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Description  |
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BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to a rapid, convenient process for distinguishing
target nucleic acid segments on the basis of nucleotide differences
wherein the nucleic acid segments may differ in size, base composition, or
both.
2. Summary of the Background
The science of genetics is based on the identification and characterization
of mutations, which are changes in DNA (DNA polymorphisms) due to
nucleotide substitution, insertion, or deletion. Thus, many techniques
have been developed to compare homologous segments of DNA to determine if
the segments are identical or if they differ at one or more nucleotides.
Practical applications of these techniques include genetic disease
diagnoses, forensic techniques, and human genome mapping.
The most definitive method for comparing DNA segments is to determine the
complete nucleotide sequence of each segment. Examples of how sequencing
has been used to study mutations in human genes are included in the
publications of Engelke, et al., Proc. Natl. Acad. Sci. U.S.A. 85:544-548
(1988) and Wong, et al., Nature 330:384-386 (1987). At the present time,
it is not practical to use extensive sequencing to compare more than just
a few DNA segments, because the effort required to determine, interpret,
and compare sequence information is time-consuming.
For genetic mapping purposes, the most commonly used screen for DNA
polymorphisms arising from mutation consists of digesting DNA with
restriction endonucleases and analyzing the rsulting fragments by means of
Southern blots, as described by Botstein, et al.,Am. J. Hum. Genet.
32:314-331 (1980); White, et al., Sci. Am. 258:40-48 (1988). Mutations
that affect the recognition sequence of the endonuclease will preclude
enzymatic cleavage at that site, thereby alterning the cleavage pattern of
that DNA. DNAs are compared by looking for differences in restriction
fragment lengths. A major problem with this method (known as restriction
fragment length polymorphism mapping or RFLP mapping) is its inability to
detect mutations that do not affect cleavage with a restriction
endonuclease. Thus, many mutations are missed with this method. One study,
by Jeffreys, Cell 18:1-18 (1979), was able to detect only 0.7% of the
mutational variants estimated to be present in a 40,000 base pair region
of human DNA. Another problem is that the methods used to detect
restriction fragment length polymorphisms are very labor intensive, in
particular, the techniques involved with Southern blot analysis.
A technique for detecting specific mutations in any segment of DNA is
described in Wallace, et al., Nucl. Acids Res. 9:879-894 (1981). It
involves hybridizing the DNA to be analyzed (target DNA) with a
complementary, labeled oligonucleotide probe. Due to the thermal
instability of DNA duplexes containing even a single base pair mismatch,
differential melting temperature can be used to distinguish target DNAs
that are perfectly complementary to the probe from target DNAs that differ
by as little as a single nucleotide. An adaptation of this technique,
described by Saiki, et al., U.S. Pat. No. 4,683,194, can be used to detect
the presence or absence of a specific restriction site. In Saiki's
adaptation, an end-labeled oligonucleotide probe spanning a restriction
site is hybridized to the target DNA. The hybridized duplex of DNA is then
appropriately incubated with the restriction enzyme for that site. Only
paired duplexes between probe and target that reform the restriction site
will be cleaved by digestion with the restriction endonuclease. Detection
of shortened probe molecules indicates that the specific restriction site
is present in the target DNA. In a related technique, described in
Landegren, et al., Science 241:1077-1080 (1988), oligonucleotide probes
are constructed in pairs such that their junction corresponds to the site
on the DNA being analyzed for mutation. These oligonucleotides are then
hybridized to the DNA being analyzed. Base pair mismatch between either
oligonucleotide and the target DNA at the junction location prevents the
efficient joining of the two oligonucleotide probes by DNA ligase. A major
problem with these and other oligonucleotide techniques is that the
mutation must already be characterized as to type and location in order to
synthesize the proper probe. Thus, techniques using oligonucleotide probes
can be used to assay for specific, known mutations, but they cannot be
used generally to identify previously undetected mutations.
In the technique described in Mundy, U.S. Pat. No. 4,656,127, specific
mutations can be detected by first hybridizing a labeled DNA probe to the
target nucleic acid in order to form a hybrid in which the 3' end of the
probe is positioned adjacent to the specific base being analyzed. Then, a
DNA polymerase is used to add a nucleotide analog, such as a
thionucleotide, to the probe strand, but only if the analog is
complementary to the specific base being analyzed. Finally, the
probe-target hybrid is treated with exonuclease III. If the nucleotide
analog has been incorporated, the labeled probe is protected from nuclease
digestion. Absence of a labeled probe indicates that the analog and the
specific base being analyzed were not complementary. As with
abovediscussed techniques involving oligonucleotides, this method detects
specific mutations, but it cannot be used in a general manner to detect
all possible nucleotide differences.
