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Description  |
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FIELD OF THE INVENTION
This invention relates to a process for distinguishing nucleic acids. More
particularly, random nucleic acid segments are distinguished by comparing
nucleotide differences in extension reaction products.
BACKGROUND OF THE INVENTION
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. 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, human genome mapping and agricultural
applications.
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 extensive.
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 resulting fragments, 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 altering the cleavage pattern of the DNA. DNAs are
compared by looking for differences in restriction fragment lengths. A
major drawback to 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
difficulty is that the methods used to detect restriction fragment length
polymorphisms are very labor intensive, in particular, the techniques
involved with Southern blot analysis.
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.
Mullis et al., U.S. Pat. No. 4,683,195, and Mullis, U.S. Pat. No. 4,683,202
disclose polymerase chain reactions which can be used to amplify any
specific segment of a nucleic acid. These analytical methods have been
used to detect polymorphisms through amplification of selected target DNA
segments from test genomes. A drawback to such methods is the requirement
that a sufficient number of bases at both ends of the specific segment be
known in sufficient detail so that two oligonucleotide primers can be
designed which will hybridize to different strands of the target segment.
It is labor intensive to obtain the necessary sequence information from
target genomes in order to design the necessary primers. M. H. Skolnic and
R. B. Wallace, Simultaneous Analysis of Multiple Polymorphic Loci Using
Amplified Sequence Polymorphisms (ASPs), Genomics 2:273-278.
Amplification using oligonucleotides of random sequence is described in a
theoretical approach for mapping a genome of a higher eukaryote in "Happy
Mapping: a Proposal for Linkage Mapping the Human Genome," P. H. Dear and
P. R. Cook, Nucleic Acids Research, Vol. 17, No. 17, 6795-6807 (1989). The
authors propose to identify the relationship between 5,000 different DNA
segments amplified from human genomic DNA by performing a polymerase chain
reaction (PCR) with various pairwise combinations of arbitrary-sequence
primers. The template DNA is isolated separately from single haploid cells
of an individual organism. Their method suggests generating a map
describing the physical location of DNA regions giving rise to the
amplified products in an individual organism. This would be accomplished
by identifying PCR products which co-amplify from the same piece of DNA,
thus establishing their physical proximity. In Dear and Cook's method,
polymorphisms would be excluded from the analysis. Their idea requires the
identification of the same DNA segment in many different haploid cells,
and polymorphisms defeat this requirement. Although efficient at
describing the physical relationship between any two PCR products, happy
mapping cannot describe the location of any genetic traits of interest.
This can only be accomplished by correlating the segregation of a genetic
trait with many different polymorphisms through a sexual cross between two
individuals. The authors teach that primer pairs longer than 9-10
nucleotides are necessary as primer pairs of 9-10 nucleotides would prime
inefficiently.
The present invention provides a process for the detection of genetic
polymorphisms in random nonspecific nucleic acid segments. The process
utilizes a reaction primed by an oligonucleotide(s) conveniently prepared
with no knowledge of the base sequence of segments amplified.
SUMMARY OF THE INVENTION
The present invention provides a process for distinguishing nucleic acids
on the basis of nucleotide differences in random segments of the nucleic
acid comprising:
(a) separately performing an extension reaction on each of at least two
nucleic acids, said reaction comprising:
(i) contacting the nucleic acids with at least one oligonucleotide primer
of greater than 7 nucleotides under conditions such that for at least one
nucleic acid an extension product of at least one primer is synthesized;
and
(b) comparing the results of the separately performed extension reactions
for differences.
In another aspect of this invention, after step (i) and before step (b),
there are provided the additional steps of (ii) dissociating the extension
product from its complement; and (iii) amplifying the random nucleic acid
segment by contacting the dissociated extension product with a primer of
step (i) under conditions such that an amplification extension product is
synthesized using the dissociated extension product as a template.
