 280.7) and 0.7 mg
of sodium acetate trihydrate in 28.5 .mu.l of water. Pour the
solution over the paper evenly, and using rubber gloves, push out
any bubbles. Rub the solution evenly over the paper with a gloved
hand, continuing until the paper is nominally dry. Dry one or more
such papers further at 60.degree. in an oven for about 10 min,
remove them, adjust the temperature of the oven to 130.degree. to
135.degree., place them back in the oven and bake them at this
temperature for 30 to 40 min. Several sheets may be baked at one
time, with as many as 3 overlapping. Wash the papers several times
with water for a total of about 20 min and three times with acetone
for a total of about 20 min, then dry them in the air. NBM-paper
(nitrobenzyloxymethyl-paper) is the most stable form and will keep
for many months in the refrigerator. It is simple to activate it
before each use. Alternatively, the less stable amino form
(ABM-paper) can be stored for 4.degree. in a vacuum for as long as
a year.
EXAMPLE II
Preparation of DBM-paper (diazobenzyloxymethyl-paper)
To reduce NBM-paper, incubate it in a hood (to eliminate SO.sub.2)
for 30 min at 60.degree. with 0.4 ml/cm.sup.2 of a 20% (w/v)
solution of sodium dithionite in water, with occasional shaking.
Wash the resulting ABM-paper several times with large amounts of
water for a few minutes, once with 30% acetic acid, then again with
several changes of water. Be sure no odor of H.sub.2 S remains.
Transfer the wet paper directly to 0.3 ml/cm.sup.2 of ice-cold 1.2
M HCl. For each 100 ml of HCl, add with mixing, 2.7 ml of a
solution of NaNO.sub.2 in water (20 mg/ml), prepared immediately
before use. Keep the paper in this solution on ice for 30 min or a
little longer, with occasional swirling. After 30 min, a drop of
the solution should still give a positive (black) reaction for
nitrous acid with starch-iodide paper. Leave the paper in the
ice-cold acid until preparation of the gel has been completed. Then
pour off the acid, wash the paper rapidly twice with ice-cold water
and twice with ice-cold transfer buffer (see below). Begin the
transfer without delay--see below for timings relative to
preparation of the gels.
EXAMPLE III
Preparation and electrophoresis of the RNA
Before electrophoresis, the RNA samples should be purified,
precipitated with ethanol, and dried. Large amounts of ribosomal
RNA compete with transfer of mRNAs from overlapping regions of the
gel. Hence, it may be advisable to reduce this competition and to
increase the concentration of a specific mRNA by selecting poly
A.sup.+ RNAs with poly-U Sepharose or oligo-dT cellulose before
electrophoresis. With two selections of oligo-dT cellulose, very
little of the isolated RNA is ribosomal. The presence of rRNA
reduces the signal in the positions of the mRNAs.
In order to disrupt secondary structure in the RNA completely,
electrophoresis should be carried out in the presence of
methylmercuric hydroxide, or after pretreatment of the RNA with
glyoxal. In either case, the distance a particular RNA migrates is
directly proportional to the logarithm of its molecular weight.
A. RNA from agarose gels containing methylmercuric hydroxide
The quantities of reagents specified are appropriate for a 150 ml
gel. Rock the gel gently for 20 to 40 min (depending on the
thickness of the gel) at room temperature in 200 ml of 50 mM NaOH
containing 5 mM 2-mercaptoethanol. Wash the gel twice for 10 min
each with 200 ml of 200 mM potassium phosphate buffer, pH6.5,
containing 7 mM iodoacetic acid at room temperature and then twice
at room temperature for 5 min each with 200 mM sodium acetate
buffer, pH4.0. Reduction of the NBM-paper should be started at the
beginning of the NaOH wash; alternatively, diazotization of the
ABM-paper should be started 0.5 hr later.
