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INTRODUCTION
1. Technical Field
Method for labeling nucleic acid fragments for detection.
2. Background
Today, biology is in many ways the science of proteins and nucleic acids.
Nucleic acids are found in all living matter. For each species or host,
unique sequences exist providing for the genotype and phenotype of that
particular host. Thus, one can use the presence of a particular sequence
as indicative of the particular strain or species. In many instances, a
number of strains will share a common sequence as distinct from other
strains or species, so that one can not only detect a particular strain
but, if desired, can detect subspecies, species or genera. In addition,
one can distinguish between RNA and DNA so as to determine whether a
particular gene is being expressed, the existence of one or more alleles,
the level of expression, and the like. Where cells, such as B-cells and
T-cells, are involved with genomic rearrangements, one can detect the
presence or absence of such rearrangements by employing probes. Thus, the
detection of particular nucleic acid sequences is a powerful tool in the
diagnosis of disease states, the presence of sets or subsets of cells, the
particular strain or species of a pathogen, such as a bacterium, protista,
virus, or the like.
The detection and isolation of sequences is also important in the field of
molecular biology. Thus, the use of probes allows for detection of a
variety of sequences of interest, including structural genes, regulatory
sequences, introns, exons, leader sequences, sequences that are both
translated and untranslated, and the like.
There is also substantial interest in detecting sequences in genetic
engineering. Monitoring levels of transcription, detecting the integrity
of constructs, monitoring levels of mutation, resection, mapping, or the
like, provide opportunities for nucleic acid screening and detection.
In many instances, the sequence of interest may be present as only a very
small fraction of the total amount of nucleic acid, and/or in very small
amount, e.g., attomole or subattomole levels. Furthermore, the sequence of
interest may be accompanied by a number of sequences having substantial
homology to the sequence of interest. Thus, relatively high stringencies
may be required to ensure the absence of unwanted hetero-duplexing, which
may further limit the effective detection of the sequence of interest.
Additionally, the same or similar sequences may appear on nucleic acid
fragments of different size and the appearance of a sequence on a
particular size fragment may be correlated to the presence of a particular
phenotype.
There is also interest in developing analytical systems which can be
automated, so as to minimize the time and energy required from
technicians, as well as minimizing errors which may result from manual
manipulation. In many systems the sample is labeled to allow for detection
of the sequence. The labeling can be time consuming and limited as to the
nature of the label as in nick translation with radioactive nucleotide
triphosphates. In other situations, the particular nature of the label may
be limited, as when using terminal deoxytransferase. Other techniques
result in random substitution. There is therefore an interest in providing
for rapid, conveniently controlled labeling and detection of sample
nucleic acids, where the labeled moiety may be commercially available and
require little, if any, technical skills, in being used to label the
sample.
Relevant Literature
Kempe et al., Nucl. Acids. Res. (1985) 13:45-57 describe biotinylated
oligonucleotide linked to DNA fragments by a ligase. Gamper et al., Nucl.
Acids Res. (1985) 14:9943-9954, employ a psoralen-functionalized oligomer
as a probe which labels target DNA when hybridization and photochemical
cross-linking occur. Zapolski et al., Electrophoresis (1987) 8:255-261
discuss a robotic system for automating Southern-type nucleic acid
hybridization analysis. Goldkorn and Prockop, Nucl. Acids Res. (1986)
14:9171-9191 describe techniques for covalent attachment of DNA probes to
cellulosic supports for hybridization-restriction analysis. Syvanen et
al., Nucl. Acids Res. (1986) 14:5037-5048 quantify nucleic acid hybrids by
affinity-based hybrid collection. Forster et al., Nucl. Acids Res. (1985)
13:745-761 covalently label nucleic acids with biotin photochemically.
Kinzler and Vogelstein (1989) Nucl. Acids Res. 17:3645-3653 describe the
application of PCR to the identification of sequences bound by gene
regulatory proteins. Roux and Dhanaragan, (1990) Biotechniques 8:48-57,
1990, Frohman et al., (1988) PNAS USA 85:8998-9002, and Loh et al. Science
(1989) 243, 217-220 describe amplification techniques.
See also EPA Serial No. 89/400220.3 and U.S. Pat. Nos. 4,683,195;
4,683,202, 4,800,159 and 4,889,818.