In another aspect of this invention, a difference in the extension product
is used as a marker to construct a genetic map, and to distinguish or
identify individuals.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 illustrates a photograph of a gel comparing amplification products
from DNA isolated from the soybeans Glvine max (Bonus) (odd-numbered
lanes) and Glycine soja (PI 81762) (even-numbered lanes) where
oligonucleotide primers of a variety of sequences were employed.
FIGS. 2A-2D illustrate a photograph of a gel comparing amplification
products, utilizing primer 10b, of 19 F2 progeny of a cross between Bonus
and PI 81762.
FIGS. 3A and 3 B illustrate a photograph of a gel comparing amplification
products from Bonus (odd-numbered lanes) and PI 81762 (even-numbered
lanes). Primer AP8 and 10 other primers differing in a single nucleotide
from AP8 were employed.
FIG. 4 illustrates a photograph of a gel comparing amplification products
employing a variety of primers on Zea mays (corn), Gossypium hirsutum
(cotton), Sacchoromyces cerevisiae (yeast) and Arabidopsis. thaliana DNA.
FIG. 5 illustrates a photograph of a gel demonstrating amplification by
priming with primers of six to ten nucleotides in length.
FIG. 6 illustrates a photograph of a gel comparing amplification products
from six transformed Homo sapien (human) lymphoblasts.
FIG. 7A-7C illustrate densitometric scans demonstrating F2 individuals of a
cross between Bonus and PI 81762 homozygous (2 copies) for a polymorphism
(FIG. 7A), heterozygous for a polymorphism (FIG. 7B) and homozygous (0
copies) for a polymorphism (FIG. 7C).
DETAILED DESCRIPTION OF THE INVENTION
The term "oligonucleotide" as used herein refers to a molecule comprised of
two or more deoxyribonucleotides or ribonucleotides.
The term "primer" as used herein refers to an oligonucleotide of any
arbitrary sequence whether occurring naturally as in a purified
restriction digest or produced synthetically, which is capable of acting
as a point of initiation of synthesis when placed under conditions in
which synthesis of a primer extension product which is complementary to a
nucleic acid strand is induced, i.e., in the presence of nucleotides and
an agent for polymerization such as DNA polymerase and at a suitable
temperature and pH. It is preferable that primers are sequences that do
not form a secondary structure by base pairing with other copies of the
primer or sequences that form a "hair pin" configuration. The sequence
conveniently can be generated by computer or selected at random from a
gene bank. The primer is preferably single stranded for maximum efficiency
in amplification, but may alternatively be double stranded. If double
stranded, the primer is first treated to separate its strands before being
used to prepare extension products. Preferably, the primer is an
oligodeoxyribonucleotide.
The term "random nucleic acid segment" as used herein refers to any
template, the segment of nucleic acid to be addressed by the primer, of
which no sequence information need be known prior to extension. Applicants
have found that it is not required, with the process of this invention,
that a sufficient number of bases at both ends of a template be known in
sufficient detail so that primers can be designed which will hybridize to
different strands of the template.
In the process of the present invention, at least one primer of greater
than 7 nucleotides is provided. Primers with less than 7 nucleotides prime
inefficiently. Primers can be synthesized by standard techniques known to
those skilled in the art. In the preferred form of the invention, at least
one primer of 9 to 10 nucleotides in length is employed. Conveniently, one
primer is employed. At least one primer employed may be biotinylated. That
is, the primer may be covalently linked to a biotin or an analog of
biotin. The linking group employed should be chosen to permit extension
under the conditions detailed herein.
The process of the present invention for distinguishing nucleic acids
compares differences in products of an extension reaction(s) separately
performed on at least two random segments of nucleic acids.
In the first step of the present process, an extension reaction is (a)
separately performed on each of at least two nucleic acids by (i)
contacting the nucleic acids with at least one oligonucleotide primer of
greater than 7 nucleotides under conditions such that for at least one
nucleic acid an extension product of at least one primer is synthesized
which is complementary to the random nucleic acid segment. A preferred
embodiment of this extension reaction exploits the ability of most DNA
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.