Nucleotide differences between two DNA sequences also can be studied by
forming a heteroduplex between the two DNAs of interest. Base pair
mismatches will occur within the heteroduplex at points where the
sequences differ. A number of methods have been developed to detect such
mismatches. Chemical probes for mismatches exist that specifically react
with those atoms in the base normally involved in hydrogen bonding, see,
e.g., Novack, et al., Proc. Natl. Acad. Sci. U.S.A. 83:586-590 (1986);
Cotton, et al., Proc. Natl. Acad. Sci. U.S.A. 85:4397-4401 (1988). These
chemically altered sites are susceptible to chemical cleavage, whereas a
perfectly paired duplex is not. Problems with this technique include: (i)
toxicity of the chemical reagents and (ii) efficiency of much less than
100% for the reactions with unpaired bases. Another approach to mismatch
detection is based upon the ability of certain nucleases to recognize and
cleave these sites. S.sub.1 nuclease and RNase A have been shown effective
in mismatch detection and cleavage, see, e.g., Shenk, et al., Proc. Natl.
Acad. Sci. U.S.A. 72:989-993 (1975); Myers, et al., Science 230:1242-1246
(1985). Neither of these enzymes, however, cleaves at all possible
mismatched base pairs. There is also considerable background associated
with nuclease cleavage at perfectly paired sites in the duplex.
Myers, et al., Nature 313:495-498 (1985), and Fischer, et al., Proc. Natl.
Acad. Sci. U.S.A. 80:1579-1583 (1983), have demonstrated that mismatched
base pairs within a heteroduplex alter its melting properties with respect
to a perfectly paired homoduplex. These altered melting properties can be
observed electrophoretically on a gel containing an exponential gradient
of denaturant. A serious drawback to this technique is the difficulty of
manipulating and processing these gels. Another problem is the inability
of this technique to detect uniformly all base pair mismatches along a
given heteroduplex. The resolving power of these gels is reduced with
increasing GC content of the heteroduplex, and thus mutations in a GC-rich
domain are more difficult to detect than mutations in a domain with a
lower GC content.
The primer extension process described in Proudfoot, et al., Science
209:1329-1336 (1980), has been widely used to study the structure of RNA
and also has been used to characterize DNA, see, e.g. Engelke, et al.,
Proc. Natl. Acad. Sci. U.S.A. 85:544-548 (1988). This process consists of
hybridizing a labeled oligonucleotide primer to a template RNA or DNA and
then using a DNA polymerase and deoxynucleoside triphosphates to extend
the primer to the 5' end of the template. The labeled primer extension
product is then fractionated on the basis of size, usually by
electrophoresis through a denaturing polyacrylamide gel. When used to
compare homologous DNA segments, this process can detect differences due
to nucleotide insertion or deletion. Because size is the sole criterion
used to characterize the primer extension product, this method cannot
detect differences due to nucleotide substitution.
In order to be useful for a wide variety of applications, a technique to
detect nucleotide differences (mutations) in DNA should be simple, fast,
and able to detect any nucleotide difference that might occur and,
additionally, should not be dependent on the prior characterization of the
nucleotide difference. The currently available detection techniques
discussed above are deficient in one or more of these areas. Many of the
problems associated with these techniques are overcome by the present
invention.
The process of the present invention exploits the fact that the
incorporation of some nucleotide analogs into DNA causes an incremental
shift in mobility when the DNA is subjected to a size fractionation
process, such as electrophoresis. Others have noted that nucleotide
analogs can cause an electrophoretic mobility shift, see, e.g., Lo, et
al., Nucl. Acids Res. 16:8719 (1988); Dattagupta, et al., European Patent
Application No. 8602766.2 (published 1986), but it has not been realized,
nor is it obvious, that this property of nucleotide analogs can be used as
the basis for a process to identify previously undetected mutations.
SUMMARY OF THE INVENTION
The present invention provides a process for distinguishing nucleic acid
segments on the basis of nucleotide differences, which comprises
synthesizing separately complementary nucleic acid strands on each of at
least two target nucleic acid templates using a nucleic acid polymerase
and nucleoside triphosphate substrates, wherein at least one of the
natural nucleoside triphosphate substrates is replaced with a
mobility-shifting analog; denaturing the synthesized strands from the
templates if necessary; and comparing the mobility of the separately
synthesized strands through a size-fractionation medium.
DETAILED DESCRIPTION OF THE INVENTION
The present invention discloses a process for distinguishing nucleic acid
segments on the basis of one or more nucleotide differences. This process
has utility as a rapid, convenient means to identify previously undetected
mutations due to nucleotide substitution, insertion, or deletion.
In sum, this process uses a nucleic acid polymerase to synthesize
separately complementary nucleic acid strands on each of at least two
target nucleic acid templates, the sequence and number of nucleotides in
the synthesized strands being determined by the sequence and length of the
target nucleic acid templates. At least one of the natural nucleoside
triphosphates is replaced with a mobility-shifting analog. The separately
synthesized strands prepared from different target nucleic acid templates
are compared by subjecting the synthesized strands to passage through a
size-fractionation medium. Synthesized strands that are the same length
but differ in the number of mobilityshifting analog molecules per strand
will exhibit different mobilities when fractionated according to size. The
detection of a mobility difference indicates that the original target
nucleic acid segments are not identical. Thus, the process of the present
invention provides a rapid assay for distinguishing target nucleic acid
segments wherein the nucleic acid segments may differ in size, base
composition, or both.