SUMMARY OF THE INVENTION
Double-stranded DNA ("dsDNA") fragments are labeled with detectable
double-stranded nucleic acid moieties that possess termini complementary
to the termini of the double-stranded DNA fragments to be labeled. The
labeling double-stranded moiety contains a sequence that can subsequently
be utilized as a primer binding site. The labeling reaction is performed
in a manner which provides for the labeling moiety to be joined to both
termini of the dsDNA fragments, wherein the 5'-3' strand of the labeling
moiety is ligated to the adjacent 5' end of the dsDNA fragment strand and
the 3'-5' strand of the labeling moiety is covalently attached by ligation
or fill-in reactions. After denaturation, strands selected by hybridizing
to a probe are separated, amplified, and the presence of the target
sequence is established and/or the DNA isolated.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1a, 1b, 1c, and 1d are diagrams of methods of covalently attaching a
labeling moiety to dsDNA sample fragments.
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
Methods and compositions are provided for producing high levels of nucleic
acid strands of a target sequence, particularly for identification of the
sequence in a complex mixture of nucleic acid fragments. The labeling
moiety will comprise a 5'-3' strand, which is normally not phosphorylated
at its 3' end, and may be ligated to the phosphorylated 5' end of the
fragments of the dsDNA. The labeling moiety will also comprise a 3'-5'
strand, which will usually be phosphorylated at its 5' end before
combining the labeling moiety with the dsDNA fragments or may be created
by filling in with a polymerase and dNTP's or may be replaced with a full
length 3'-5' strand which is phosphorylated at its 5' end which is
subsequently ligated. The labeling procedure results in target dsDNA
fragments joined at both ends to both strands of dsDNA labeling moieties
to provide symmetrical termini. After selecting those labeled sequences
having a target sequence by means of a probe, such selected sequences are
then separated and amplified and the presence of the target sequence may
then be further established and/or the DNA isolated.
The strategy for analysis of a dsDNA sample provides for simultaneous or
consecutive restriction of a dsDNA sample and tagging or labeling with
oligomeric labeling dsDNA moieties as described above. The labeling
moieties comprise a primer binding site. DNA to be used as a probe is
joined to a separation means, which may be one member of a specific
binding pair, with the other member bound to a separable support. The
probe DNA and the labeled sample DNA are denatured and hybridized and
sequence homologous to the probe DNA separated. After remaining
non-specifically bound DNA is removed, the target DNA may be directly
detected by a gel scanner, but will usually be amplified and detected.
The subject method finds use in a number of situations. The subject method
can be used for detection of a target sequence to identify the presence of
pathogens in a sample, the presence of a mutation, the presence of a
particular length polymorphism or isozyme, the isolation of homologous
sequences from different species, and the like. See the discussion in the
Background section.
Usually, the sample employed will be genomic or cDNA which can be digested
into a plurality of different restricted fragments by use of restriction
endonucleases, generally at least two different fragments, usually five or
more different fragments, and the mixture may be fifty or more or
thousands or millions of different fragments. The restriction digestion
may be prior to or concurrent with the mixing with the labeling moieties
and ligation.
The method described above for labeling double-stranded DNA fragments with
a detectable moiety covalently joining the labeled DNA to the fragments,
separating sequences binding to a predetermined probe sequence and then
further amplifying these sequences is very general and can be used in any
situation where one wishes to amplify and or detect a particular sequence,
even if the actual sequence of nucleic acids of such target has not been
established.
The source of the sample may be any material or substance comprising
nucleic acid. The nucleic acid need not be a naturally occurring nucleic
acid, but may be synthesized chemically, enzymatically, or biologically
and may have other than naturally occurring purines and pyrimidines. The
sample source may be cellular or non-cellular, may be a clinical sample or
isolate, may be derived from such physiological media as blood, serum,
plasma, stool, pus, scrapings, washing, urine, or the like; may be
associated with a set or subset of cells, such as neoplastic cells,
lymphocytes, e.g., T-cells or B-cells, monocytes, neutrophils, etc.;
pathogens, including viruses, bacteria, mycoplasma, fungi, protozoa, etc.;
may include constructs, involving plasmid, viruses or DNA or RNA
fragments, or the like. The nucleic acid sample may involve DNA, which may
be chromosomal or extrachromosomal, e.g., plasmid, viruses, synthetic
constructs, etc. or RNA, such as messenger RNA, transfer RNA, ribosomal
RNA, viruses, or the like, where the RNA may be transcribed into dsDNA.
