U.S. patent number 5,468,610 [Application Number 08/074,275] was granted by the patent office on 1995-11-21 for three highly informative microsatellite repeat polymorphic dna markers.
This patent grant is currently assigned to The Government of the United States of America as represented by the. Invention is credited to Carl R. Merril, Mihael H. Polymeropoulos.
United States Patent |
5,468,610 |
Polymeropoulos , et
al. |
November 21, 1995 |
Three highly informative microsatellite repeat polymorphic DNA
markers
Abstract
The invention relates to polymorphic markers (two
tetranucleotide and one dinucleotide repeat polymorphisms) that are
useful for human individualization. Applications are in forensic
medicine and for paternity and prenatal screening as well as
genetic mapping. These markers are characterized by sets of
oligonucleotide primers according to the invention useful in PCR
amplification and DNA segment resolution. The invention further
relates to an assay for measuring the subtle differences in genetic
material regarding an added or omitted set of dinucleotide or
tetranucleotide repeat polymorphisms which comprises obtaining an
amount of nucleotide segments effective for testing, amplifying the
segments by the PCR procedure using at least one primer nucleotide
sequence according to the present invention, resolving the
amplified segments using gel electrophoresis, and comparing the
resolved segments by autoradiography to observe the differences in
migration patterns due to structural differences. The assay
according to the invention is easy to perform and results can be
obtained within 24 hours. It is not uncommon for results to be
available within 3-4 hours. Accordingly, the invention also relates
to an improved PCR procedure and a PCR assay kit which comprise
nucleotides according to the invention.
Inventors: |
Polymeropoulos; Mihael H.
(Bethesda, MD), Merril; Carl R. (Rockville, MD) |
Assignee: |
The Government of the United States
of America as represented by the (Washington, DC)
|
Family
ID: |
24841960 |
Appl.
No.: |
08/074,275 |
Filed: |
June 9, 1993 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
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707501 |
May 29, 1991 |
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Current U.S.
Class: |
435/6.12;
435/91.2; 536/24.31; 536/24.33 |
Current CPC
Class: |
C12Q
1/6858 (20130101); C12Q 1/6876 (20130101); C12Q
1/6858 (20130101); C12Q 2600/156 (20130101); C12Q
2525/151 (20130101) |
Current International
Class: |
C12Q
1/68 (20060101); C12Q 001/68 (); C12P 019/34 ();
C07H 021/04 () |
Field of
Search: |
;435/6,91.2
;536/24.33 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Polymeropoulos et al. (Feb. 11, 1991) Nucleic Acids Res., vol.
19(3), p. 689. .
Weber et al "Abundant Class of Human DNA Polymorphisms Which Can Be
Typed Using the Polymerase Chain Reaction", pp. 388-396, Am. J.
Hum. Genet 44, 1989. .
Tautz et al, Nucleic Acids Research, vol. 12, No. 10, "Simple
Sequences Are Ubiquitous Repetitive Components of Eukaryotic
Genomes", pp. 4127-4138, 1984. .
Nakamura et al, "Variable Number of Tandem Repeat (VNTR) Markers
for Human Gene Mapping", Science vol. 235 pp. 1616-1622, 1987.
.
Overhauser et al, Nucleic Acids Research, vol. 15, No. 11,
"Identification of 28 DNA Fragments That Detect RFLPs in 13
Distinct Physical Regions of the Short Arm of Chromosome 5", pp.
4617-4627, 1987. .
Jeffreys et al, "Hypervariable `minisatellite` Regions in Human
DNA", vol. 314, pp. 67-73, 1985. .
Weber et al, "Dinucleotide Repeat Polymorphism at the DIOS89
Locus", Nucleic Acids Research, vol. 18, No. 15, p. 4637. .
Dariavach et al. (1988) European Journal of Immunology, vol. 18,
pp. 1901-1905. .
Moos et al. (1983) The EMBO Journal, vol. 2(5), pp. 757-761. .
Chen et al. (1989) Genomics, vol. 4, pp. 479-497..
|
Primary Examiner: Jones; W. Gary
Assistant Examiner: Arthur; Lisa
Attorney, Agent or Firm: Lowe, Price, LeBlanc &
Becker
Parent Case Text
This application is a continuation of application Ser. No.