B. RNA pretreated with glyoxal from agarose gels
Place the gel in 200 ml of 50 mM NaOH with or without ethidium
bromide (1 .mu.g/ml) for 1 hr at room temperature. Neutralize the
gel by washing it twice for 15 min each with 200 mM sodium acetate
buffer, pH4.0, (the ethidium bromide staining can now be observed).
Reduction of the NBM-paper should be started about 0.5 hr after the
NaOH wash; alternatively, diazotization of the ABM-paper should be
started at the beginning of the first buffer wash.
EXAMPLE IV
Transfer to DBM-paper
Saturate two or three sheets of Whatman 3 MM paper with the same
buffer used for the final wash of the gels, then place them in
contact with a source of additional buffer. Place the gel on top of
the wet paper and place the fresh DBM-paper on top of the gel,
using Saran.RTM. wrap at the edges of the gel to prevent the
DBM-paper from touching the wet 3 MM paper below. Add two or three
layers of dry 3 MM paper, several layers of paper towels and a
weight. Allow the buffer to blot through the gel and DBM-paper
overnight, either at room temperature or at 4.degree..
EXAMPLE V
Pretreatment and hybridization
For RNA pretreatment and hybridization, the same procedure may be
employed as for DNA, described in Example VIII, except that 0.1%
SDS (sodium dodecyl sulfate) is included in the medium both during
the pretreatment and hybridization.
EXAMPLE VI
Transfer of small DNA fragments from composite gels
A. Gel electrophoresis
Restriction fragments are separated on polyacrylamide-agarose slab
gels (23.times.14.times.0.15 cm) using Tris-acetate buffer (40 mM
Tris hydrochloride, pH7.8, 20 mM sodium acetate, 2 mM EDTA). The
same buffer is used in the electrode reservoirs. To prepare the
gels, mix 8 ml of 10.times. concentrated gel buffer, 59 ml of water
and 560 mg of agarose (BioRad) and dissolve the agarose by boiling.
To the solution cooled to 50.degree., and an appropriate volume of
30% acrylamide stock solution (27.78 g of acrylamide plus 2.22 g of
N,N'-diallyltartardiamide (BioRad) per 100 ml) and 0.25 ml of 10%
ammonium persulfate. Gels containing a single concentration of
polyacrylamide between 5 and 12% are used to resolve fragments in
the size range 2000 to 10 base pairs, increasing polyacryamide with
decreasing sizes. DNA samples should be precipitated with ethanol
before electrophoresis. The electrophoresis is carried out at room
temperature at 15 to 20 mA.
B. Preparation of the gels and transfer
Place the gel into 20 ml of 2% periodic acid and rock it gently for
15 min at 37.degree. to cleave the cross-links. Rinse the gel with
water and put it into 250 ml of 0.5 M NaOH for 10 min at room
temperature to denature the DNA. Rinse the gel with water and
neutralize it in 250 ml of 0.5 M sodium phosphate buffer, pH5.5,
for 10 min at room temperature, and then put it into 250 ml of
ice-cold 50 mM sodium phosphate buffer, pH5.5, until the DBM-paper
is ready (no longer than 15 min). Diazotiazation of ABM-paper
should start at the same time as the treatment with periodic acid;
alternatively, reduction of NBM-paper should start about 0.5 hr
sooner. Do the transfer as described in the procedure for RNA,
except use 50 mM sodium phosphate buffer, pH5.5, at 4.degree..
EXAMPLE VII
Transfer of larger DNA fragments from agarose gels
A. Gel electrophoresis
Restriction fragments are separated on 0.5% agarose slab gels
containing ethidium bromide (0.5 .mu.g/ml) in both the gel and the
buffer reservoirs. Use the electrophoresis buffer described in the
previous example. Add the ethidium bromide to the molten agarose
just before pouring the gel. Perform the electrophoresis at room
temperature until the bromcresol purple dye marker has migrated
about 12 cm (8 to 12 hrs).