The nucleic acid sequences may involve structural genes, untranslated
regions, regulatory regions, introns, exons, or the like.
The detection may be for a wide variety of purposes. Detection may involve
diagnosis of a diseased state in plant or animal species, such as
neoplasia or other aberrant cellular state, the detection of sets or
subsets of cells, such as lymphocytes at various stages of
differentiation, the detection of strains or species of pathogens, the
monitoring of genetically engineered expression, or the like.
Prior to use of the sample in the subject invention, the sample may have
been subjected to a variety of chemical or physical treatments, such as
proteolysis, extraction, precipitation, separation of nucleic acid from
other components, such as lipids, proteins, or the like, hydrolysis of
RNA, inactivation of nuclease, concentration, chromatography, dehydration,
heating, etc. The sample may be manipulated for a variety of reasons, such
as removal of interfering materials, preparation for storage or shipment,
concentration, or the like.
The sample will normally be subjected to fragmentation by employing
restriction enzymes. The restriction enzymes will cleave at the
recognition site, usually, but not necessarily, having a symmetrical 4 to
8 bp, usually resulting in termini, blunt or overhangs, of known sequence.
One or more restriction enzymes may be employed, normally not more than
two, preferably one. Depending upon the nature of the sample for the
amplification, as a result of the restriction process fragments may be
provided usually varying from abut 50 bp to 200 kbp. Various restriction
enzymes may be used depending upon the size of fragments desired, the
nature of the sample, and the like.
In some instances, the sample may involve the reverse transcription product
of messenger RNA, where the mixture may be relatively small sequences of
DNA and RNA. If desired, the RNA may be hydrolyzed or digested, leaving
substantially only the DNA sequences. In this manner, one would have a
composition of single stranded DNA. The single stranded DNA may then be
converted into dsDNA using an enzyme such as DNA polymerase.
One may provide for simultaneous restriction and ligation of the 5'-3'
strand of the labeling moiety to the 5' end of the sample dsDNA as
described in U.S. Pat. No. 5,093,245 or EPA 89400220.3. Thus, the
restriction enzyme(s) and ligase may be combined in the same reaction
mixture. Normally not more than one restriction enzyme will be used. The
labeling moiety is usually designed so that it will not be capable of self
ligation, e.g. by the lack of terminal phosphates, and, when the moiety is
ligated to the sample dsDNA, the ligated product will not recreate the
restriction enzyme recognition sequence at the site of ligation. Any
sample dsDNA restriction fragments that are ligated to each other will be
subsequently cleaved until a labeling moiety is ligated on to the sample
dsDNA fragment. Thus, the system ensures the substantially complete
cleavage and labelling of all of the restriction enzyme recognition sites
in the sample DNA.
In reviewing the process of providing for the labeling moieties comprising
the primer binding site joined to the termini of the sample dsDNA
fragments, reference will be made to the drawings.
The labeling DNA moiety will have two-strands, one, referred to as the
first 5'-3' strand, will be capable of ligation to the abutting sample
strand, or may be provided by an alternative process. Either strand or
both may include one or more tags or ligands, but tags or ligands are not
required. In addition, the 3'-5' strand will serve as a primer binding
site in the amplification reaction resulting in replication of the strand
to which the 3'-5' strand is bound. Usually, each sequence will be of at
least six nucleotides, usually at least eight nucleotides, and at least
one sequence, usually the ligaid will be at least twelve nucleotides, and
may be fifteen nucleotides or more, usually not more than one hundred
nucleotides.
Because of the manner in which restriction enzymes cleave, the first strand
may be readily ligated to its associated strand (adjacent 5' termini) of
the dsDNA fragment as depicted in FIG. 1a. U.S. Pat. No. 5,093,245
describes various methods for performing such ligations, including
situations when the restriction results in blunt ends or 3' overhangs.