Target nucleic acid segments to be analyzed by this process may be DNA or
RNA, and the DNA or RNA may be double stranded or single stranded. As
described in more detail in Mullis, et al., U.S. Pat. No. 4,683,195 and
Mullis, U.S. Pat. No. 4,683,202, any source of nucleic acid, in purified
or nonpurified form, can be utilized as the starting nucleic acid or
acids, if it contains, or is suspected of containing, the target nucleic
acid. The target nucleic acid can be only a fraction of a larger molecule
or can be present initially as a discrete molecule. Additionally, the
target nucleic acid may constitute the entire nucleic acid or may be a
fraction of a complex mixture of nucleic acids.
In the first step of the present process, complementary nucleic acid
strands are synthesized separately on at least two target nucleic acid
templates using a nucleic acid polymerase and nucleoside triphosphate
substrates, wherein at least one of the natural nucleoside triphosphate
substrates is replaced with a mobility-shifting analog. A preferred
embodiment of this process exploits the ability of most DNA polymerases
and some RNA polymerases to perform primer extension reactions. Execution
of a primer extension reaction requires a nucleic acid substrate
consisting of a primer hybridized to a template strand such that the 3'
end of the primer is recessed relative to the 5' end of the template
strand. As primer extension methods are most commonly practiced, the
primer is DNA, the template strand is either DNA or RNA, and a DNA
polymerase is used to extend the primer. The basic elements required for
execution of primer extension reactions are reviewed in Mullis, et al.,
U.S. Pat. No. 4,683,195, and Mullis, U.S. Pat. No. 4,683,202, and include
definition of a primer, size of primers, preparation of oligonucleotide
primers, methods for separating strands of double stranded nucleic acid,
preferable ratio of primer to template, conditions for mixing and
annealing primer to template strand, and conditions for extending the
primer to produce synthesized strands.
Any target nucleic acid segment can be used as a template and analyzed by
the primer extension process. It is only necessary that sufficient
sequence information at one or both ends of the target nucleic acid be
known so that an oligonucleotide primer or primers can be prepared that
will hybridize to the template strand containing the target nucleic acid
segment at a position such that the extension product synthesized from the
primer will use the target nucleic acid segent as template. For single
stranded target nucleic acid segments, the template strand is the strand
that contains the target nucleic acid segment. For double stranded target
nucleic acid segments, the template strand may be either of the two
strands that contains the target nucleic acid segment, and which strand is
used as a template is determined by the selection of the primer.
Additionally, if double stranded, the nucleic acid containing the template
strand is treated to separate the strands before being used to prepare
primer extension products. The primer and template strands are mixed,
allowed to anneal, and then treated with a nucleic acid polymerase.
For DNA template strands, suitable commercially available DNA polymerases
include the DNA polymerase obtained from the thermophilic bacterium
Thermus aquaticus (Taq polymerase), E. coli DNA polymerase I, Klenow
fragment of E. coli DNA polymerase I, reverse transcriptase, phage T4 DNA
polymerase, and phage T7 DNA polymerase. Structural variants and modified
forms of these and other DNA polymerases also can be used. For RNA
templates, reverse transcriptase is an example of a DNA polymerase that
can be used. In the presence of the four natural deoxyribonucleoside
triphosphates (dNTPs), or analogs for one or more of the dNTPs, the DNA
polymerase will extend the length of the oligonucleotide primer in the 3'
direction. For the method of this invention at least one of the natural
dNTPs is replaced with a mobility-shifting analog. Extension is achieved
by the attachment of the 5' phosphate group of an incorporating nucleotide
to the 3' hydroxyl of the previously incorporated nucleotide. The sequence
of the extension product will be complementary to the corresponding
sequence of the template strand. The reaction terminates when every base
in the template strand is represented in the primer extension product.
Adaptations and alternatives of the primer extension technique can also be
used with the process of the present invention. Double stranded nucleic
acid targets can be used to generate both the template and primer strands,
thereby eliminating the primer-template annealing step. By enzymatic or
chemical treatment of the double stranded nucleic acid, molecules can be
produced that have a recessed 3' strand and an overhanging 5' strand and
thus are substrates for nucleotide addition by a DNA polymerase. For
example, cleavage of DNA with many restriction enzymes generates 5'
overhangs that are substrates for DNA polymerases. Also, there are 3'
exonucleases that remove 3' nucleotides from double-stranded DNA,
producing molecules with 3' recessed strands and 5' overhanging strands.
By using an RNA-dependent RNA polymerase, such as QB replicase, primer
extension reactions can be performed using an RNA template and an RNA,
rather than DNA, primer. In this case, the mobility-shifting analogs would
be ribonucleotides rather than the deoxyribonucleotides used in
conventional primer extension.