07/707,501 now abandoned filed May 29, 1991.
Claims
We claim:
1. A DNA fragment consisting of a nucleotide sequence selected from
the group consisting of a sequence according to SEQ ID NO:1, a
sequence according to SEQ ID NO:2, a sequence according to SEQ ID
NO:3, a sequence according to SEQ ID NO:4, a sequence according to
SEQ ID NO:5, or a sequence according to SEQ ID NO:6.
2. A DNA fragment according to claim 1, wherein the sequence is a
sequence according to SEQ ID NO:1.
3. A DNA fragment according to claim 1, wherein the sequence is a
sequence according to SEQ ID NO:2.
4. A DNA fragment according to claim 1, wherein the sequence is a
sequence according to SEQ ID NO:3.
5. A DNA fragment according to claim 1, wherein the sequence is a
sequence according to SEQ ID NO:4.
6. A DNA fragment according to claim 1, wherein the sequence is a
sequence according to SEQ ID NO:5.
7. A DNA fragment according to claim 1, wherein the sequence is a
sequence according to SEQ ID NO:6.
8. An assay kit for conducting a polymerase chain reaction
procedure to detect an added or omitted set of dinucleotide or
tetranucleotide repeat polymorphisms, wherein said assay results in
a polymorphic information content of about 0.9 comprising an
effective amount for amplification by polymerase chain reaction of
a pair of oligonucleotide primers selected from the group
consisting of
a) a sequence as set forth in SEQ ID NO:1 and a sequence as set
forth in SEQ ID NO:2;
b) a sequence as set forth in SEQ ID NO:3 and a sequence as set
forth in SEQ ID NO:4; and
c) a sequence as set forth in SEQ ID NO:5 and a sequence as set
forth in SEQ ID NO:6, in combination with an effective amount of
ancillary polymerase chain reaction reagents.
9. A method for conducting a polymerase chain reaction procedure to
detect an added or omitted set of dinucleotide or tetranucleotide
repeat polymorphisms, wherein said method results in a polymorphic
information content of about 0.9 comprising
a) obtaining a DNA fragment comprising said repeat polymorphisms in
an amount sufficient for amplification by polymerase chain
reaction,
b) amplifying the repeat polymorphisms by polymerase chain reaction
using a pair of oligonucleotide primers selected from the group
consisting of (1) a primer having the sequence of SEQ ID NO:1 and a
primer having the sequence of SEQ ID NO:2; (2) a primer having the
sequence of SEQ ID NO:3 and a primer substantially having the
sequence of SEQ ID NO:4; and (3) a primer substantially having the
sequence of SEQ ID NO:5 and a primer having the sequence of SEQ ID
NO:6;
(c) determining the length of the amplification product containing
the repeat polymorphisms of step (b) in order to detect genetic
differences which result from added or omitted sets of dinucleotide
or tetranucleotide repeat polymorphisms.
10. An assay for measuring differences in genetic material to
detect an added or omitted set of dinucleotide or tetranucleotide
repeat polymorphisms in forensic screening, paternity, prenatal
screening, or genetic mapping, wherein said assay results in a
polymorphic information content of about 0.9, and wherein said
genetic material comprises a DNA fragment comprising a nucleotide
sequence selected from the group consisting of a sequence according
to SEQ ID NO: 7, a sequence according to SEQ ID NO:8 and a sequence
according to SEQ ID NO:9, which assay comprises
a) obtaining a DNA fragment comprising repeat polymorphisms
comprising a nucleotide sequence selected from the group consisting
of a sequence according to SEQ ID NO:7, a sequence according to SEQ
ID NO: 8 and a sequence substantially according to SEQ ID NO:9 in
an amount sufficient for amplification by polymerase chain
reaction,
b) amplifying the repeat polymorphisms by polymerase chain reaction
using a pair of oligonucleotide primers capable of detecting said
repeat polymorphism selected from the group consisting of (1) a
primer having the sequence of SEQ ID NO:1 and a primer having the
sequence of SEQ ID NO:2; (2) a primer having the sequence of SEQ ID
NO:3 and a primer having the sequence of SEQ ID NO:4; and (3) a
primer having the sequence of SEQ ID NO:5 and a primer having the
sequence of SEQ ID NO:6;
(c) determining the length of the amplification product containing
the repeat polymorphisms of step (b) in order to detect genetic
differences which result from added or omitted sets of dinucleotide
or tetranucleotide repeat polymorphisms.