Various techniques may be employed for forming or joining the ligaid at or
to the 3' termini of the dsDNA fragments. The drawings depict some of
these techniques. As depicted in FIG. 1b, one method is referred to as the
"fill-in" reaction, where after combination of the dsDNA with the labeling
DNA moiety, the first 5'-3' strand is joined to the associated dsDNA
strand by ligation. (By "associated" is intended the strands of different
entities which have the bonding of the sugars in the same direction, e.g.,
5'-3' or 3'-5'. Thus, the associated strands allow for ligation while
maintaining the same directional order). Then, by filling in with DNA
polymerase employing dNTPs, one can extend the 3' end of the dsDNA to
replicate any overhang and the first strand sequence, while displacing the
ligaid. In this manner, a blunt end can be obtained with a recreated
ligaid.
An additional way, shown in FIG. 1c, is to use a truncated ligaid as part
of the labeling moiety. The truncated ligaid would be in register with the
associated strand of the sample dsDNA to which the truncated ligaid is
adjacent. The dsDNA and labeling moiety may have blunt or staggered ends
where the ends of the different entities may be associated or in register.
(In register intends that each of the ends of the strands of the labeling
moiety will be adjacent to the associated ends of the dsDNA fragment
strands.) After ligating the first strands to the 5' termini of the
associated strands, one could then mildly denature the mixture, in the
presence of a longer ligaid which would extend at least through the primer
sequence, where the ligaid would have a phosphorylated 5' terminus (a 5'
phosphate or triphosphate). After displacement and hybridization occurred,
the temperature could be lowered and the ligaid ligated to the 3' terminus
of its associated fragment strand. Alternatively, ligation could be
attempted at the elevated temperature, because ligation can proceed at
mildly denaturing temperatures at which the ligase enzymatic activity is
not lost.
Generally, the truncated ligaid will be at least about 5 nucleotides, more
usually at least about eight nucleotides, and not more than about thirty
nucleotides, more usually not more than about twenty nucleotides. The
phosphorylated ligaid will be at least twenty number percent larger than
the truncated ligaid more usually at least about thirty number percent
larger and may be one hundred number percent or greater than the truncated
ligaid.
Yet another additional way to accomplish labeling is to utilize a
phosphorylated ligaid in the initial ligase labeling reaction instead of
the unphosphorylated ligaid. See Figure 1d. Phosphorylation of the ligaid
can be achieved by using a kinase enzyme with an appropriate nucleotide
triphosphate, or, preferably, it can be chemically added during ligaid
synthesis. Lower yields will be encountered, based on the amount of
labeling moiety in relation to the amount of labeling achieved, because
ligaids will then ligate directly to first strands in a reaction that
competes with the desired labeling. A low yield at this stage may be
satisfactory if sufficient amplification is gained at a later stage.
The particular manner of ligation is not critical to this invention and any
convenient ligating conditions may be employed for either enzymatic or
chemical ligation.
The labeling moiety dsDNA will have a sequence (primer binding sequence)
which will serve for hybridization to a primer sequence for use in an
amplifying system. Any convenient sequence may be employed, particularly
sequences which are not likely to be encountered in the sample. While as
few as five nucleotides may suffice, normally at least eight nucleotides
will be employed, more usually at least twelve nucleotides, and in many
instances, one may employ thirty or more nucleotides. Various arbitrary
sequences may be used, where the sequences will be selected to minimize
binding of primers to other than the ligaid primer binding site. If
desired, one may have a plurality of primer binding sites present in the
ligaid, so that one may use different sequences as primers, depending upon
the nature of the sample. By having a plurality of primer binding sites,
one can determine which of the primer binding sites may be best suited for
a particular type of sample or target DNA. Alternatively, these primer
sites could be used in series to further reduce background. By providing
for a primer sequence foreign to the sample DNA, a target of unknown
sequence may be amplified.
Once the labeling moiety has been ligated to or filled-in adjacent to the
two strands of the dsDNA fragments at both termini, the resulting primer
binding site labeled fragments can be probed for the presence of a
particular sequence. See U.S. patent application Ser. No. 276,139, filed
Nov. 23, 1988 or EPA 88403266.5 for a discussion of possible means for
selection by hybridization prior to amplification. Denaturation is usually
the first step in such means for selection. The particular manner of
denaturation is not critical to this invention and any technique may be
employed. Elevated pH, employing a convenient hydroxide, low ionic
strength, elevated temperatures, chaotropes or denaturants, may be used
individually or in combination. The resulting single stranded DNA may then
be probed for the presence of a target sequence of interest for isolation
of hybridizing strands. A single probe may be employed, or a mixture of
probes, for example, where the protein sequence is known, but the DNA is
not. In this situation, one prepares a mixture of probes, based on the
redundancy of the codons. Various techniques may be used to identify the
strands which bind to the probe, so as to reduce the complexity of the
mixture which is subsequently to be amplified. Techniques include gel
electrophoresis, cesium chloride gradient centrifugation, particle
separation, filtration, etc.
