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CA2110059C - Three highly informative microsatellite repeat polymorphic dna markers - Google Patents

Three highly informative microsatellite repeat polymorphic dna markers Download PDF

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Publication number
CA2110059C
CA2110059C CA002110059A CA2110059A CA2110059C CA 2110059 C CA2110059 C CA 2110059C CA 002110059 A CA002110059 A CA 002110059A CA 2110059 A CA2110059 A CA 2110059A CA 2110059 C CA2110059 C CA 2110059C
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set forth
nucleotide sequence
sequence
nucleotide
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CA2110059A1 (en
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Mihael H. Polymeropoulos
Carl R. Merrill
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US Department of Health and Human Services
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6858—Allele-specific amplification
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes

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Abstract

The invention relates to polymorphic markers (two tetranucleotide, one dinucleotide repeat polymorphisms and 27 markers characterized by primer pairs 1A-27A) that are useful for human individualization. Applications are in forensic medicine and for paternity and prenatal screening as well as genetic mapping. These markers ae 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 re-peat 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 migra-tion 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 im-proved PCR procedure and a PCR assay kit which comprise nucleotides according to the invention.

Description

i THREE HIGHLY INFORMATIVE MICROSATELLITE REPEAT
POLYMORPHIC DNA MARKERS

Technical Field This application relates to genetic testing with poiymorphic DNA markers having=.repeat seqpences to 1=! 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, "nucleoticies") 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 ..... . .. t,..,, ....,. . . . .... . .... . . . . ...,. ..r.. . .. . . . ...
. . ' , .. . . ' W(' 92/21693 PCT/iJS92/04195 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 i.nf ormati.ve 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 ( RF'LPS ) 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 = WO 92/21693 PC7'/US92/04195 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
segm'ents 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. Patent 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 Drobe 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.Q., by electrophoresis via a denaturing polyacrylamide gel.

W092/21693 PC'f/US92/04195 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. Nucleot3.de analogs can be used to identify changes since they can cause an electro-phoretic mobility shift. See, U.S.. Patent 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 is a need in this art for a rapid, simple, inexpensive and accurate technique having 5 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 Tnvention An object of an aspect of the present invention is to provide a fast and accurate test for measurir.ig the subtle differences in individuals by way of genetic testing.
Another object of an aspect of the invention relates to polymorphic markers that can be used for human individualization.
A further object of an aspect of the invention is to provide a fast and accurate technique for measuririg the subtle differences iri 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 of an aspect of the invention 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.
According to an aspect of the present invention, there is provided an isolated 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, and a sequence according to SEQ ID NO:6.
According to another aspect of the present invention, there is provided 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 polymorphism information content of about 0.9, the method comprising the steps of:
i) obtaining a DNA fragment comprising the repeat polymorphisms in an amount sufficient for amplification by polymerase chain reaction;
ii) amplifying the DNA fragment by polymerase chain reaction using a pair of oligonucleotide primers comprising at least one DNA fragment as described in the above paragraph, wherein said pair of oligonucleotide primers is 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 to produce amplification; and iii) determining the length of the amplification products of step (ii) in order to detect the added or 6a omitted set of dinucleotide or tetranucleotide repeat polymorphisms.

According to a further aspect of the present invention, there is provided 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 polymorphism information content of about 0.9 comprising an amount of a pair of oligonucleotide primers sufficient for amplification by polymerase chain reaction comprising at least one DNA fragment having a sequence selected from the group consisting of a sequence according to SEQ ID
NO:l, 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, and a sequence according to SEQ ID NO:6, wherein said pair of oligonucleotide primers is 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.