The nucleic acids to be analyzed by this process may be DNA or RNA, and the
DNA or RNA may be double stranded or single stranded. Any source of
nucleic acid, in purified or nonpurified form, can be utilized as the
starting nucleic acid. For example the nucleic acid may be from a natural
DNA or RNA from any source, including virus, bacteria, and higher
organisms such as plants, animals and microbes or from cloned DNA or RNA.
Additionally, the nucleic acid may constitute the entire nucleic acid or
may be a fraction of a complex mixture of nucleic acids. Preferably the
nucleic acid is deoxyribonucleic acid. Surprisingly, Applicants have found
the process of the present invention useful in distinguishing nucleic
acids from different individual organisms containing small genomic DNA
such as yeast DNA (2.times.10.sup.7 base pairs per genome) as well as
different individual organisms containing large genomic DNA such as human
DNA (3.0.times.10.sup.9 base pairs per genome).
The choice of nucleic acid polymerase used in the extension reaction,
depends on the nature of the template. For DNA template strands, suitable
commercially available DNA polymerase includes DNA polymerase obtained
from the thermophilic bacterium Thermus aquaticus (Taq polymerase) or
other thermostable polymerases. Structural variants and modified forms of
this and other DNA polymerases would also be expected to be useful in the
process of the present invention. For RNA templates, reverse transcriptase
is an example of a DNA polymerase that would also be expected to be
useful. In the presence of the nucleoside triphosphate substrates, natural
or analogues, the polymerase extends the length of the primer in the 3'
direction. The sequence of the extension product will generally be
complementary to the corresponding sequence of the template strand.
The nucleoside triphosphate substrates are employed as described in PCR
Protocols, A Guide to Methods and Applications, M. A. Innis, D. H.
Gelfand, J.-J. Sninsky and T. J. White, eds. pp. 3-12, Academic Press
(1989), which is incorporated by reference, and U.S. Pat. Nos. 4,683,195
and 4,683,202. The substrates can be modified for a variety of
experimental purposes in ways known to those skilled in the art. As an
example, at least one of the natural nucleoside triphosphate substrates
may be replaced by a mobility-shifting analogue as taught in U.S. Pat. No.
4,879,214 which patent is incorporated by reference.
In another aspect of this invention, after step (i) is performed, the
additional steps of (ii) dissociating the extension product from its
complement; and (iii) amplifying the random nucleic acid segment by
contacting the dissociated extension product with a primer of step (i)
under conditions such that an amplification extension product is
synthesized using the dissociated extension product as a template are
performed.
An amplification reaction is generally described in Mullis et al., U.S.
Pat. No. 4,683,195 and Mullis, U.S. Pat. No. 4,683,202, which patents are
incorporated by reference. Specific conditions for amplifying a nucleic
acid template are described in M. A. Innis and D. H. Gelfand, PCR
Protocols, A Guide to Methods and Applications, M. A. Innis, D. H.
Gelfand, J.-J. Sninsky and T. J. White, eds. pp. 3-12, Academic Press
(1989), which is incorporated by reference.
Specifically, Mullis is directed to a process for amplifying any desired
specific nucleic acid sequence contained in a nucleic acid or mixture
thereof. The process of Mullis comprises treating separate complementary
strands of the nucleic acid with a molar excess of two oligonucleotide
primers, and extending the primers to form complementary primer extension
products which act as templates for synthesizing the desired nucleic acid
sequence. The primers of Mullis are designed to be sufficiently
complementary to different strands of each specific sequence to be
amplified. The steps of the reaction may be carried out stepwise or
simultaneously and can be repeated as often as desired.
In the extension reactions of the present invention, a nucleic acid is
contacted with at least one oligonucleotide primer as described herein.
The extension product is dissociated from the complementary random nucleic
acid on which it was synthesized to produce a single-stranded molecule;
and the random nucleic acid segment is amplified by contacting the
single-stranded extension product with a primer from above under
conditions as for example disclosed in PCR Protocols and U.S. Pat. No.
4,683,202 such that an amplification extension product is synthesized
using the single strand produced (i.e., the dissociated extension product)
as a template.