Of particular interest is particle separation, where the probe has a
specific binding pair member, which member is a ligand or receptor,
normally a ligand, or is directly linked, covalently or non-covalently, to
a support which allows for separation, e.g., particles, well walls, etc. A
variety of complementary specific binding pair members are available, such
as biotin and avidin or streptavidin (strept/avidin), haptens and
antibodies, substrate and enzymes, ligand and surface membrane protein
receptors, complementary nucleic acid sequences and the like. The
biotin-strept/avidin combination is of particular interest because of the
high binding affinity of the pair.
The probe may be labeled in a wide variety of ways. For example,
nucleotides conjugated to, or which can be conjugated to biotin or other
ligands may be included during synthesis of the probe. Particles may then
be conjugated with the reciprocal specific binding pair member, so that
separation can be achieved by binding of the sample strands to the
particles by means of the probe and the specific binding pair. Among
particles, paramagnetic particles are preferred since they may be
separated by means of a magnetic field. Particularly, by employing tubular
vessels, the magnetic field may be used to orient the particles along the
side(s) of the tubular vessel for ease of removing all of the liquid from
the vessel without disturbing the particles and than washing the particles
vigorously to remove non-specifically bound nucleic acid.
Once the separation and removal of non-specifically bound nucleic acids has
been performed, the sample strands may be released by employing
denaturation conditions or by breaking the link joining the support to the
probe (e.g., reducing a disulfide link) and the liquid removed from the
vessel for further processing. Alternatively, the DNA can be amplified
directly off the support.
The primers may or may not be labelled, depending on the ultimate mode of
detection preferred. If the primer comprises a fluorescent label, the
amplification product fragment(s) will be fluorescent and can be detected
on a fluorescent gel scanner or other appropriate fluorescent detection
systems. Such labeling would also permit the analysis of fragments of
different samples in the same lane of a gel if different fluorescent dyes
were used for the amplification primers of different samples. Also, size
standards with unique fluorescent labels can be run in the same lane in
order to provide for in-lane size calibration. If the primer is not
labeled, the strands can be identified or detected by means of a probe or
probe mixture of known sequence and comprising a detectable entity or by
means of some other identifying molecule, such as a fluorescent molecule,
such as ethidium bromide which binds to DNA, or silver staining the DNA.
The next step is amplification, where the selected sample strands are
hybridized with the primer. Since both ends of the strands have been
attached to the same dsDNA labelling moiety, only a single primer is
required.
The sample DNA may be amplified by any means. The particular manner of
amplification is not critical to this invention, so long as the method
provides for sufficient reliability and production of a large enough
amount of DNA to be analyzed.
After sufficient cycles of denaturation and hybridization with the primers
and primer extension, the resultant DNA may be used in a variety of ways
for analysis. Where one is interested in small differences in sequences,
one may denature the double-stranded DNA and probe under stringent
conditions with a specific probe. Various techniques exist for identifying
the presence of a single site mutation, such as hybridization using
allele-specific oligonucleotide. In this manner, one may distinguish
between various polymorphs, pathogen strains, or the like.
In many other cases, one may be solely interested in the size of the
fragments as diagnostic of the sample. In this situation, one may employ
electrophoresis, separating the fragments by size and identifying the
nature of the sample by the size of the amplified DNA. In some instances,
it may be useful to do restriction analysis, where one may use one or more
restriction enzymes, particularly one restriction enzyme, and identify the
nature of the sample by the size of the resulting fragments or the
presence or absence of a particular restriction site. Other techniques may
also be employed.
The following examples are offered by way of illustration and not by way of
limitation.
EXPERIMENTAL
The following are exemplary labeling protocols.
Labeling Protocol
Described below is a protocol for labeling a sample of DNA with desired
priming sequences when the DNA of interest is restricted with the enzyme
HindIII. Analogous protocols would be used for other restriction enzymes.
Note the three possible variations of Step 4.