According to another aspect of the present invention, there is provided 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 the assay results in a polymorphism information content of about 0.9 and 6b wherein the 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 the repeat polymorphisms in an amount sufficient for amplification by polymerase chain, b. amplifying the DNA fragment by a polymerase chain reaction procedure using a pair of oligonucleotide primers which amplify the DNA fragment, c. resolving the amplified DNA fragment using polyacrylamide gel electrophoresis, and d. comparing the resolved DNA fragment by autoradiography to observe the differences in migration patterns due to length variation.
According to a further aspect of the present invention, there is provided 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 beta-actin related pseudogene, wherein the method results in a polymorphism information content of about 0.9, and wherein the genetic material comprises a DNA fragment comprising a nucleotide 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 method comprises a. obtaining a DNA fragment comprising the repeat polymorphisms in an amount sufficient for amplification by polymerase chain, b. amplifying the DNA fragment by a polymerase chain reaction procedure using a pair of oligonucleotide primers which amplify the DNA fragment, 6c c. resolving the amplified DNA fragment using polyacrylamide gel electrophoresis, and d. comparing the resolved DNA fragment by autoradiography to observe the differences in migration patterns due to length variation.
According to another aspect of the present invention, there is provided an assay for measuring the subtle differences in genetic material regarding an added or omitted set of dinucleotide or tetranucleotide repeat polymorphisms wherein the genetic material comprises a sequence characterized by primer pairs 1A-27A, which comprises a. obtaining nucleotide segments comprising the repeat polymorphisms in an amount sufficient for amplification by polymerase chain, b. amplifying the segments by a PCR procedure using a pair of oligonucleotide primers capable of amplifying the polymorphisms containing segments, c. resolving the amplified segments using polyacrylamide gel electrophoresis, and d. comparing the resolved segments by autoradiography to observe the differences in migration patterns due to length variation.

An assay kit for conducting a PCR procedure to detect an added or omitted set of dinucleotide or tetranucleotide repeat polymorphisms, comprising an amount sufficient for amplification by polymerase chain reaction of at least one nucleotide sequence having a sequence according to claim 14, wherein the nucleotide sequence is part of a primer pair selected from the group of primer pairs consisting of d) a nucleotide sequence having the sequence as set forth in SEQ ID NO:10 and a nucleotide sequence as set forth in SEQ ID NO:11;

6d e) a nucleotide sequence having the sequence as set forth in SEQ ID NO:12 and a nucleotide sequence as set forth in SEQ ID NO:13;
f) a nucleotide sequence having the sequence as set forth in SEQ ID NO:14 and a nucleotide sequence as set forth in SEQ ID NO:15;
g) a nucleotide sequence having the sequence as set forth in SEQ ID NO:16 and a nucleotide sequence as set forth in SEQ ID NO:17;
h) a nucleotide sequence having the sequence as set forth in SEQ ID NO:18 and a nucleotide sequence as set forth in SEQ ID NO:19;
i) a nucleotide sequence having the sequence as set forth in SEQ ID NO:20 and a nucleotide sequence as set forth in SEQ ID NO:21;

j) a nucleotide sequence having the sequence as set forth in SEQ ID NO:22 and a nucleotide sequence as set forth in SEQ ID NO:23;

k) a nucleotide sequence having the sequence as set forth in SEQ ID NO:24 and a nucleotide sequence as set forth in SEQ ID NO:25;

1) a nucleotide sequence having the sequence as set forth in SEQ ID NO:26 and a nucleotide sequence as set forth in SEQ ID NO;27;
m) a nucleotide sequence having the sequence as set forth in SEQ ID NO:28 and a nucleotide sequence as set forth in SEQ ID NO:29;
n) a nucleotide sequence having the sequence as set forth in SEQ ID NO:30 and a nucleotide sequence as set forth in SEQ ID NO:31;
o) a nucleotide sequence having the sequence as set forth in SEQ ID NO:32 and a nucleotide sequence as set forth in SEQ ID NO:33;

6e p) a nucleotide sequence having the sequence as set forth in SEQ ID NO:34 and a nucleotide sequence as set forth in SEQ ID NO:35;

q) a nucleotide sequence having the sequence as set forth in SEQ ID NO:36 and a nucleotide sequence as set forth in SEQ ID NO:37;

r) a nucleotide sequence having the sequence as set forth in SEQ ID NO:38 and a nucleotide sequence as set forth in SEQ ID NO:39;
s) a nucleotide sequence having the sequence as set forth in SEQ ID NO:40 and a nucleotide sequence as set forth in SEQ ID NO:41;

t) a nucleotide sequence having the sequence as set forth in SEQ ID NO:42 and a nucleotide sequence as set forth in SEQ ID NO:43;

u) a nucleotide sequence having the sequence as set forth in SEQ ID NO:44 and a nucleotide sequence as set forth in SEQ ID NO:45;