The steps described herein may be conducted sequentially or simultaneously
as taught by U.S. Pat. Nos. 4,683,195 and 4,683,202. In addition, the
steps may be repeated until the desired level of sequence amplification is
obtained as taught by U.S. Pat. Nos. 4,683,195 and 4,683,202.
Following extension, the results of step (b) are compared for differences.
Generally the results are products or an absence of product. Specifically,
the products are compared to determine if the segments are identical or
for differences in one or more nucleotides, by nucleotide substitution,
insertion or deletion. Differences in the products can take the form of,
for example, difference in size of each product, difference in the
sequence of each product, difference in the quantity of each product
produced, and differences in the number of products.
Comparison of the product can be accomplished by a variety of techniques
known to those skilled in the art. Examples include nucleotide sequence
determination of the amplified segment and RFLP analysis. Conveniently,
comparison can be through a size fractionation medium. If the resolution
of the size fractionation process is sufficient, differences of a single
nucleotide can be detected. The preferred process for size fractionation
is electrophoresis through a polyacrylamide or agarose gel matrix.
Simple inspection of the electrophoresis gel of the product can reveal
polymorphisms that affect the size and quantity of the amplified segment
and polymorphisms that determine whether a segment is amplified.
Polymorphisms revealed by the present invention can be quantified by known
densitometric procedures performed on the amplified product in the gel.
Quantification is important in certain applications where it is necessary
to distinguish homozygous from heterozygous polymorphisms. Applicants have
found that a homozygote with two copies of the polymorphism generates
twice as much of the product than does a heterozygote.
The process of the present invention has practical applications. Examples
of such applications include genetic mapping and distinguishing and
identifying individuals.
Applicants have found that the polymorphisms or differences identified
through the process of this invention can have utility in genetic mapping.
Commonly, a genetic map is constructed by through RLFP analysis. A genetic
map of an organism could be constructed, e.g. by substituting
polymorphisms, identified using different primers on the same organism,
detected by the present invention for the RFLPs used as genetic markers as
described in the art, e.g., Botsein, D., "Construction of a Genetic
Linkage Map in Man Using Restriction Fragment Length Polymorphisms", Am.
J. Hum. Genet. 32:314-331, (1980) which reference is incorporated herein.
Genetic mapping using the polymorphisms of the present invention as
markers has important plant breeding applications as Applicants have
determined that polymorphisms detected by the present invention are
heritable genetic markers. These markers, just as RFLP markers in
Tanksley, S. D., "RFLP Mapping in Plant Breeding: New Tools for an Old
Science", Bio/Technology Vol. 7 March, 1989, pp.257-264, can be used as
probes for the presence or absence of certain chromosome segments as
Applicants have found that the polymorphisms detected by the process of
the present invention can co-segregate with standard RFLP markers.
Further, the process of the present invention can be used to construct a
nucleic acid `fingerprint`. Such fingerprints are specific to individuals
and can be applied to problems of identification or distinguishing of
individuals. Such a `fingerprint` would be constructed using multiple
polymorphisms generated by different primers and detected by the present
invention, just as the polymorphisms are used to create a fingerprint in
Jeffreys, A. J., "Individual-Specific `Fingerprints ` of Human DNA",
Nature, Vol. 316 Jul. 4, 1985 which reference is incorporated herein. That
is, genomes are compared for the presence of absence of polymorphisms.
EXAMPLES
Example 1 demonstrates synthesis and purification of oligonucleotide
primers. Example 2 shows that a variety of 10 nucleotide primers are able
to amplify random nucleic acid segments, and that polymorphisms can be
detected between genomic DNA samples from two soybean cultivars. Example 3
illustrates that polymorphisms revealed by this invention are inherited in
normal fashion by the progeny of a cross between two soybean lines. This
example establishes the utility of the polymorphisms as genetic markers.