B. Preparation of gels and transfer of DNA to DBM-Paper
The following protocol is designed for a 150 ml
(14.5.times.13.5.times.0.8 cm) agarose gel and should be changed
accordingly for smaller volumes. It is advantageous to use
bromcresol purple as the tracking dye during electrophoresis since
it provides a convenient indicator for monitoring pH changes during
the later washes. All the procedures are done at room temperature.
Place the gel in an enamal pan and shake it gently with two 250 ml
portions of 0.25 M HCL for 15 min each. Decant the acid, wash the
gel briefly with distilled water, and shake the gel with two 250 ml
portions of 0.5 M NaOH, 1.0 M NaCl for 15 min each. Decant the
NaOH-NaCl solution and shake the gel with two 250 ml portions of 1
M sodium acetate buffer, pH4.0, for 30 min each. Wash the
diazo-paper with ice-cold 20 mM sodium acetate buffer, pH4.0, just
before transfer, and perform the transfer in 1 M sodium acetate
buffer, pH4.0 as follows.
Place the gel on top of two sheets of Whatman 3 MM paper
(approximately 20.times.30 cm each) saturated with 1 M sodium
acetate buffer (pH4.0) (or 20x SSC for transfer to nitrocellulose).
Place sheets of Saran.RTM. wrap on the Whatman paper around the
perimeter of the gel to prevent contact between the paper layers to
be placed above the gel and the saturated paper beneath the gel.
Position the DBM-paper (or nitrocellulose) on top of the gel.
Regions where the gel and paper are in contact should be free of
air bubbles which may interfere with the transfer. Place two sheets
of dry Whatman 3 MM paper on top of the DBM-paper (or
nitrocellulose), then a 3-inch layer of paper towels, and finally a
light weight, to insure even contact between the different layers.
Allow the transfer to occur for 2 hrs or longer. It is not
necessary to add buffer to the saturated paper during transfer.
EXAMPLE VIII
Pretreatment, Hybridization, and Detection of Specific DNA
Sequences Bound to DNA-paper ot DNA-Nitrocellulose
The sporadic appearance of high backgrounds, a major problem in
two-phase hybridizations, is minimized by the following procedure.
It is very important to follow the procedure exactly. The protocol
is designed for a 9.times.13.5 cm paper.
Place DNA-solid support in 10 ml of 50% formamide (reagent grade),
5.times.SSC, 5.times.Denhardt's reagent, 50 mM sodium phosphate
buffer (pH6.5), 1% glycine and 250-500 .mu.g/ml sonicated denatured
salmon sperm DNA (Sigma) in a polyethylene bag. Incubate at
42.degree. for at least 1 hr. Remove as much of this solution from
the bag as possible, but do not blot the filter. (The easiest
method is to draw a rod over the open bag to extrude the liquid.)
Prepare 10 ml of a solution of 50% formamide, 5.times.SSC,
1.times.Denhardt's reagent, 20 mM sodium phosphate buffer (pH6.5)
and 100 .mu.g/ml sonicated, denaturated salmon sperm DNA, and 10%
sodium dextran sulfate 500 (Pharmacia). (The dextran sulfate is
added most conveniently as a 50% (wt/vol) aqueous solution, which
is slightly yellow and quite viscous.) Add 9 ml of the complete
mixture to the bag, wetting the paper thoroughly. Heat the
remaining 1 ml to 65.degree. for a few minutes to reduce the
viscosity, then add the probe. Mix vigorously in a voetex and add
to the bag. Seal the bag close to the paper and avoid trapping
large air bubbles. Mix the solution in the bag thoroughly to insure
uniform distribution of probe. Incubate the bag at 42.degree. for
4-16 hrs. depending on the source and amount of the DNA being
analyzed and quantity of probe being used. This procedure may also
be used for hybridization probes to RNA-paper. In thise case, 0.1%
sodium dodecyl sulfate should be included in the prehybridization
and hybridization solutions to inhibit ribonuclease. Sodium dodecyl
sulfate is not advantageous in hybridizations to DNA-paper.