v) a nucleotide sequence having the sequence as set forth in SEQ ID NO:46 and a nucleotide sequence as set forth in SEQ ID NO:47;

w) a nucleotide sequence having the sequence as set forth in SEQ ID NO:48 and a nucleotide sequence as set forth in SEQ ID NO:49;

x) a nucleotide sequence having the sequence as set forth in SEQ ID NO:50 and a nucleotide sequence as set forth in SEQ ID NO:51;

y) a nucleotide sequence having the sequence as set forth in SEQ ID NO:52 and a nucleotide sequence as set forth in SEQ ID NO:53;

z) a nucleotide sequence having the sequence as set forth in SEQ ID NO:54 and a nucleotide sequence as set forth in SEQ ID NO:55;

6f aa) a nucleotide sequence having the sequence as set forth in SEQ ID NO:56 and a nucleotide sequence as set forth in SEQ ID NO:57;

bb) a nucleotide sequence having the sequence as set forth in SEQ ID NO:58 and a nucleotide sequence as set forth in SEQ ID NO:59;

cc) a nucleotide sequence having the sequence as set forth in SEQ ID NO:60 and a nucleotide sequence as set forth in SEQ ID NO:61; and dd) a nucleotide sequence having the sequence as set forth in SEQ ID NO:62 and a nucleotide sequence as set forth in SEQ ID NO:63;

wherein said amount of said nucleotide sequence is in combination with an effective amount of ancillary PCR
reagents.
A method for conducting a PCR procedure to detect an added or omitted set of dinucleotide or tetranucleotide repeat polymorphisms, comprising using an amount sufficient for amplification by polymerase chain reaction of at least one nucleotide sequence having a sequence according to claim 14, wherein the nucleotide sequence is part of a primer pair selected from the group of primer pairs consisting of d) a nucleotide sequence having the sequence as set forth in SEQ ID NO:10 and a nucleotide sequence as set forth in SEQ ID NO:11;

e) a nucleotide sequence having the sequence as set forth in SEQ ID NO:12 and a nucleotide sequence as set forth in SEQ ID NO:13;

f) a nucleotide sequence having the sequence as set forth in SEQ ID NO:14 and a nucleotide sequence as set forth in SEQ ID NO:15;

g) a nucleotide sequence having the sequence as 6g set forth in SEQ ID NO:16 and a nucleotide sequence as set forth in SEQ ID NO:17;

h) a nucleotide sequence having the sequence as set forth in SEQ ID NO:18 and a nucleotide sequence as set forth in SEQ ID NO:19;

i) a nucleotide sequence having the sequence as set forth in SEQ ID NO:20 and a nucleotide sequence as set forth in SEQ ID NO:21;

j) a nucleotide sequence having the sequence as set forth in SEQ ID NO:22 and a nucleotide sequence as set forth in SEQ ID NO:23;

k) a nucleotide sequence having the sequence as set forth in SEQ ID NO:24 and a nucleotide sequence as set forth in SEQ ID NO:25;

1) a nucleotide sequence having the sequence as set forth in SEQ ID NO:26 and a nucleotide sequence as set forth in SEQ ID NO:27;

m) a nucleotide sequence having the sequence as set forth in SEQ ID NO:28 and a nucleotide sequence as set forth in SEQ ID NO:29;

n) a nucleotide sequence having the sequence as set forth in SEQ ID NO:30 and a nucleotide sequence as set forth in SEQ ID NO:31;

o) a nucleotide sequence having the sequence as set forth in SEQ ID NO:32 and a nucleotide sequence as set forth in SEQ ID NO:33;

p) a nucleotide sequence having the sequence as set forth in SEQ ID NO:34 and a nucleotide sequence as set forth in SEQ ID NO:35;

q) a nucleotide sequence having the sequence as set forth in SEQ ID NO:36 and a nucleotide sequence as set forth in SEQ ID NO:37;

r) a nucleotide sequence having the sequence as 6h set forth in SEQ ID. NO:38 and a nucleotide sequence as set forth in SEQ ID NO:39;

s) a nucleotide sequence having the sequence as set forth in SEQ ID NO:40 and a nucleotide sequence as set forth in SEQ ID NO:41;

t) a nucleotide sequence having the sequence as set forth in SEQ ID NO:42 and a nucleotide sequence as set forth in SEQ ID NO:43;