Example 4 shows that the pattern of amplified bands is specific to a given
primer sequence. Most changes involving a single nucleotide in the primer
sequence cause a complete alteration in the pattern of amplified DNA
segments. This suggests that most single-nucleotide polymorphisms at
corresponding priming sites in individual genomes will be seen by the
process of the present invention as a difference in the pattern of
amplified DNA segments. Example 5 illustrates that 10 nucleotide primers
amplify generally equivalent numbers of DNA segments from both small and
large genomes. Example 6 demonstrates the utility of primers of from 6 to
10 nucleotides in length. Example 8 shows the process of the present
invention detects a polymorphism that can be used to distinguish human DNA
samples. Example 9 shows quantitation of the amplification products.
EXAMPLE 1
Synthesis and purification of oligonucleotide primers
Eight oligonucleotide primers (Table I) were synthesized by standard
solid-phase methods with phosphoramidite chemistry in a Du Pont Model
Coder 300 automated DNA synthesizer (E. I. du Pont de Nemours and Company,
Wilmington, Del.). The eight primers were of arbitrary nucleotide
sequence. Each primer was selected to contain the same proportion of the
bases A and T as the bases G and C for generally equal stability when
attached to the template.
After deprotection in ammonium hydroxide according to standard practice,
the samples were dried under vacuum, dissolved in 0.2 ml of TEN (10 mM
Tris-Cl, pH 7.5, 1 mM NaEDTA, 10 mM NaCl) and purified by gel filtration
chromatography on Sephadex G25 in NAP-5 cartridges (Pharmacia, Piscataway,
N.J.) equilbrated in TEN. Gel filtration was accomplished by applying 0.2
ml of sample; followed by 0.6 ml of TEN, followed by 0.5 ml of TEN applied
to the top of the column and collecting 0.5 ml of eluant. The
concentration of purified oligonucleotide was calculated by absorbance at
260 nm on a spectrophotometer. An absorbance value of 1 corresponding to
33 .mu.g/ml was assumed.
TABLE I
______________________________________
Primer Primer Concentration of
sequence name stock solution .mu.g
______________________________________
5'-AGCACTGTCA 10b 10
5'-TCGTAGCCAA AP3 10
5'-TCACGATGCA AP4 10
5'-CTGATGCTAC AP5 10
5'-GCAAGTAGCT AP6 10
5'-CTGATACGGA AP7 10
5'-TGGTCACTGA AP8 10
5'-ACGGTACACT AP9 10
______________________________________
EXAMPLE 2
Primers (Table I) detect polymorphisms in soybean DNA templates
Genomic DNA was isolated from the soybean cultivars PI 81762 and Bonus by
centrifugation in CSCl/ethidium bromide as described in Murray, M.G.
"Rapid Isolation of High Molecular Weight Plant DNA", Nucleic Acids Res.
Vol. 8 (1980) pp. 4321-4326 which reference is incorporated herein. The
source of the soybean cultivars was Professor Theodore Hymowitz,
University of Illinois.
Eight different oligonucleotides, each 10 nucleotides in length (Table I)
were tested separately as primers in the amplification reaction detailed
below for their ability to amplify DNA segments from the two varieties of
soybean DNA.
The process of the present invention was performed in a reaction volume of
50 .mu.l containing genomic DNA cocktails of 10 mM Tris-Cl, pH 8.3, 50 mM
KCl, 2.5 mM MgCl.sub.2, 0.001% gelatin (Sigma, St. Louis, Mo. cat. (1990)
no. G-2500), 0.1 .mu.g of soybean DNA, 20 pmole of oligonucleotide, 200
.mu.M each of the deoxynucleotide triphosphates, dATP, dCTP, dGTP, dTTP,
and 1.25 units of Amplitaq DNA polymerase (Perkin-Elmer Cetus cat. no.
N801-0060, Norwalk, Conn.). The genomic DNA cocktail was prepared by
mixing ingredients 1-5 (Table II) and heating this mixture at 99.degree.
C. for 4 minutes in the heating block of a DNA thermal cycler. This
mixture was then cooled to room temperature before the addition of
ingredients 6 and 7 (Table II).
Amplification was conducted in 0.6 ml microcentrifuge tubes (cat. no.