If a DNA-dependent RNA polymerase is used, such as SP6 RNA polymerase, T3
RNA polymerase, T7 RNA polymerase, or E. coli RNA polymerase, the need for
a primer is eliminated entirely. What is required is a double stranded DNA
template that contains a specialized sequence element called a promoter.
Each RNA polymerase has its own specific set of promoter sequences,
typically 15 to 35 nucleotides in length. In Mullis, et al., U.S. Pat. No.
4,683,195, and Mullis, U.S. Pat. No. 4,683,202, a method is described for
attaching a noncomplementary sequence, such as an RNA polymerase promoter
sequence, to any segment being amplified by the polymerase chain reaction
(PCR) disclosed in these patents. A procedure for preparing RNA
transcripts using PCR-amplified templates is described in Stoflet, et al.,
Science 239:491-497 (1988). The RNA polymerase binds to the promoter
element and initiates RNA synthesis at a defined site on the DNA template,
thus generating an RNA transcript with a defined 5' end. By replacing at
least one of the four natural ribonucleoside triphosphates with a
mobility-shifting analog, these analogs can be incorporated into the RNA
transcript. The 3' end of the RNA transcript is usually determined by
having a defined end to the DNA template. RNA transcripts can be analyzed
by size fractionation in the same manner as primer extension products.
Mullis, et al., U.S. Pat. No. 4,683,195,and Mullis, U.S. Pat. No.
4,683,202, disclose PCR, which can can be used to amplify any specific
segment of nucleic acid. PCR is basically a series of successive primer
extension reactions, and it is an excellent and preferred process to use
for preparing target nucleic acid templates for the process of the present
invention because the final products of PCR are double stranded DNA
segments with defined ends. The ends of each of the amplified DNA segments
are determined by the 5' ends of the two oligonucleotide primers used to
initiate and maintain the PCR process. Mobility-shifting nucleotide
analogs can be incorporated into DNA as part of the PCR process by
replacing at least one of the four natural deoxyribonucleoside
triphosphates normally used in PCR with a mobility-shifting analog. By
performing PCR in this manner, homologous DNA segments can be
simultaneously amplified and prepared to detect mobility differences that
would indicate nucleotide substitutions, insertions, or deletions.
In the present process, the critical feature is the synthesis of nucleic
acid strands complementary to target nucleic acid segments wherein at
least one of the natural nucleoside triphosphate substrates is replaced
with a mobility-shifting nucleotide analog that changes the migration
properties of the synthesized strand through size-fractionation media. It
is known in the prior art that when nucleic acid strands synthesized with
natural nucleotides are fractionated on the basis of size, the mobility of
the synthesized strand through a separation medium is determined by the
total number of nucleotides present in the synthesized strand. When
comparing synthesized strands prepared from homologous target nucleic
acids and containing only natural nucleotides, a difference in mobility
indicates that the target nucleic acids contain different numbers of
nucleotides. Thus, target nucleic acids with nucleotide insertions or
deletions can be distinguished by techniques known in the art, however
nucleotide substitutions cannot be detected by such techniques.
The present invention discloses the substitution of mobility-shifting
analogs for natural nucleotides. A mobility-shifting nucleotide analog is
any nucleotide analog (i) that can be incorporated specifically into a
growing nucleic acid strand in place of one of the natural nucleotides,
(ii) that after being incorporated can function as substrate for further
additions of nucleotides to the growing nucleic acid strand without
terminating synthesis of the growing strand, and (iii) that causes the
nucleic acid to migrate at a position different than that expected from
its length when analyzed by passage through a size-fractionation medium.
For example, incorporation of biotin-11-dUTP (Enzo Diagnostics, Inc., New
York, N.Y.), an analog of TTP, into a DNA strand causes a one nucleotide
mobility shift when the DNA is fractionated on a sequencing gel. This
means that, for each biotin-11-dUTP residue incorporated, the DNA strand
migrates at a position one nucleotide slower than is expected based on the
length of the DNA strand.
Examples of commercially available compounds that can be used as
mobility-shifting analogs include biotin-11-dUTP, biotin-11-dCTP, and
biotin-11-UTP (Enzo Diagnostics, Inc., New York, N.Y.); biotin-7-dATP
(Bethesda Research Laboratories, Gaithersburg, Md.); digoxigenin-11-dUTP
(Boeringer Mannheim Biochemicals, Indianapolis, Ind.); and
5-([N-biotinyl]-3-amino-allyl)-2'-deoxyuridine 5'-triphosphate
(Bio-4-dUTP),
8-(N-[N-biotinyl-.epsilon.-aminocaproyl]-8-aminohexylamino)adenosine
5'-triphosphate (Bio-14-ATP), and N.sup.6
-(N-[N-biotinyl-.epsilon.-aminocaproyl]-6-aminohexyl-carbamoylmethyl)adeno
sine 5'-triphosphate (Bio-17-ATP) (Sigma Chemical Co., St. Louis, Mo.).