u) a nucleotide sequence having the sequence as set forth in SEQ ID NO:44 and a nucleotide sequence as set forth in SEQ ID NO:45;

v) a nucleotide sequence having the sequence as set forth in SEQ ID NO:46 and a nucleotide sequence as set forth in SEQ ID NO:47;

w) a nucleotide sequence having the sequence as set forth in SEQ ID NO:48 and a nucleotide sequence as set forth in SEQ ID NO:49;

x) a nucleotide sequence having the sequence as set forth in SEQ ID NO:50 and a nucleotide sequence as set forth in SEQ ID NO:51;

y) a nucleotide sequence having the sequence as set forth in SEQ ID NO:52 and a nucleotide sequence as set forth in SEQ ID NO:53;

z) a nucleotide sequence having the sequence as set forth in SEQ ID NO:54 and a nucleotide sequence as set forth in SEQ ID NO:55;

aa) a nucleotide sequence having the sequence as set forth in SEQ ID NO:56 and a nucleotide sequence as set forth in SEQ ID NO:57;

bb) a nucleotide sequence having the sequence as set forth in SEQ ID NO:58 and a nucleotide sequence as set forth in SEQ ID NO:59;

cc) a nucleotide sequence having the sequence as 6i set forth in SEQ ID NO:60 and a nucleotide sequence as set forth in SEQ ID NO:61; and dd) a nucleotide sequence having the sequence as set forth in SEQ ID NO:62 and a nucleotide sequence as set forth in SEQ ID NO:63.

Brief Description of the Drawings Figure 1 relates to a nucleotide sequence according to SEQ ID NO:1.
Figure 2 relates to a nucleotide sequence according to SEQ ID NO:2.
Figure 3 relates to a nucleotide sequence according to SEQ ID NO:3.
Figure 4 relates to a nucleotide sequence according to SEQ ID NO:4.
Figure 5 relates to a nucleotide sequence according to SEQ ID NO:5.
Figure 6 relates to a nucleotide sequence according to SEQ ID NO:6.
Figure 7 relates to a nucleotide sequence accordin g to SEQ ID NO:7.
Figure 8 relates to a nucleotide sequence according to SEQ ID NO:8.
Figure 9 relates to a nucleotide sequence according to SEQ ID NO:9.
Figure 10 relates to a nucleotide sequence according to SEQ ID NO:10.
Figure 11 relates to a nucle otide sequence accordirig to SEQ ID NO:11.
Figure 12 relates to a nucleotide sequence according to SEQ ID NO:12.
Figure 13 relates to a nucleotide sequence according to SEQ ID NO:13.
Figure 14 relates to a nucleotide sequence according 6j Figure 10 relates to a nucleotide sequence according to SEQ ID NO:10.
Figure 11 relates to a nucleotide sequence according to SEQ ID NO:11.
Figure 12 relates to a nucleotide sequence according to SEQ ID NO:12.
Figure 13 relates to a nucleotide sequence according to SEQ ID NO:13.
Figure 14 relates to a nucleotide sequence according to SEQ ID NO:14.

W092/21693 PC$'/US92/04195 J, Figure 15 relates to a nucleotide sequence according to SEQ ID NO:15.
Figure 16 relates to a nucleotide sequence according to SEQ ID NO:16.
Figure 17 relates to a nucleotide sequence according to SEQ ID NO:17.
Figure 18 relates to a nucleotide sequence according to SEQ ID NO:18.
Figure 19 relates to a nucleotide sequence according to SEQ ID NO:19.
Fi.gure 20 relates to a nucleotide sequence according to SEQ ID NO:20.
Fa.gure 21 relates to a nucleotide sequence according to SEQ ID NO:21.
Figure 22 relates to a nucleotide sequence according to SEQ ID NO:22.
Figure 23 relates to a nucleotide sequence according to SEQ ID NO:23.
Figure 24 re?.ates to a nucleotide sequence according to SEQ ID NO:24.
Figure 25 relates to a nucleotide secruence according to SEQ ID NO:25.
Figure 26 relates to a nucleotide sequence according to SEQ ID NO:26.
Figure 27 relates to a nucleotide sequence according to SEQ ID NO:27.
Figure 28 relates to a nucleotide sequence according to SEQ ID NO:28.
Fs.gure 29 relates to a nucleotide sequence according to SEQ ID NO:29.
Figure 30 relates to a nucleotide sequence according to SEQ ID NO:30.
Figure 31 relates to a nucleotide sequence according to SEQ ID NO:31.