1048-00-0 from Robbins Scientific Corp., 1280 Space Park Way, Mtn. View,
Calif. 94943 (1990)) and the reaction was initiated by adding 48 .mu.l of
a genomic DNA cocktail (Table II) to 2 .mu.l of each primer from Table I
and overlayed with 40 .mu.l of mineral oil (Sigma cat. no. M3516).
TABLE II
______________________________________
Volume
# Genomic DNA cocktails
PI81762 .mu.l
Bonus .mu.l
______________________________________
1 Deionized water 309.0 308.0
2 10x Buffer* 45.0 45.0
3 MgCl.sub.2, 200 mM 2.2 2.2
4 PI 81762 DNA, 672 .mu.g/ml
1.3 --
5 Bonus DNA, 448 .mu.g/ml
-- 2.0
6 Mixture of dATP, dCTP, dGTP,
72.0 72.0
TTP, 1.25 mM each
7 Amplitaq polymerase, 2.2 2.2
5 units/.mu.l
______________________________________
*10x Buffer consists of 100 mM TrisCl (pH 8.3), 500 mM KCl, 15 mM
MgCl.sub.2 and 0.01% Gelatin as directed in PerkinElmer Cetus cat. no. N
8010060 Amplitaq DNA polymerase.
Temperature cycling in the amplification reaction was performed in a DNA
Thermal Cycler (Perkin-Elmer Cetus, Norwalk, Conn.) for cycles with
parameters set at: hold 94.degree. C. for 1 min, hold 35.degree. C. for
sec, ramp to 72.degree. C. in 1:51 min, hold 72.degree. C. for 2 min.
Upon completion of the 16 separate amplification reactions performed above,
5 .mu.l aliquots of each sample were analyzed by electrophoresis on a 1.4%
agarose gel at 8.5 volt/cm for 60 minutes. Electrophoresis was conducted
according to standard practice as described in J. Sambrook, E. F. Fritsch
and T. Maniatis, Molecular Cloning A Laboratory Manual, Cold Spring-Harbor
Laboratory Press, pp. 6.3-6.19 (1989), which is incorporated by reference.
The gel was stained with ethidium bromide and was photographed.
Polymorphisms, i.e., difference in the reaction product in the DNA patterns
were evident for all primers except AP6 by comparing DNA bands amplified
by a given primer from both Bonus and PI 81762 (FIG. 1). FIG. 1 is a
photograph of the stained electrophoresis gel. Lane 0 is a molecular
weight standard. Odd numbered lanes are the Bonus DNA amplification
products from the eight primers (Table I) and even numbered Lanes are the
PI 81762 DNA amplification products from the primers. It can be seen from
the figure that random nucleic acid segments can be distinguished on the
bases of nucleotide differences. As an example, two bands denoted "A" and
"B" amplified by primer 10b were seen in DNA from PI 81762 (lane 2) but
not from Bonus (lane 1).
EXAMPLE 3
Amplified polymorphisms are useful for genetic mapping
To determine whether such polymorphisms as seen in Example 2 have utility
in genetic mapping, the band A polymorphism detected with primer 10b (FIG.
2) was mapped to the soybean genome. This was accomplished by scoring each
of 66 segregating F.sub.2 individuals (T. Hymowitz, U. of Ill.), for their
parental pattern of inheritance of the band A polymorphism and correlating
this with segregation data from the same 66 individuals for 430 RFLP
markers previously mapped to the soybean genome (J. A. Rafalski and S. V.
Tingey, Abstract, Genome Mapping and Sequencing, Apr. 26-30, 1989).
Samples of the F.sub.2 genomic DNA were subjected to the process of the
present invention with primer 10b under the conditions described in
Example 2 and analyzed by electrophoresis in a 1.4% agarose gel. Lane 0 is
a molecular weight standard, and the remaining lanes are the reaction
products of the F.sub.2 individuals. Table 3 presents the parental pattern
of inheritance for the primer 10b band A polymorphism. Since band A is
amplified from PI81762 and not Bonus (FIG. 2), the presence of band A in a
particular sample indicates that it received at least one copy of that DNA
segment from PI81762. At first inspection it is difficult to distinguish
between homozygotes that have received two copies of band A from PI81762
and heterozygotes that have received only one copy of band A from PI81762.