11. A method for detecting differences in genetic material in (a)
an added or omitted set of (AT)n polymorphisms in the human CTLA-4
gene, (b) a (GAAA)n polymorphism in the human growth hormone loci,
or (c) a G(AAA)n polymorphism in the human cytoplasmic betaactin
related pseudogene, wherein said method results in a polymorphic
information content of about 0.9, which method comprises
a) obtaining a DNA fragment comprising repeat polymorphisms in an
amount sufficient for amplification by polymerase chain
reaction,
b) amplifying the DNA fragment by polymerase chain reaction using a
pair of oligonucleotide primers capable of detecting said
polymorphisms selected from the group consisting of (1) a primer
having the sequence of SEQ ID NO:1 and a primer having the sequence
of SEQ ID NO:2; (2) a primer having the sequence of SEQ ID NO:3 and
a primer having the sequence of SEQ ID NO:4; and (3) a primer
having the sequence of SEQ ID NO:5 and a primer having the sequence
of SEQ ID NO:6;
(c) performing a polymerase chain reaction using a primer pair from
step (b) to produce amplification products; and
(d) detecting the length of the amplification product of step (c)
in order to detect genetic differences which result from added or
omitted sets of dinucleotide or tetranucleotide repeat
polymorphisms.
12. An oligonucleotide primer consisting of a nucleotide sequence
selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ
ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6.
Description
TECHNICAL FIELD
This application relates to genetic testing with polymorphic DNA
markers having repeat sequences to provide a rapid and convenient
high resolution process for distinguishing target nucleic acid
segments on the basis of nucleotide differences according to human
individualization wherein the nucleic acid segments differ in
size.
BACKGROUND ART
The science of genetics has taken a keen interest in the
identification of human individualization and genetic relationships
between individuals. Each individual has hereditary material (DNA,
"nucleotides") which is unique to that individual and hereditary
material which is related to that of others. Procedures have been
developed which are based on identification and characterization of
changes in DNAs, which are changes in DNA (DNA polymorphisms) due
to nucleotide substitution, insertion, or deletion within the
chains of DNAs.
In the field of forensic medicine, for example, there is a keen
interest in such polymorphisms for identification purposes.
Forensic geneticist have developed many techniques to compare
homologous segments of DNA to determine if the segments are
identical or if they differ in one or more nucleotides. Practical
applications of these techniques relate to fields other than
forensic medicine, for example, genetic disease diagnosis and human
genome mapping.
At the present time in this art, the most accurate and informative
way to compare DNA segments requires a method which provides the
complete nucleotide sequence for each DNA segment. Particular
techniques have been developed for determining actual sequences in
order to study mutation in human genes. See, for example, Proc.
Natl. Acad. Sci. U.S.A. 85, 544-548 (1988) and Nature 330, 384-386
(1987). However, because of the extensive amounts of time and high
costs to determine, interpret, and compare sequence information,
presently it is not practical to use extensive sequencing for
compare more than just a few DNA segments.
In genetic mapping, the most frequently used screening for DNA
polymorphisms arising from mutations consist of digesting the DNA
strand with restriction endonucleases and analyzing the resulting
fragments by means of Southern blots. See Am. J. Hum. Genet. 32,
314-331 (1980) or Sci. Am. 258, 40-48 (1988). Since mutations often
occur randomly they may affect the recognition sequence of the
endonuclease and preclude the enzymatic cleavage at that cite.
Restriction fragment length polymorphism mappings (RFLPS) are based
on changes at the restriction site. They are accurate but not very
informative (PIC [0.3). The major problem with RFLPs is the
inability of a test to detect changes that do not affect cleavage
with a restriction endonuclease. As in many of the test methods in
the DNA art, the methods used to detect RFLPs are very labor
intensive and expensive, especially the techniques which includes
Southern blot analysis.