Wash the paper with three 250 ml portions of 2.times.SSC, 0.1%
sodium dodecyl sulfate for 5 min each at room temperature, then
with two 250 ml portions of 0.1.times.SSC, 0.1% sodium dodecyl
sulfate at 50.degree. for a total of 30 min. The background
(detected with a monitor) should be very low. If the background is
unacceptably high at this point, continue washing with this buffer
for an additional 30 min. Expose the x-ray film to the paper at
-70.degree., using a Dupont Lighting Plus intensifying screen.
EXAMPLE IX
Determination of DNA Fragment Lengths Following Partial
Depurination and Strand Cleavage in Agarose Gels
DNA samples were separated by electrophoresis through a 0.8%
agarose gel until the bromcresol purple dye marker was 1 cm from
the origin. The DNA samples in one-half of the gel were then
depurinated partially and cleaved by sequential treatment with acid
and alkali as described below. A sample of .gamma. DNA from strain
J.sup.-.sub.am Z.sup.-.sub.am Vir, digested with restriction
endonuclease HindIII and run in the other half of the gel, was
treated with alkali alone to provide single-stranded molecular
weight markers. Both halves were equilibrated with 30 mM NAOH, 2 mM
EDTA (8 changes for 15 min each), and electrophoresis was resumed
with this solvent until the dye marker was approximately 6 cm from
the origin (16 hr). Fragments were visualized with 254 nm light
after equilibrating the gel with 0.2 M sodium phosphate (pH6.5)
containing 1 .mu.g/ml of ethidum bromide.
EXAMPLE X
Preparation of End-labeled .gamma. DNA Fragments
Ten .mu.g of .gamma. DNA from J.sup.-.sub.am Z.sup.-.sub.am Vir
were cleaved with HindIII in a buffer containing 20 mM Tris-HCl
(pH7.4), 60 mM CaCl.sub.2, 7 mM MgCl.sub.2, 100 .mu.g/ml bovine
serum albumin (Bethesda Research Laboratories) and 2 mM
dithiothreitol in a total volume of 60 .mu.l. Reverse transcriptase
from avian myeloblastosis virus was then used to catalyze addition
of [.alpha.-.sup.32 P]dCTP and [.alpha.-.sup.32 P]dGTP to the
staggered ends of the restriction fragments. The HindIII
restriction digest was diluted with an equal volume of 20 mM
Tris-HCl (pH7.4), 20 mM NaCl, 400 .mu.M dATP, 400 .mu.M dTTP, 50
.mu.Ci each of the .sup.32 P-labeled triphosphates
(Amersham/Searle, 300 Ci/mmole), and 16 units of reverse
transcriptase (Life Sciences, Inc., St. Petersburg, Fla.). The
reaction mixture was incubated at 37.degree. for 1.5 hrs and
reaction was stopped by adding 0.1 volume of a solution 1% in
Sarkosyl and 125 mM in EDTA, followed by heating to 70.degree. for
5 min. Unincorporated nucleotides were removed by filtering the
mixture through a column of Biogel P-60, equilibrated with 10 mM
Tris-HCl (pH7.4), 1 mM EDTA.
EXAMPLE XI
Preparation of End-labeled .PHI.X174 Viral DNA
.PHI.X174 viral DNA (5 .mu.g, was incubated at room temperature
with 0.20 M HCl for 5 min, followed by 0.50 M NaOH for 30 min to
yield fragments 100-1000 bases long. The fragments were collected
by ethanol preciptation and dissolved in 200 .mu.l of 10 mM
Tris-HCl (pH8.7), 1 mM MgCl.sub.2. The 5'-phosphoryl groups were
removed by incubation for 3 hrs at 37.degree. with calf intestine
alkaline phosphatase. Proteins were removed by extraction with
phenol and the DNA was collected by precipitation with ethanol. The
5'-termini of the fragments were labeled with [.gamma.-.sup.32
P]ATP (3000 ci/mmole, Amersham/Searle) using T4 polynuceotide
kinase (PL Biochemicals).