Therefore this polymorphism is scored as a dominant marker (see legend to
Table 3). Individuals that do not contain band A have inherited this
segment of the genome from Bonus.
In order to determine the position of the band A locus in the soybean
genome it was necessary to correlate the inheritance of this locus with
that of several RFLP markers previously mapped to the soybean genome (J.
A. Rafalski and S. V. Tingey, Abstract, Genome Mapping and Sequencing,
Apr. 26-30, 1989). This genetic map (data not shown) was constructed by
standard RFLP methodology from analysis of the segregation patterns of
many RFLP markers in the same F.sub.2 population as used in this example
(for standard RFLP mapping technology, see T. Helentjaris, M. Slocum, S.
Wright, A. Schaefer and J. Nienhuis, 1986, Theor. Appl. Genet., 72:761-769
which reference is incorporated herein). The basis for genetic mapping
analysis is that markers located near to each other in the genome are
inherited together in the F.sub.2 progeny, while markers located farther
apart are co-inherited less frequently. Segregation analysis and marker
map positions were calculated using the MapMaker program, incorporated
herein (E. S. Lander, P. Green, J. Abrahamson, A. Barlow, J. F. Daly, S.
E. Lincoln and L. Newburg, 1987, Genomics 1:174-181). The results indicate
that the primer 10b band A polymorphism maps to linkage group 17 in
between RFLP markers 249 and 7315 at a distance from each marker of 9.4
and 9.2 centimorgans, respectively. The probability that the band A
polymorphism segregated purely by chance in the observed data was only 1
chance in 10.sup.-11.8 indicating the strength of this map position. This
example demonstrates that polymorphisms as revealed by the present
invention have utility as genetic markers.
TABLE III
__________________________________________________________________________
F.sub.2 Individuals
Marker Name
1 2 4 5 6 7 8 9 12
13
14
15
16
17
18
19
20
21
22
23
25
26
27
28
29
30
31
__________________________________________________________________________
RFLP Marker
B A H H H H A H B H H H A H H H A H A
H
m
H A H A B A
249
RFLP Marker
B A H m H A H H B B H A H H H H A H H
m
H
m m H A B A
7315
Primer 10b
b A b m b b m b b b b A A A b b A b A
m
b
m A b A b A
Band A
__________________________________________________________________________
F.sub.2 Individuals
Marker Name
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
5 54
55
56
57
58
59
__________________________________________________________________________
RFLP Marker
B A H H B a H A A m A B B H B H A B A
H
B
H B A A B m
249
RFLP Marker
B H H H B H H A A A A H H H B H H B H
H
B
H H A A B H
7315
Primer 10b
b b b b b b b A A A A b b b b b b b A
b
b
b b A A b b
Band A
__________________________________________________________________________
F.sub.2 Individuals
Marker Name 61
62
63
64
65
68
70
71
72
73
74
75
76
77
78
80
82
83
84
85
86
88
90
91
96
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RFLP Marker m m m m m H A H B A A H B B H H A B A
A
B
m m m m
249
RFLP Marker H A H A m H m A m m A m B B H H H H A
A
A
m m B A
7315
Primer 10b b m m A m b m m m m A m b b A b b b A
A
b
m m b A
Band A
__________________________________________________________________________
Legend for Table III
DNA isolated from 88 F.sub.2 individuals segregating from a cross between
Bonus and PI81762. The genotype of each individual at each of the above
marker loci was determined by either RFLP analysis or by the process of
this invention (see text). Individual scores indicate the parental
genotype of the marker locus. A score of "A" or "B" designates that the
locus was inherited from Bonus or PI81762, respectively. A score of "H"
designates that the locus was inherited both from Bonus and PI81762. A
score of "a" designates that the locus was either inherited only from
Bonus or that it was inherited from both Bonus and PI81762. A score of "b"
designates that the locus was either inherited from PI81762 or that it was
inherited from both Bonus and PI81762. A score of "m" designates missing
data.