Another technique for detecting specific mutations in particular
DNA segment involves hybridizing DNA segments which are being
analyzed (target DNA) with a complimentary, labeled oligonucleotide
probe. See Nucl. Acids Res. 9, 879-894 (1981). Since DNA duplexes
containing even a single base pair mismatch exhibit high thermal
instability, the differential melting temperature can be used to
distinguish target DNAs that are perfectly complimentary to the
probe from target DNAs that only differ by a single nucleotide.
This method has been adapted to detect the presence or absence of a
specific restriction site, U.S. Pat. No. 4,683,194. The method
involves using an end-labeled oligonucleotide probe spanning a
restriction site which is hybridized to a target DNA. The
hybridized duplex of DNA is then incubated with the restriction
enzyme appropriate for that site. Reformed restriction sites will
be cleaved by digestion in the pair of duplexes between the probe
and target by using the restriction endonuclease. The specific
restriction site is present in the target DNA if shortened probe
molecules are detected.
Another process for studying differences in DNA structure is the
primer extension process which 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. Resolution of the labeled
primer extension product is then done by fractionating on the basis
of size, e.g., by electrophoresis via a denaturing polyacrylamide
gel. This process is often used to compare homologous DNA segments
and to detect differences due to nucleotide insertion or deletion.
Differences due to nucleotide substitution are not detected since
size is the sole criterion used to characterize the primer
extension product.
Another process exploits the fact that the incorporation of some
nucleotide analogs into DNA causes an incremental shift of mobility
when the DNA is subjected to a size fractionation process, such as
electrophoresis. Nucleotide analogs can be used to identify changes
since they can cause an electrophoretic mobility shift. See, U.S.
Pat. No. 4,879,214.
Unfortunately, the above techniques used for identification of
polymorphisms are either not very informative or take a long period
of time to perform. For example, techniques which identify changes
in individual nucleotides on a particular DNA strand often take at
least three to four days to perform. Accordingly, such tests are
very labor intensive and expensive to perform.
Further, subtle genetic differences among related individuals
regarding nucleotides which are substituted in the DNA chains are
difficult to detect. VNTR's or Jeffrey's probes (which the FBI is
using to test and identify DNA chains) are very informative but
labor intensive, in distinction to microsatellites as our which are
equally informative PCR based polymormismic.
The use of certain nucleotide repeat polymorphisms for identifying
or comparing DNA segments have been described by Weber & May 89
Am Hum Genet 44:388, Litt & Luthy '89 Am) Hum Genet 44:397).
However the particular polymorphism genetic segments and primers
used to identify the polymorphisms (for identification and
comparison purposes) of the present invention have not been
previously known or suspected.
Accordingly, there a need in this art for a rapid, simple,
inexpensive and accurate technique having a very high resolution
value to determine relationships between individuals and
differences in degree of relationships. Also, there is a need in
the art for a very accurate genetic relationship test procedure
which uses very small amounts of an original DNA sample, yet
produces very accurate results. This is particularly true in the
forensic medicine area and criminology, since often times very
small samples of DNA are available for testing.
DISCLOSURE OF THE INVENTION
An object of the present invention is to provide a fast and
accurate test for measuring the subtle differences in individuals
by way of genetic testing.
Another object of the invention relates to polymorphic markers (two
tetranucleotide and one dinucleotide repeat polymorphisms) that can
be used for human individualization.
A further object of the invention is to provide a fast and accurate
technique for measuring the subtle differences in individuals by
way of genetic testing that can be applied in multiple areas, e.g.,
forensic screening, paternity and prenatal screening and genetic
mapping.
A still further object is to provide an improved method for
conducting a PCR procedure using an effective amount of a
nucleotide according to the present invention and to provide an PCR
assay kit comprising an effective amount of a nucleotide according
to the present invention and ancillary PCR reagents.
BRIEF DESCRIPTION OF DRAWINGS
FIG. 1 relates to a nucleotide sequence according to SEQ ID
NO:1.
FIG. 2 relates to a nucleotide sequence according to SEQ ID
NO:2.
FIG. 3 relates to a nucleotide sequence according to SEQ ID
NO:3.
FIG. 4 relates to a nucleotide sequence according to SEQ ID
NO:4.
FIG. 5 relates to a nucleotide sequence according to SEQ ID
NO:5.
FIG. 6 relates to a nucleotide sequence according to SEQ ID
NO:6.