The efficiency of transfer was assessed employing restriction
fragments obtained as described in Example X, i.e. .gamma.
J.sup.-.sub.am Z.sup.-.sub.am Vir DNA with HindIII. Transfer was
found to be complete in 2 hrs and fragments in the size range
0.56-22.7 kb are all transferred at the same high efficiency to
either DBM paper or to nitrocellulose. It should be noted, that
fragments smaller than about 1 kb can be transferred to DBM-paper,
but not effectively to nitrocellulose.
In order to test the use of dextran sulfate, DNA from a
PALA-resistant mutant with approximately 7 times with wild-type
number of CAD genes was digested with EcoR1, fractionated on an
agrose gel and transferred to DBM-paper. Identical DNA-paper strips
were hybridized with the same quantity of nick-translated probe in
the presence of different levels of dextran sulfate. The time for
the hybridization was 16 hrs and 5.times.10.sup.6 cpm of
nick-translated probe (1.times.10.sup.6 cpm/ml, 5.times.10.sup.7
cpm/.mu.g) was employed. The washed filters were autoradiographed
for 10 hrs.
Comparing the signals obtained in the presence of 10% sodium
dextran sulfate and in its absence as a function of time of
hybridization, reveals that the signal obtained after only 2 hrs in
the presence of dextran sulfate is 3-4 times greater than the
signal obtained after 72 hrs in its absence. While enhanced
background is observed by the use of dextran sulfate, by employing
5.times.Denhardt's reagent prior to hybridization with the probe,
the background is reduced substantially.
Also studied were the effects of dextran sulfate on the rates of
hybridization to DNA-paper of single-stranded and double-stranded
probes. Labeled single-stranded .PHI.X-174 viral DNA, average
length approximately 250 bases and nick-translated double-stranded
.PHI.X-174 replicative form DNA were hybridized to .PHI.X-174
DNA-paper in the presence and absence of 10% dextran sulfate. Three
to four times more single-stranded probe binds the DNA-paper in 12
hrs in the presence of dextran sulfate than in its absence. With
the double-stranded probe, the rate in the presence of dextran
sulfate was at least 15 times the rate in its absence, although
enhancement of the absolute rate was less than usually
observed.
Dextran sulfate can also be employed with hybridization to
immobilized RNA, as previously indicated. Dextran sulfate also
increases rates of hybridization in in situ hybridizations used to
locate specific gene sequences in polytene chromosomes and to
detect recombinant mammalian viruses in plaques. Detection of
recombinant molecules in the plaque-filter and colony-filter
methods should also be facilitated by dextran sulfate. The
hybridization employing dextran sulfate need not be limited to
DBM-paper, but may also be used with nucleic acid bonded to any
substrate.
It is evident from the above results that novel and useful
techniques have been provided for rapid determination of nucleic
acids by appropriately immobilizing nucleic acids on an appropriate
vehicle, followed by hybridization with detectable probes. While
hybridization has involved the use of radioactive labels, it is
evident that other lables could also be employed, such as
fluorescers, enzymes or the like. By employing polymeric materials
in the hybridization medium, the rate of hybridization is greatly
enhanced, so that determinations can be quickly and accurately made
as to the presence or absence of particular nucleotide
sequences.
The subject method also allows for a rapid and accurate analysis of
large DNA molecules, greater than about 1 kb. By electrophoretic
separation of a DNA mixture, which includes large DNA molecules,
the DNA in zones of high molecular weight are fragmented and
denatured to provide moderate to small DNA molecular weight
fragments (10 to 2000 kb). These DNA molecules are then readily
transferred to the reactive substrate for subsequent hybridization
and analysis with labeled probes or may themselves be labeled and
hybridized with DNA-substrate of known composition.
Although the foregoing invention has been described in some detail
by way of illustration and example for purposes of clarity of
understanding, it is obvious that certain changes and modifications
may be practiced within the scope of the appended claims.
* * * * *