1. Oligonucleotide of the following sequences are synthesized using a Model
381A DNA Synthesizer (Applied Biosystems, Foster City, Calif.) and
standard protocols as recommended by the manufacture:
a. The "First Strand" oligo (see FIG. 1a), with the sequence 5'GGT GTG GTT
TGG TTG TGT TTG GTT GGT TTG TGG TGT T 3' (SEQ. ID. NO: 1:)
b. The "Ligaid" oligo (see FIG. 1a), with the sequence 5'AGC TAA CAC CAC
AAA C 3' (SEQ. ID. NO: 2:)
c. The "Phosphorylated ligaid" (see FIG. 1c), with the sequence 5'p-AGC TAA
CAC CAC AAA CCA ACC AAA CAC AAC CAA ACC ACA CC 3' (SEQ. ID. NO: 3:)
If desired, a fluorescent dye such as fluorescein may be added to the 5'
end of the First Strand oligo to track the labeling reaction. This would
be done using standard protocols for producing fluorescent labeled
oligonucleotide using the Amino-Link 2 compound from Applied Biosystems.
The Phosphorylated ligaid should be phosphorylated using the standard
kinasing protocols (see Maniatis et al., Handbook of Molecular Biology,
Cold Spring Harbor Press, 1982) or using a compound like 5' Phosphate-ON
from Clontech, Palo Alto, Calif.
2. In a 1.5 ml polypropylene tube, the following items are mixed together:
5.2 .mu.l of a 20 picomole per .mu.l of an aqueous solution of the First
Strand oligo.
5.2 .mu.l of a 20 picomole per .mu.l of an aqueous solution of the ligaid
oligo.
11.5 .mu.l of water
10.0 .mu.l of a buffer which is 100 mM Tris HCl, pH 7.5, 600 mM NaCl, 70 mM
MgCl.sub.2, and 1 mg/ml bovine serum albumin
5.0 .mu.l of 20 mM ribose ATP
3.30 .mu.l of 300 mM dithiothreitol
50 .mu.l of a 200 microgram per ml aqueous solution of the DNA to be
labeled
5.8 .mu.l of a 12 unit per .mu.l solution of HindIII enzyme
4.0 .mu.l of a 2 unit per .mu.l solution of T4 DNA ligase
3. The entire mixture is incubated at 37.degree. C. for 4 hours, resulting
in the digestion of the sample into HindIII restriction fragments that are
end-labeled with the First Strand oligo on the 5' end of each strand of
each restriction fragment.
4. The amplification primer binding site is then attached to the 3' end of
each restriction fragment using one of the three following techniques:
a. A fill-in protocol is accomplished by performing the following steps:
i. add the four deoxynucleotide triphosphates (A, T, C and G) to a final
concentration of 200 .mu.M each to the labeling mixture described above
following the incubation step.
ii. Klenow polymerase I is added to the mixture, using 1.5 units of enzyme
per .mu.g of DNA being labeled.
iii. Incubate for 10 minutes at 37.degree. C. The reaction is then stopped
with addition of EDTA to a final concentration of 20 mM or by freezing.
b. A displacement protocol is accomplished by performing the following
steps:
i. 6.5 .mu.l of a 20 picomole per .mu.l aqueous solution of the
Phosphorylated ligaid (see step 1) is added to the labeling mixture
following the initial restriction/ligation incubation.
ii. Heat the resulting mixture to 50.degree. C. for 10 minutes to
destabilize the Ligaid oligo and have it displaced by the phosphorylated
ligaid which will bind to the first strand, abutting the 3' end of each
strand of the restriction fragment.
iii. Continue the ligation procedure at 37.degree. C. for 30 minutes.
Sufficient ligase activity should still exist from the step 3 incubation
to allow ligation of the Phosphorylated ligaid to 3' end of each strand of
the restriction fragment; if not, additional ligase can be added.
C. a direct labeling protocol is accomplished by substituting the
Phosphorylated ligaid for the Ligaid oligo in step 2 above. This direct
labeling protocol is useful if inefficient labeling can be tolerated.
5. The labeled reaction products can then be used in the next step
(typically the hybridization and selection protocol) or else stored for
future use at 4.degree. C.
The following is an exemplary protocol for hybridization and selection of
sample DNA.