These compounds are commercially available because they contain biotin or
digoxigenin, which are small molecules that can be detected by convenient
and sensitive assay. For the purposes of this invention, it is not
required that the mobility-shifting nucleotide analog also contain a
moiety that is used for detection such as biotin, digoxigenin, or some
other reporter. The commercially available compounds listed above are a
small subset of the following families of modified nucleoside
triphosphates that can be used as mobility-shifting analogs.
##STR1##
described by Klevan, et al., WO 86/02929 (published 1986);
##STR2##
described by Ruth, WO 84/03285 (published 1984); and
##STR3##
described by Langer, et al., Proc. Natl. Acad. Sci. U.S.A. 78:6633-6637
(1981); Ward, et al., U.S. 4,711,955; Hobbs, et al., European Patent
Application No. 87305844.0 (published 1988). In the above structures,
=double or triple bond
Q=O, S, or NH
Y=H or OH
Z=H or NH.sub.2
R=H, acyl, aryl, heterocyclic, or alkyl radical, wherein the alkyl radical
can be straight-chained, branched, or cyclic. R can optionally contain
double bonds, triple bonds, aryl groups, or heteroatoms, such as N, O, S,
or halogens. The heteroatoms can be part of such functional groups as
ethers, thioethers, esters, amines, amides, or heterocycles.
R'=substituted or unsubstituted diradical moiety of 1-20 atoms. R' can be
straight-chained C.sub.1 -C.sub.20 alkylene and optionally can contain
double bonds, triple bonds, aryl groups, or heteroatoms, such as N, O, or
S. The heteroatoms can be part of such functional groups as ethers,
thioethers, esters, amines, or amides. Substituents on R' can include
C.sub.1 -C.sub.6 alkyl, aryl, ester, ether, amine, amide, or chloro
groups.
For generality, the above nucleotide analogs are shown with the
triphosphate group in the tetraacid form. When dissolved in water, the
tetraacid form of these compounds slowly decomposes due to the acidity of
the resulting solution (pH approximately 2). These compounds are therefore
generally prepared and used as tri- or tetrabasic salts at about pH 4 to
10.
A single mobility-shifting analog cannot be used to detect all possible
nucleotide substitutions. One way to detect all possible substitutions is
to have separate mobility-shifting analogs for each of the four natural
nucleotides. Then, by performing four separate strand synthesis reactions,
one with each of the analogs, target nucleic acids can be assayed for
differences involving any one of the four natural nucleotides. For double
stranded nucleic acid targets, all possible nucleotide substitutions can
be analyzed with just two mobility-shifting analogs by using each of the
two strands as template strand in separate strand synthesis reactions. For
synthesized DNA strands, one analog must be a dATP or TTP analog, and the
other analog must be a dCTP or dGTP analog. For synthesized RNA strands,
one analog must be an ATP or UTP analog, and the other analog must be a
CTP or GTP analog. Also, for primer extension reactions, a separate primer
complementary to each of the template strands is required. This analysis
of double stranded targets with just two analogs is possible because of
the complementary nature of double stranded nucleic acids. Any change that
affects an A in one strand must necessarily affect a T in the opposite
strand, and any change that affects a G in one strand must necessarily
affect a C in the opposite strand. Thus, by using two sets of two separate
strand synthesis reactions to test both strands of the target nucleic acid
with, for example, a T and a C analog, all possible single nucleotide
substitutions can be detected.
After the complementary nucleic acid strands are separately synthesized on
at least two target nucleic acid templates, wherein at least one of the
natural nucleotides is replaced with a mobilityshifting analog, the next
step of the present process is to denature the synthesized strands from
their respective templates, if the method of preparation results in
synthesized strands remaining hybridized to template strands. The duplex
must be denatured so that the synthesized strands can be analyzed by size
fractionation. Mullis, et al., U.S. Pat. No. 4,683,195, and Mullis, U.S.
Pat. No. 4,683,202, describe a variety of treatments that can be used to
denature or separate the strands of double stranded nucleic acid.
Next, the mobility of the synthesized strands is compared through a
size-fractionation medium. The synthesized strands prepared from target
nucleic acid templates are presumed identical but may, in fact, differ by
one or more nucleotide variations. When a mobility-shifting analog is used
in place of one of the natural nucleotides, synthesized strands that
contain different numbers of analog residues can be distinguished on the
basis of differential mobility. If the synthesized strands from the
different target nucleic acid templates are identical, they will all
contain the same number of mobility-shifting analogs and, therefore, will
migrate identically. Synthesized strands that differ in the number of
mobility-shifting analog molecules per strand (because some of the target
nucleic acids had a different sequence of bases) will exhibit different
mobilities when fractionated according to size. Thus, use of a
mobility-shifting analog makes base composition a major factor affecting
mobility through a sizefractionation medium.