FIG. 7 relates to a nucleotide sequence according to SEQ ID NO:
7.
FIG. 8 relates to a nucleotide sequence according to SEQ ID
NO:8.
FIG. 9 relates to a nucleotide sequence according to SEQ ID
NO:9.
BEST MODE FOR CARRYING OUT THE INVENTION
The present invention provides a fast and accurate test for
measuring subtle genetic differences in individuals by way of
genetic testing. The invention further relates to polymorphic
markers (two tetranucleotide and one dinucleotide repeat
polymorphisms) that can be used for human individualization.
Applications for the technique and markers according to the
invention are for example, in forensic screening, in paternity and
prenatal screening as well as in genetic mapping.
The invention relates to polymorphic markers (two tetranucleotide
and one dinucleotide repeat polymorphisms) that are useful for
human individualization of forensic screen, and for paternity and
prenatal screening as well as genetic mapping. The markers
according to the present invention have high polymorphism
information content (PIC) values. These markers are characterized
by sets of oligonucleotide primers as follows:
1. Set 1, PIC 0.92
a. A nucleotide sequence according to SEQ ID NO:1
b. A nucleotide sequence according to SEQ ID NO:2
2. Set 2, PIC 0.91
a. A nucleotide sequence according to SEQ ID NO:3
b. A nucleotide sequence according to SEQ ID NO:4
3. Set 3, PIC 0.92
a. A nucleotide sequence according to SEQ ID NO:5
b. A nucleotide sequence according to SEQ ID NO:6.
These polymorphic markers (two tetranucleotide and one dinucleotide
repeat polymorphisms which are also accompanied by beginning and
ending nucleotide sequences) that can be used for human
individualization are further characterized by the following marker
sequences.
1. A nucleotide sequence having a repeat polymorphism according to
SEQ ID NO:7.
2. A nucleotide sequence having a repeat polymorphism according to
SEQ ID NO:8.
3. A nucleotide sequence having a repeat polymorphism according to
SEQ ID NO:9.
Since a polymorphic marker and an index locus occur as a "pair",
attaching a primer oligonucleotide according to the present
invention to the polymorphic marker allows PCR amplification of the
segment pair. The amplified DNA segment can then be resolved by
electrophoresis and autoradiography. A resulting autoradiography
can then be analyzed for its similarity to another DNA segment
autoradiography. Following the PCR amplification procedure,
electrophoretic motility enhancing DNA analogs may optionally be
used to increase the accuracy of the electrophoresis step.
Also, the invention relates to a method for conducting a PCR
procedure comprising using an effective amount of at least one
nucleotide according to according to the invention as set forth
above, wherein the nucleotide is part of a primer pair of
nucleotides selected from the group of nucleotide pairs consisting
of
a) a nucleotide sequence having the sequence as set forth in SEQ ID
NO:1 and a nucleotide sequence as set forth in SEQ ID NO:2;
b) a nucleotide sequence having the sequence as set forth in SEQ ID
NO:3 and a nucleotide sequence as set forth in SEQ ID NO:4; and
c) a nucleotide sequence having the sequence as set forth in SEQ ID
NO:5 and a nucleotide sequence as set forth in SEQ ID NO:6.
Therefore, the invention further relates to an assay for measuring
the subtle differences in genetic material regarding an added or
omitted set of dinucleotide or tetranucleotide repeat polymorphisms
selected from the group consisting of a sequence according to SEQ
ID NO:7, a sequence according to SEQ ID NO:8 and a sequence
according to SEQ ID NO:9, which comprises
a. obtaining nucleotide segments comprising said repeat
polymorphisms in an amount effective for testing,
b. amplifying said segments by a PCR procedure using a pair of
oligonucleotide primers capable of amplifying said polymorphism
containing segments,
c. resolving the amplified segments using page gels
electrophoresis, and
d. comparing the resolved segments by autoradiography to observe
the differences in migration patterns due to length variation.