EXAMPLE 4
The pattern of amplification products of DNA segments is sensitive to
single-nucleotide changes in the primer sequence
A series of primers were synthesized based on the oligonucleotide primer
AP8 of Example 2 (Table I). Each variant differed from the canonical
sequence at a single nucleotide, progressing stepwise through the primer
sequence. Nucleotide composition in each variant was maintained at 50% A+T
as in Example 2. In Table IV, the nucleotide site that differs from the
canonical sequence is shown in lower-case type.
TABLE IV
______________________________________
Primer Primer Concentration of
sequence name stock solution
______________________________________
5'-TGGTCACTGA AP8 (canonical)
10 .mu.M
5'-aGGTCACTGA AP8-a 10 .mu.M
5'-TcGTCACTGA AP8-b 10 .mu.M
5'-TGcTCACTGA AP8-c 10 .mu.M
5'-TGGaCACTGA AP8-d 10 .mu.M
5'-TGGTgACTGA AP8-e 10 .mu.M
5'-TGGTCtCTGA AP8-f 10 .mu.M
5'-TGGTCAgTGA AP8-g 10 .mu.M
5'-TGGTCACaGA AP8-h 10 .mu.M
5'-TGGTCACTcA AP8-i 10 .mu.M
5'-TGGTCACTRGt
AP8-j 10 .mu.M
______________________________________
Cocktails containing genomic DNA from Bonus and PI 81762 were prepared as
described in Example 2 (Table II). However, instead of using
microcentrifuge tubes as the reaction vessels for amplification, the
samples were placed in the wells of a polyvinyl chloride (PVC) 96-well
microtiter plate (Falcon, 1989, 3911, Becton Dickinson Labware, Oxnard,
Calif.) and were overlaid with 40 .mu.l of mineral oil (Sigma, St. Louis,
Mo, cat. no. M3516 1990). Unused wells were filled with 50 .mu.l of water
plus 40 .mu.l of oil. The 96-well plate was placed without a lid in an
air-driven cycling oven (BSC 1000 with temperature probe BSC-2005, Bios
Corp., 291 Whitney Ave., New Haven, Conn. 06511) atop a metal test tube
rack with a 1-inch square grid. The rack was made 2" high by clipping the
top tier from a three-tiered rack (4.75" wide.times.11.5" long.times.4"
high). The freed tier was laid on top of the 96-well plate. The oven was
in a room at an ambient temperature of 22.degree. C. and was programmed
for 35 cycles with the parameters: hold 93.degree. C. for 1 min, hold
35.degree. C. for 1 min, ramp to 72.degree. C. in 2 min, hold 72.degree.
C. for 2 min. The soak tolerance was set at 2.degree. C.
After the amplification reaction of the present invention was complete, 12
.mu.l of each sample was analyzed by electrophoresis on a 1.4% agarose gel
as described in Example 2.
FIG. 3 is a photograph of the stained electrophoresis gel. Lane 0 is a
molecular weight standard. DNA amplification products are shown for Bonus
(odd numbered lanes) and for PI 81762 (even numbered lanes). Arrows mark
the location in each primer that differs from the sequence of primer AP8.
Each primer generated very different patterns of amplified DNA segments
with the exception of AP8 and AP8-a which appeared to differ qualitatively
in only 1 band (see 250 bp bands found only with AP8-a; FIG. 3, lanes
1-4). Whereas the overall pattern of bands differed for the majority of
primers, there were at least some bands that may be common to different
primers (note polymorphic 1 kb bands that appear to be amplified by AP8,
AP8-a and AP8-b; FIG. 3, lanes 1-6). Except for primers AP8 and AP8-a,
different polymorphisms between Bonus and PI 81762 were revealed with each
primer. Among the patterns generated by all 11 primers, there were about
22 distinct polymorphisms as defined by the presence of a band in one
genome and its corresponding absence in the other genome.
A few exceptions notwithstanding, a single nucleotide change in a primer
generally produced a significant change in the pattern of amplified bands
and revealed new polymorphisms. This suggests that the conditions of this
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