If the resolution of the size fractionation process is sufficient, then
differences of a single nucleotide can be detected. The preferred process
for size fractionation is electrophoresis through a polyacrylamide gel
matrix containing a denaturant. One such denaturant is urea, although
others such as formamide or sodium hydroxide may be used. This type of gel
electrophoresis is used routinely in methods to determine the nucleotide
sequence of DNA, and it is preferably used in the present process because
it can separate nucleic acid fragments smaller than about 500 nucleotides
with single nucleotide resolution. Thus, if the incremental mobility shift
produced by a single analog residue is equivalent to the shift produced by
an additional natural nucleotide, then nucleotide substitutions as small
as a single nucleotide can be detected on such a sequencing gel.
Interpretation of mobility results requires that the synthesized strands
prepared from each target nucleic acid be of discrete size. The 5' end of
a primer extension product is defined by the 5' end of the primer strand
used to initiate synthesis. The 3' end of a primer extension product is
usually determined by having a defined 5' end on the template strand. The
defined 5' end of the template strand can be a natural defined end as in
mRNA; an end produced by sequence-specific enzymatic or chemical cleavage,
such as the type of end produced by cleavage with a restriction
endonuclease; or an end determined by the method used to produce the
template strand, as is the case when the polymerase chain reaction is used
to prepare target DNA. Alternatively, the 3' end of the primer extension
produce can be determined by cleaving the primer extension product with a
sequence-specific enzymatic or chemical cleaving agent.
In order to compare the mobility of the synthesized strands, the strands
must be detected. Any method used to detect nucleic acid can be used to
detect the synthesized strands containing mobility-shifting nucleotide
analogs. One method is to perform the primer extension reaction with a
labeled primer. Typically, oligonucleotide primers are labeled by
attaching a reporter to the 5' end of the oligonucleotide. This reporter
can be a radioactive isotope; a group that takes part in an enzyme or
fluorescent or chemiluminescent reaction; or some other small molecule,
such as biotin, that can be detected by a convenient method.
Alternatively, labeling can be performed by incorporating reporter-tagged
nucleotides into the synthesized strands as they are synthesized. The
reporter can be attached either to a mobility-shifting nucleotide analog
or to one of the other nucleotides used as substrates in the preparation
of synthesized strands. The same type of reporters used to label primers
can be used to label the nucleotide substrates. Another method of
detection entails hybridizing a reporter-labeled probe to the synthesized
strands after the size fractionation step, as in the Southern blot
procedure.
The detection of newly synthesized nucleic acid strands with mobility
differences indicates that the original target nucleic acid segments are
not identical. When comparing synthesized strands prepared from presumed
identical target nucleic acid templates, a difference in mobility
indicates that the target nucleic acid templates contain different numbers
of total nucleotides and/or different numbers of nucleotides complementary
to the mobility-shifting analogs. Thus, as illustrated in the following
Examples, the process of the present invention can be used to distinguish
homologous DNAs or RNAs differing by nucleotide substitutions that affect
the number of mobility-shifting analog residues incorporated into
synthesized strands, as well as to distinguish differences due to
nucleotide insertion or deletion.
EXAMPLES
The following examples illustrate, but do not limit, the process of the
present invention. Example 1 discloses the preparation of a preferred
mobility-shifting analog, 5-(Bio-AC-AP3)dCTP for the present process.
Examples 2 and 3 demonstrate detection of a single nucleotide substitution
that distinguishes two alleles of the human insulin receptor gene through
the use of the present pocess. In Example 2, a commercially available
nucleotide analog is used as the mobility-shifting analog. In Example 3,
the preferred mobility-shifting analog prepared in Example 1 is used.
EXAMPLE 1
Preparation of
5-(3-(6-Biotinamido(Hexanoylamido))-1-Propynyl)-2'-Deoxycytidine
5'-Triphosphate. 5-(Bio-AC-AP3)dCTP
The triethylammonium salt of 5-(3-amino-1-propynyl)-2'-deoxycytidine
5'-triphosphate, 5-(AP3)dCTP,
##STR4##
is prepared as follows.
5-(3-Trifluoroacetamido-1-propynyl)-2'-deoxycytidine [100.9 mg; 248 umol
(corrected for isopropanol content)] prepared as described in Example 8 of
Hobbs, et al., European Patent Application No. 87305844.0 (published
1988), was dissolved in trimethyl phosphate (0.5 mL). Phosphorus
oxychloride (47 .mu.L; 500 .mu.mol) was added and the mixture was stirred
at ambient temperature under an argon atmosphere for 30 min. The reaction
mixture was added dropwise to a 1.0 M solution of
tris(tri-n-butylammonium) pyrophosphate in DMF (1.5 mL; 1.5 mmol) and the
resulting solution was stirred at ambient temperature under argon for 10
min. The reaction mixture was quenched by adding it dropwise to an
ice-cooled solution of triethylamine (350 .mu.L) in water (5 mL).