Preferably, the invention further relates to an assay for measuring
the subtle differences in genetic material regarding an added or
omitted set of dinucleotide or tetranucleotide repeat polymorphisms
selected from the group consisting of a sequence according to SEQ
ID NO:7, a sequence according to SEQ ID NO:8 and a sequence
according to SEQ ID NO:9, which comprises
a. obtaining nucleotide segments comprising said repeat
polymorphisms in an amount effective for testing,
b. amplifying said segments by a PCR procedure using the pair of
oligonucleotide primers selected from the group consisting of a
sequence according to SEQ ID NO:1, a sequence according to SEQ ID
NO:2, a sequence according to SEQ ID NO:3, a sequence according to
SEQ ID NO:4, a sequence according to SEQ ID NO:5, or a sequence
according to SEQ ID NO:6,
c. resolving the amplified segments using page gels
electrophoresis, and
d. comparing the resolved segments by autoradiography to observe
the differences in migration patterns due to length variation.
Still further, the invention relates to an assay kit for conducting
a PCR procedure comprising an effective amount of at least one
nucleotide having a sequence according to the invention as set
forth above, wherein the nucleotide is part of a primer pair of
nucleotides selected from the group of nucleotide pairs consisting
of
a) a nucleotide sequence having the sequence as set forth in SEQ ID
NO:1 and a nucleotide sequence as set forth in SEQ ID NO:2;
b) a nucleotide sequence having the sequence as set forth in SEQ ID
NO:3 and a nucleotide sequence as set forth in SEQ ID NO:4; and
c) a nucleotide sequence having the sequence as set forth in SEQ ID
NO:5 and a nucleotide sequence as set forth in SEQ ID NO:6, in
combination with an effective amount of ancillary PCR reagents.
Accordingly, the above described polymorphisms are useful for human
sample individualization, because of their high PIC values. Since
the described polymorphic systems are based on the polymerase chain
reaction (PCR), only minute (40 nanograms) amounts of genomic DNA
are required for each test. The target sequences range from 92 to
310 base pairs so that high molecular weight DNA is not necessary,
and common problems such as shearing of DNA will have minimal
impact on the performance of the assay. The assay is easy to
perform and results can be obtained within 24 hours. It is not
uncommon for results to be available within 3-4 hours. By
comparison, the prior art methods require a number of days before
results are available, usually 3-4 days are required.
Further, the assay according to the invention is able to detect
very small differences in nucleotide sequences. A single omission
or addition of the repeat sequence will change the mobility due to
the electrical nature and molecular weight of the target nucleotide
sequence. These differences are clearly visible on the
autoradiographs after electrophoresis.
Microsatellite repeat polymorphisms have been shown to be useful
tools in DNA analysis. The three polymorphisms described here are
original and are based on previously sequenced genes. The two
tetranucleotide repeat markers described, can be scored easily
since allele sizes differ by four base pairs. The most commonly
used technique used in forensic screening is based on minisatellite
markers, in distinction to the PCR able microsatellites described
in the present invention.
The general PCR technique step is conducted generally as described
in U.S. Pat. No. 4,683,195 to Mullis et al and U.S. Pat. No.
4,683,202 to Mullis et al, which are hereby incorporated by
reference thereto. Further, electrical motility enhancing DNA
analogs can optionally be used during the replication and
amplification PCR procedure.
The degree of polymorphism in the genetic segments according to the
present invention, which polymorphisms yield highly informative
identification test results, is surprising and unexpected. The high
PIC value (approximately 0.9) is totally unexpected.
Accordingly, the use of a PCR procedure and PCR primers pairs, such
as those primer sequences according to SEQ ID NO:1 to SEQ ID NO:6,
to detect the polymorphism DNA segment according to the present
invention yields excellent results. Such results are sufficiently
accurate and informative to accurately identify DNA segments and
determine degrees of relationship between DNA segments of
individuals.
Moreover, conducting three sets of PCR procedures on the same DNA
segment samples while using a different PCR primer pair according
to the present invention for each of the three procedures yields
extraordinarily accurate and informative test results. Comparison
of the three sets of test results data provides extremely accurate
DNA segment identification.
The following examples are provided to more specifically describe
the invention which is not limited to the following examples.
The described oligonucleotide primers are used to amplify the
target sequences using PCR, under the following conditions:
Example 1
The samples are of DNA are prepared as follows.