WO 92/21693 PCT/US92/0419.5 ?1~ Q~ -), :9 Figure 32 relates to a nucleotide sequence according to SEQ ID NO:32.
Figure 33 relates to a nucleotide sequence according to SEQ ID NO:33.
Figure 34 relates to a nucleotide sequence according to SEQ ID NO:34.
Figure 35 relates to a nucleotide sequence according to SEQ ID NO:35.
Figure 36 relates to a nucleotide sequence according to SEQ ID NO:36.
Figure 37 relates to a nucleotide sequence according to SEQ ID NO:37.
Figure 38 relates to a nucleotide sequence according to SEQ ID NO:38.
Figure 39 relates to a nucleotide sequence according to SEQ ID NO:39.
Figure 40 relates to a nucleotide sequence according to SEQ ID NO:40.
Figure 41 relates to a nucleotide sequence according to SEQ ID NO:41.
Figure 42 relates to a nucleotide sequence according to SEQ ID NO:42.
Figure 43 relates to a nucleotide seauence according to SEQ ID NO:43.
Figure 44 relates to a nucleotide sequence according to SEQ ID NO:44.
Figure 45 relates to a nucleotide sequence according to SEQ ID NO:45.
Figure 46 relates to a nucleotide sequence according to SEQ ID-NO:46>
Figure 47 relates to a nucleotide sequence according to SEQ ID NO:47.
Figure 48 relates to a nucleotide sequence according to SEQ ID NO:48.
2 1~

Figure 49 relates to a nucleotide sequence according to SEQ ID:N0:49.
Figure 50 relates to a nucleotide sequence according to SEQ ID N0:50.
Figure 51 relates to a nucleotide sequence according to SEQ ID N0:51.
Figure 52 relates to a nucleotide sequence according to SEQ ID N0:52.
Figure 53 relates to a nucleotide sequence according to SEQ ID NOo53.
Figure 54 relates to a nucleotide sequence according to SEQ ID N :54.
Figure 55 relates to a nucleotide sequence according to SEQ ID N0:55.
Figure 56 relates to a nucleotide sequence according to SEQ ID N0:56.
Figure 57 relates to a nucleotide sequence according to SEQ ID N0:57.
Figure 58 relates to a nucleotide sequence according to SEQ ID NO:58.
Figure 59 relates to a nucleotide sequence according to SEQ ID N :59.
Figure 60 relates to a nucleotide sequence according to SEQ ID NC:60.
Figure 61 relates to a nucleotide sequence according to SEQ ID NO:61.
Figure 62 relates to a nucleotide sequence according to SEQ ID N :62.
Figure 63 relates to a nucleotide sequence according to SEQ ID NC}:63.

Best Mode for Carryinc out the Invention The present invention provides a fast and accurate test for measuring subtle genetic differences in . ...., _ .._... _.., , .. ,. , WO 92/21693 PC'I'/YJS92/04195 ~114 00 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 5 individualization. The invention further relates to 27 other polymorphic markers useful for human individualization. Applications for the technique and markers according to the invention are for example, in forensic screening, in paternity and prenatal screening 10 as well as in genetic mapping.
The invention relates to polymorphic markers (two tetranucleotide, one dinucleotide- repeat polymorphisms and 27 other unique polymorphic markers) 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. The first three markers are characterized by sets of oligonucleotide primers as f.ollJws:
1. Set i, PIC 0.92 a. A nucleotide sequence according to SEQ ID
N0:1 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
P1C s 4 3. Set 3, PIC 0.92 A. A nucleotide sequence according to SEQ ID
N :5 WO 92/21693 P(:T/tJS92/04195 ~ ~_U~/