After standing overnight at 0.degree. C., the solution was stripped down,
redissolved in water (25 mL), and loaded onto a DEAE Sephadex A-25-120
column (1.6.times.55 cm) that had been equilibrated with 0.1 M aqueous
triethylammonium bicarbonate (TEAB), pH 7.6. The column was eluted with a
linear gradient of TEAB, pH 7.6, from 0.1 M (300 mL) to 1.0 M (300 mL),
running at c. 100 mL/hr, and collecting fractions every 6 min. The eluent
was monitored by absorbance at 270 nm, and the fractions corresponding to
the major band (#45-50) were ooled, stripped, and co-evaporated (2x) with
ethanol.
The residue was taken up in water (0.70 mL), concentrated aqueous ammonia
(0.70 mL) was added, and the solution was stirred in a stoppered flask at
ambient temperature for 1 hour. The solution was bubbled with argon,
lyophilized, taken up in 0.1 M TEAB, and loaded onto a DEAE Sephadex
column (1.times.30 cm). The column was eluted with a linear gradient of
TEAB, pH 7.6, from 0.1 M (150 mL) to 1.0 M (150 mL), running at c. 100
mL/hr, and collecting fractions every 3 min. Again, the eluent was
monitored at 270 nm and the fractions corresponding to the major peak
(#28-35) were pooled, stripped, and coevaporated (2x) with ethanol. The
product was assayed by UV absorbance assuming an absorption coefficient at
294 nm equal to that of the starting material in water (10,100). The yield
was thus 54 .mu.mol (21%). The product was shown to be 97.5% pure by HPLC
on Zorbax SAX eluting with aqueous potassium phosphate, pH 6.5.
Next, the triethylammonium salt of
5-(3-biotinamido(hexanoylamido))-1-propynyl)-2'-deoxycytidine
5'-triphosphate, 5-(Bio-AC-AP3)dCTP,
##STR5##
was prepared from 5-(3-amino-1-propynyl)-2'-deoxycytidine 5'-triphosphate,
5-(AP3)dCTP. 5-(3-Amino-1-propynyl)-2'-deoxycytidine 5'-triphosphate (30
umol) was dissolved in 1 M aqueous TEAB, pH 7.6, (600 .mu.L) and
sulfosuccinimidyl 6-(biotinamido)-hexanoate Na salt (Piece Chemical Co.,
Rockford, IL; 33 mg; 60 .mu.mol) was added. The solution was held at
50.degree. C. for 90 min and then diluted to 6 mL with water. The solution
was loaded onto a DEAE Sephadex A-25-120 column (1.times.19 cm) that had
been equilibrated with 1.0 M aqueous TEAB, pH 7.6. The column was eluted
with a linear gradient of TEAB, pH 7.6, from 0.1 M (150 mL) to 1.0 M (150
mL), running at c. 100 mL/hr, collecting fractions every 3 min. The eluent
was monitored by absorbance at 270 nm, and the fractions corresponding to
the second peak (#22-33) were pooled, stripped, and co-evaporated (2x)
with ethanol. The residue was subjected to reverse phase chromatography
(Baker 7025-00) on a column (1.times.8 cm) poured in acetonitrile/1 M
aqueous TEAB, pH 7.6. The column was eluted with a step gradient of
acetonitrile/1 M TEAB, pH 7.6, (0-30% acetonitrile, 2%/step, 2 mL/step, 1
fraction/step). The fractions were assayed by HPLC on a reverse phase
column under conditions analogous to those used in the above separation.
Starting material eluted first (#2-6) followed by the product (#7-11). The
product fractions were pooled, stripped, and coevaporated (2x) with
ethanol. The yield, assuming an absorption coefficient of 10,100 at 295.5
nm, was 10.5 umol (35%). HPLC on Zorbax SAX and on reverse phase showed
the material to be >99% pure. The .sup.1 H- and .sup.31 P-NMR spectra are
fully consistent with the title structure.
EXAMPLE 2
Comparison of a Segment of Two Alleles of the Human Insulin Receptor Gene
that Differ by a Single Nucleotide Substitution (Primer Extension Analysis
Using the Mobility-Shifting Analog, Biotin-11-dUTP)
The sequence of a 140-bp region of the structural gene encoding the human
insulin receptor gene is shown below.
##STR6##
This sequence was determined from the cDNA sequence reported in Ullrich,
et al., Nature 313:756 (1985). By analyzing patient DNA, Kadowaki, et al.,
Science 240:787-790 (1988) characterized a nonsense mutation resulting
from a C to T base change at nucleotide 2143. A "normal" individual herein
is heterozygous for a C at position 2143. The other individual is
heteroxygous at this locus. In the heteroxygote, the paternal copy of this
gene has the C to T nonsense mutation at position 2143, whereas the
maternal copy has the normal C at this position. (The genomic DNA samples
were obtained from Dr. Domenico Accili, National Institutes of Health,
Bethesda, Md.)
PCR reactions to amplify this region from the genomic DNAs of the two
individuals were performed in 0.5-mL Eppendorf microfuge tubes as follows.