60 ng of genomic DNA are used as template for PCR with 80 ng of
each oligonucleotide primer, 0.6 units of Taq Polymerase 50 mM KCL,
10 mM Tris (pH 8.3), 1.5 mM MgCl.sub.2, 0.01% gelatin, 200 uM of
each dGTP, dATP, dTTP, 2.5 uM dCTP and 10 microcuries of alpha P32
dCTP., in a final reaction volume of 15 microliters. The samples
are overlayed with 15 microliters of mineral oil to prevent
evaporation.
EXAMPLE 2
PCR is performed for each of the samples and primers described in
Example 1, above.
PCR is performed in a Techne MW-1 microplate thermocycler under the
following conditions denaturation of 94 degrees C for 1.4 min.,
annealing at 55 degrees C. for 2 min., and extension at 72 degrees
C. for 2 min. The cycle is repeated 30 times with a final extension
at 72 degrees C for 10 min.
EXAMPLE 3
The amplified DNA segments from each of the samples described in
Example 2 above are resolved by electrophoresis as follows.
Two microliters of each PCR reaction mixture sample are
electrophoresed on a 6% PAGE sequencing gel and visualized by
autoradiography. Exposure times for the autoradiography range from
3-16 hours.
__________________________________________________________________________
SEQUENCE LISTING (1) GENERAL INFORMATION: (iii) NUMBER OF
SEQUENCES: 9 (2) INFORMATION FOR SEQ ID NO:1: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 20 (B) TYPE: nucleic acid (C)
STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
AATCTGGGCGACAAGAGTGA20 (2) INFORMATION FOR SEQ ID NO:2: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 (B) TYPE: nucleic acid (C)
STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(genomic) (x i) SEQUENCE DESCRIPTION: SEQ ID NO:2:
ACATCTCCCCTACCGCTATA20 (2) INFORMATION FOR SEQ ID NO:3: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 (B) TYPE: nucleic acid (C)
STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
TCCAGCCTCGGAGACAGAAT20 (2) INFORMATION FOR SEQ ID NO:4: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 (B) TYPE: nucleic acid (C)
STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
AGTCCTTTCTCCAGAGCAGGT21 (2) INFORMATION FOR SEQ ID NO:5: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 (B) TYPE: nucleic acid (C)
STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
GCCAGTGATGCTAAAGGTTG20 (2) INFORMATION FOR SEQ ID NO:6: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 (B) TYPE: nucleic acid (C)
STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
AACATACGTGGCTCTATGCA20 (2) INFORMATION FOR SEQ ID NO:7: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 291 (B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
AATCTGGGCGACAAGAGTGAAACTCCGTCAAAAGAAAGAAAGAAAGAGACAAAGAGAGTT60
AGAAAGAAAGAAAGAGAGAGAGAGAGAAAGGAAGGAAGGAAGAAAAAGAAAGAAAAAGAA 120
AGAAAGAGAAAGAAAGAAAGAGAAAGAAAGAAAGAAAGAAAGAAAGAAAGAAAGAAAGAA180
AGAAAGAAAAAGAAAGAAAGAAAGAAAGAAAGAAAGAAAGAAAGAAAGAAAGAAAGAAAG240
AAAGAAAGGAAGGAAAGAAAGAGCAAGTTACTATAGC GGTAGGGGAGATGT291 (2)
INFORMATION FOR SEQ ID NO:8: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 128 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)
TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE
DESCRIPTION: SEQ ID NO:8: GCCAGTGATGCTAAAGGTTGTATTGCATA
TATACATATATATATATATATATATATATAT60
ATATATATATATATATATATATATATATATTTTAATTTGATAGTATTGTGCATAGAGCCA120
CGTATGTT128 (2 ) INFORMATION FOR SEQ ID NO:9: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 243 (B) TYPE: nucleic acid (C)
STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
TCCAGCCTCGGAGACAGAATGAGACTCCATCAAAAACAAGAAAGAAAGAAAGACAAAGAG 60
AGAAAGAAAGAAAGAAAGAAAGAAAGAAAGAGAGAGAGAGAGAGAGAGAGAGAAAGAAAG120
AAAGAAAGAAAGAAAGAAAGAAAGAAAGAAAGAAAGAAAGAAAGAAAGAAGGAAAGAAAG180
AAAGGAAACTAAAATAACTAAATAACTGAGTAGCACCAC ACCACCTGCTCTGGAGAAAGG240
ACT243
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