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 polymorpha.sm according to SEQ ID NO:8.
e-P 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 he 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.
Twenty-seven other primary pair sequences for detecting unique polymorphisms are sequences according to SEQ ID NU:10 through SEQ ID NO:63.
The described polymorphisms are useful for human sample individualization, because of their high F'IC
values. Since the described polymorphisms are based on the polymerase chain reaction, only minute amounts of genomic DNA are required for each test. The target sequences range from 69-260 bps in length 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 .5 hours. Microsatellite repeat polymorphisms have been shown to be useful tools in DNA analysis. The 27 polymorphisms described here are original and are based on previously sequenced human genes. The most commonly used technique in forensic screening is based on minisatellite markers in distinction to the PCR able microsatellites described here.
The 27 markers are characterized by sets of oligonucleotide primers as follows:

tJ
~..Chzcmceonal Primer SEQ ~
Pair Locus Location ID N i Heteroz PIC Size Repeat No. of alleles lA Int-2 E1q13 10,11 84.6% 0.79 161-177 (TG)5TC(TG)16 9 2A PLA-AZ 12 12,13 73.3% 0.76 122-137 (TTA)16 6 3A FABP2 4q28-q31 14,15 644 0.64 99-117 (TTA) 13 6 4A THEt 1 15q15 16,17 601 0.58 165-181 (CT)14 9 5A CYARP450 15Fq21.1 18,19 91.3% 0.67 175-199 (TTTA) 5 6A GCG 2q36-q37 20,21 88% 0.77 142-172 (GA)198 11 7A IL-9 5q 22,23 62.5% 0.75 127-139 (TG)20 7 8A CSTPI 20 24,25 61% 0.58 123-141 (GT)15 9A ANKYRIN 8pl1.1-21.1 26,27 54% 0.45 107-113 (AC)14 4 IOA CD-19 16 28,29 401 0.39 79-91 (GT)17 7 11A c-fms 5q33.3-34 30,31 86% 0.85 95-127 (GT)26 10 12A CD 8 2p12 32,33 711 0.66 138-170 (AC)14 7 13A CYP2D7-8 22 34,35 80l 0.78 98-116 (GT)18 10 --w 14A w 30 7q 36,37 74% 0.72 11 15A H14G-14 21 38,39 69% 0.67 69-93 (GT)19 10 16A RHO 3 40,41 72% 0.68 145-169 5 y~=
17A PFKL 21q22.3 42,43 701 0.66 129-145 (AC)16 7 18A HSFLT 13q12 44,45 51% 0.49 164-186 (TG)21 8 Y~~
C=) 19A liSP4YH(11 14 46,47 664 0.60 90-102 (GT) 15 6 20A HSATPSY1 12p13-qter 48y49 60% 0.54 111-117 (GT)li 4 21A CFES PPS 15q25-qter 50,51 75% 0.70 143-163 (ATTT 6 )11 22A DHFHP2 6 52,53 70% 0.66 157-173 (AAAC)7 5 23A CR7G1 2q34-35 54,55 68% 0.61 117-126 (AAC)9 4 24A F13A1 6p24-25 56,57 781 0.75 180-230 (AAAG)7 8 25A TRH1 6p23-c112 58,59 541 0.50 174-186 (AAC)8 5 26A II-D 6 60,61 81% 0.78 185-206 (CAG) 27A Tli 11p15.5-p15 62,63 78% 0.75 244-260 (TCAT)9 5 WO 92/23693 PCT/iJS92/04195 14 _, .

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 nucleotid.e 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 SEQID NO:2;
b) a nucleotide sequence having the sequence as set forth in SEQ ID NCJ.3 and a nucleotide sequence as set forth in SEQ ID NO:4;
c) a nucleotide sequence having the sequence as set f orth in SEQ ID NO: 5 and a nucleotide sequence as set forth in SEQ ID NO:6;
d) a nucleotide sequence having the sequence as set forth in SEQ ID AIO:10 and a nucleotide sequence as set forth in SEQ ID NO:11;
e) a nucleotide sequence having the sequence as set forth in SEQ ID NO:12 and a nucleotide sequence as set forth in SEQID NO:13;
f) a nucleotide sequence having the sequence as set forth in SEQ ID NO:14 and a nucleotide sequence as set forth in SEQ ID Nfl:15;
g) a nucleotide sequence having the sequence as set forth in SEQ ID NO:16 and a nucleotide sequence as set forth in SEQ ID N :17 ;
h) a nucleotide sequence having the sequence as set forth in SEQ ID ND:18 and a nucleotide sequence as set forth in SEQ ID N0:19 ;
i) a nucleotide sequence having the sequence as set forth in SEQ ID NO:20 and a nucleotide sequence as set forth in SEQ ID NO:21;