One microgram of genomic DNA in 5 .mu.L Te buffer (10 mM Tris-HC1, pH 8.0;
1 mM EDTA) was placed in a microfuge tube. Separate tubes contained DNAs
from individuals of different genotypes. To each tube was added 40 ng of
primer A (5'-CCTGGTCTCCACCATTCG-3') in 10 .mu.L and 40 ng of primer B
(5'-CTTCCTAAACGAGGACTCCT-3') in 10 .mu.L, bringing the volume to 25 .mu.L.
Four microliters of 10X Taq polymerase reaction buffer (166 mM
(NH.sub.4).sub.2 SO.sub.4 ; 670 mM Tris-HC1, pH 8.8; 45 mM MgC1.sub.2 ;
100 mM 2-mercaptoethanol; 1700 ug/mL bovine serum albumin) and five units
(1 .mu.L) Taq polymerase (Perkin-Elmer Cetus, Norwalk, Conn.) were added
to the reaction mix. Six microliters of stock dNTPs were added to a final
concentration of 187 .mu.M each for dATP, dGTP, dCTP, and TTP.
Filter-sterilized, deionized water was added to bring the reaction volume
to 40 .mu.L. The reaction was then overlayed with mineral oil. Reactions
were performed in a DNA Thermal Cycler (Perkin-Elmer Cetus, Norwalk,
Conn.) as follows: 94.degree. C. (7 min), 50.degree. C. (3 min),
72.degree. C. (4 min) for one cycle and then 30 cycles of 94.degree. C. (2
min), 50.degree. C. (3 min), 72.degree. C. (4 min) with one second
transition steps.
The PCR-amplified template was then applied to parafilm, which absorbs the
mineral oil, leaving the aqueous reaction phase. Unincorporated dNTPs were
removed from this aqueous phase by passing it over a G-50 spin column (New
England Nuclear, Boston, Mass.). The effluent was collected in a 1.5-mL
Eppendorf microfuge tube, precipitated by the addition of 1 volume of 5 M
ammonium acetate and 3 volumes of absolute ethanol, and incubated in a
methanol:dry ice slurry for 20 minutes. DNA was collected by
centrifugation at 12,200 rpm (12,400.times.g) for 20 minutes at room
temperature. The supernatant was pipetted off and the pellet was washed
with 200 .mu.L 70% (v/v) ethanol, and dried under vacuum for 15 min in a
Speed-Vac concentrator (Savant Instruments, Inc., Hicksville, N.Y.). Each
DNA sample was resuspended in 20 .mu.L of filter-sterilized, deionized
water and stored at 4.degree. C.
Primer C (5'-CTGAAGATTCTCAGAAGCAC-3'), shown above, was selected to prime
the extension reaction. Using the PCR-amplified DNA as template, this
primer should yield an extension product 119 nucleotides long that
includes the C to T nonsense mutation. The primer extension product from
the normal allele found in the homozygote and the heteroxygote should
contain 20 Ts or T analogs. The mutant allele from the heterozygote should
generate a primer extension product containing 21 Ts or T analogs.
Primer C was radiolabeled with .gamma.-.sup.32 P-ATP as follows. Twenty
microliters (200 .mu.Ci) of .gamma.-.sup.32 P-ATP (6000 Ci/mmol; New
England Nuclear, Boston, Mass.) was placed in a 1.50-mL Eppendorf
microfuge tube and dried under vacuum. One microliter of 10X kinase buffer
(700 mM Tris-HC1, pH 7.5; 100 mM MgC1.sub.2 ; 50 mM dithiothreitol) was
combined with 8 .mu.L (320 ng) of primer C in the presence of 5 units (1
.mu.L) of T4 polynucleotide kinase (New England BioLabs, Beverly, Mass.)
and added to the microfuge tube containing the radiolabel. The reaction
was conducted at 37.degree. C. for one hour. The reaction was stopped with
200 .mu.L 100 mM TrisHC1, pH 7.8/1 mM EDTA and subjected to NENSORB.RTM.
20 chromatography (New England Nuclear, Boston, Mass.) to remove unreacted
.gamma.-.sup.32 P-ATP from the reaction. This chromatographic step
involves activating the column with methanol, washing the activated column
with 100 mM Tris-HC1, pH 7.8/1 mM EDTA, applying the kinased sample,
washing the column with buffer and filter-sterilized, deionized water, and
eluting the kinased primer from the column with 20% (v/v) ethanol. The
liquid portion of the eluate was removed by vacuum in a Speed-Vac
concentrator; the resulting residue was resuspended in 100 .mu.L of
filter-sterilized, deionized water, and stored at -20.degree. C.
For the DNA from each individual of different genotype, 10 .mu.L of
PCR-amplified template was combined with 20 .mu.L of phosphorylated primer
(64 ng; 9 pmol; .about.10.sup.7 cpm). This mixture was heated to
94.degree. C. for approximately 5 minutes, iced, and centrifuged at 12,200
rpm (12,400.times.g) for 60 seconds to sediment any condensate to the
bottom of the centrifuge tube. The template:primer mixture was stored on
ice until all other reactants were combine | | |