Hybridization/Selection
After the sample has been labeled with the desired priming sequences, a
probe is used to select out those particular fragments in the sample that
contain sequence homologous to the probe. Described below is an exemplary
protocol for the hybridization and selection process:
1. Biotinylated probe is made using a variant of the protocol described
above for ligating on priming sequences. The following mixture is
prepared:
14.0 .mu.l of 25 picomoles/.mu.l biotinylated oligo (synthesized with the
sequence 5' TNN NTT TTT TTT TTT TCA GTT ATG ATG TTG T 3', (SEQ. ID. NO:
5:), where N is a linker arm nucleotide (Molecular Biosystems, Inc., San
Diego, Calif.) to which biotin N-hydroxysuccinimide is coupled).
14.0 .mu.l of 25 picomoles/.mu.l of a complementary oligonucleotide
(synthesized with the sequence 5' ACA ACA TCA TAA CTG AAA 340 ) (SEQ. ID.
NO: 6:)
60 .mu.l of water
13.0 .mu.l of 10X Restriction Enzyme Buffer A (Boehringer Mannheim,
Indianapolis, Ind.)
6.5 .mu.l of 20 mM ATP
10.0 .mu.l of 1 .mu.g/.mu.l of purified probe DNA to be labelled, e.g., if
a probe for the plasmid pSP64 is made, purified pSP64 DNA is used.
10.0 .mu.l of AluI restriction enzyme (@8 units/.mu.l)
3.5 .mu.l of T4 DNA ligase (@3 units/.mu.l)
The total reaction volume of 130 .mu.l is incubated for 4 hours at
37.degree. C. and then quenched with the addition of 6.5 .mu.l of 0.2M
EDTA.
2. Streptavidin coated paramagnetic particles are prepared from
biotinylated particles (Advanced Magnetic Cambridge, Mass.;) 20 ml of the
commercial particles at 5 mg/ml are washed and then resuspended at 5 mg/ml
in 500 mM HEPES, pH 7.8 with 0.25% Tween-20. 0.1 ml of a 25% solution
(w/v) of succinic anhydride in dimethylformamide is added to each ml of
particle suspension. The mixture is placed on ice for 15 minutes, shaken,
and then put on a shaker bath at 37.degree. C. for 60 minutes. The
particles are magnetically separated, supernatant is removed and then the
particles are resuspended with a buffer which is 10 mM sodium phosphate,
pH 7.4, 0.15M sodium chloride, 1 mM EDTA, and 0.25% Tween-20. This
separation, supernatant removal and resuspension process (a "wash") is
then repeated two more times and then the particles are resuspended at 5
mg/ml in the same buffer. (This buffer is referred to as 1X SSPE plus
0.25% Tween- 20). 0.1 ml of a 10 mg/ml solution of streptavidin in the
same phosphate buffer is then added to each ml of the particle suspension.
The particles are then shaken at 30.degree. C. for one hour, magnetically
separated, and then washed three more times with 1X SSPE plus 0.25%
Tween-20 and finally resuspended in this buffer at 5 mg/ml of particles
and stored at 4.degree. C. Just prior to use, the particles are washed
with 1X SSPE plus 1.% Tween-20 followed by a wash in 160 mM sodium
carbonate, pH 10.2 with 1.0% Tween-20, (referred to as the alkaline wash
buffer), and finally resuspended to a concentration of 5 mg/ml in the
alkaline wash buffer. Alternatively, M280 streptavidin Dynabeads, Dynal
Great Neck, N.Y., may be used as the particles and do not require the
above derivitization steps.
3. A two-part hybridization mixture is prepared as follows:
3a. Solution A--115 ml of 1.0M sodium carbonate is mixed with 29 ml of 1.0M
sodium hydroxide and 43 ml of 0.2M trisodium EDTA.
3.b. Solution B--557 ml of 9.0M sodium perchlorate is mixed with 257 ml of
a 51.5% (w/v) aqueous solution of sodium polyacrylate. (The sodium
polyacrylate solution is prepared by slowing adding 50% (w/w) NaOH into
65% polyacrylic acid (Aldrich, Milwaukee, Wis.) on ice until a diluted
solution gives a pH of 8.0 to 8.5).
4. The hybridization mix is prepared by combining 10 .mu.l of solution A
and 60 .mu.l of solution B and then adding the following:
10 .mu.l of biotinylated probe, prepared as above and diluted to 100
femtomoles of biotinylated ends per .mu.l.
100 .mu.l of the DNA (which has been labeled with the priming sequences as
described previously) at a concentration of about 0.1 .mu.g/.mu.l.
5. The solution is then mixed well, incubated at 90.degree. C. for 15
minutes to denature the DNA, and then incubated for 15 minutes at
47.degree. C. to allow hybridization to occur.
6. After cooling to 37.degree. C., 40 .mu.l of the streptavidin coated
particles are added, mixed in the DNA solution, and allowed to incubate
for 15 minutes at 37.degree. C.
7. The particles are then pulled to one side of the 1.5 ml Eppendorf tube
with a magnet, the supernatant is removed, and the particles are
resuspended with fresh alkaline wash buffer at 60.degree. C. This washing
procedure is then done five more times. A final wash is then done in 100
mM NaCl with 0.5% Tween-20 at room temperature, supernatant is removed,
and the particles are ready for the amplification steps.
Amplification
In preparation for the amplification step, the primer sequence, 5' G TTT
GGT TGG TTT GTG GTG T (SEQ. ID. NO: 4:), is synthesized and diluted to a
concentration of 10 picomoles/.mu.l in water. An exemplary protocol for
the amplification of the labeled DNA which has been selected by the probe
and bound to the particles is performed as follows:
1. The particles are resuspended in 70 .mu.l of water and 20 .mu.l of 25%
chelex in water, allowed to incubate for 10 minutes as follows:
2. The particles are magnetically pulled to the side of the tube and 20
.mu.l of the supernatant is then mixed with the following:
28.5 .mu.l H.sub.2 O
10 .mu.l of buffer which is 100 mM Tris-HCl, pH 8.4, 500 mM KCl, 15 mM
MgCl.sub.2, 1 mg/ml gelatin (Perkin Elmer-Cetus 10.times. Amplification
Buffer)
1.0 .mu.l of 10 mM MgCl.sub.2
12 .mu.l of a mixture containing the deoxynucleotides dATP, dCTP, dGTP and
dTTP, each at a concentration of 2.5 mM.
24 .mu.l of the 10 picomole/.mu.l solution of the amplification primer
described above.
The mixture is then overlayed with mineral oil in a 500 .mu.l polypropylene
tube and heated to 90.degree. C. Then, 5 units of Taq polymerase (1 .mu.l
) is added and mixed into the aqueous solution.
3. Samples are placed in a Perkin Elmer-Cetus Thermal Cycler and amplified
using 25 of the following cycles: 94.degree. C. for 1 minute for
denaturation, 65.degree. C. for 2 minutes of annealing, and 72.degree. C.
for 6 minutes for extension. A 5 .mu.l aliquot of the sample is then
taken, 2 .mu.l of 15% Ficoll loading buffer is added, and the
amplification products are loaded onto a 0.8% agarose gel for separation
and detection. The gel is run for 4 hours at 3 volts/cm using TBE running
buffer including ethidium bromide at 0.1 .mu.g/ml in the buffer and gel
for staining the DNA. The particular band or bands corresponding to the
fragment length(s) which are selected by the probe are then detected.
Actual detection may be visual or with the use of a fluorescence gel
scanner. In the case of the probe being pSP64 prepared as described above
and the specific target being pSP64 in a sample cut by Hind III enzyme,
the detected band would be 3.0 kilobases long.
In accordance with the subject invention, numerous advantages are achieved.
The requirement for high efficiency at individual steps in order to detect
rare sequences is substantially reduced. Smaller amounts of sample are
required. A single oligo primer binding site is employed, so that only one
primer needs to be used during each cycle of the amplification. The
protocols may be automated using conventional materials, whereby
technician error may be substantially minimized and higher sensitivities
and accuracy achieved. By employing purified sequences, longer strands may
be copied with higher fidelity. The subject method provides for
amplification of DNA where the sequence of the target DNA may be only
partially known or unknown.
All publications and patent applications cited in this specification are
herein incorporated by reference as if each individual publication or
patent application were specifically and individually indicated to be
incorporated by reference.
Although the foregoing invention has been described in some detail by way
of illustration and example for purposes of clarity of understanding, it
will be readily apparent to those of ordinary skill in the art in light of
the teachings of this invention that certain changes and modifications may
be made thereto without departing from the spirit or scope of the appended
claims.
__________________________________________________________________________
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(iii) NUMBER OF SEQUENCES: 6
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 37 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
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