2t j) a nucleotide sequence having the sequence as set forth in SEQ ID NO:22 and a nucleotide sequence as set forth in SEQ ID NO:23;
k) a nucleotide sequence having the sequence as 5 set forth in SEQ ID NO:24 and a nucleotide sequence as set forth in SEQ ID NO:25;
1) a nucleotide sequence having the sequence as set forth in SEQ ID NO:26 and a nucleotide sequence as set forth in SEQ ID NO:27;
10 xn) a nucleotide sequence having the sequence as set forth in SEQ ID NO:28 and a nucleotide sequence as set forth in SEQ ID NO:29;
A~.
n) a nucleotide sequence having the sequence as set forth in SEQ ID NO:30 and a nucleotide sequence as 15 set forth in SEQ ID NO:31;
o) a nucleotide sequence having the sequence as set forth in SEQ ID NO:32 and a nucleotide sequence as set forth in SEQ ID NO:33;
p) a nucleotide sequence having the sequence as set forth in SEQ ID N0:34 and a nucleotide sequence as set forth in SEQ ID NO:35;
q) a nucleotide secruence having the sequence as set forth in SEQ ID NO:36 and a nucleotide sequence as set forth in SEQ ID NO:37;
r) a nucleotide sequence having the sequence as set forth in SEQ ID NO:38 and a nucleotide sequence as set forth in SEQ ID NO:39;
s) a nucleotide sequence having the sequence as set forth in SEQ ID NO:40 and a nucleotide sequence as set forth in SEQ ID NO:41;
t) a nucleotide sequence having the sequence as set forth in SEQ ID NO:42 and a nucleotide sequence as set forth in SEQ ID NO:43;

.._.., :;, ., =x.: _ : ; -, ...=. :-,.,. . .t._ . :~f . ;.;<., ......, . , . , _ _ .

Gd ~
1 0 0 . ' r.!

u) a nucleotide sequence having the sequence as set forth in SEQ ID NO:44 and a nucleotide sequence as set forth in SEQ ID NO:45;
v) a nucleotide sequence having the sequence as set forth in SEQ ID NO:46 and a nucleotide sequence as set forth in SEQ ID NO:47;
w) a nucleotide sequence having the sequence as set forth in SEQ ID NO:48 and a nucleotide sequence as set forth in SEQ ID NO: 4 9;, x) a nucleotide sequence having the sequence as set forth in SEQ ID NO:50 and a nucleotide sequence as set forth in SEQ ID NO:51;
y) a nucleotide seauence having the sequence as set forth in SEQ ID NO:52 and a nucleotide sequence as set forth in SEQ ID NO:53;
z) a nucleotide sequence having the sequence as set forth in SEQ ID NO:54 and a nucleotide sequence as set forth in SEQ ID NO:55;
aa) a nucleotide sequence having the sequence as set forth in SEQ ID NOc56 and a nucleotide sequence as set forth in SEQ ID NO:57;
bb) a nucleotide sequence having the sequence as set forth in SEQ ID NO:58 and a nucleotide seQuence as set forth in SEQ ID NO:59; _ .25 cc) anucleotide sequence having the sequence as set forth in SEQ ID NO:60 and a nucleotide sequence as set forth in SEQ ID NO:61;
dd) a nucleotide sequence hava.ng the sequence as set forth;in SEQ ID NO:62 and a nucleotide sequence as set forth in SEQ ID NO:63 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 ., . .5.. ' .,. , ,'.. .. .~õ . ., '..~: . _ selected from the group consisting of a sequence according to SEQ ID N0:7, a sequence according to SEQ
ID NO:8 and a sequence according to SEQ ID N0:9, 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. coanuaring 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 selec-~ed from the group consisting of a sequence according to SEQ ID NO:7, a seauence 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 amoun-L 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 seauence according to SEQ ID NO:5, or a sequence according to SEQ ID NO:b, WO 92/21693 P("T/iJS92/04195 2'1 if1~

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 seguence having the seauence 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, d) a nucleotide sequence having the sequence as set forth in SEQ ID NO:10 and a nucleotide sequence as set forth in SEQ ID NO:11;
e) a nucleotide sequence having the sequence as set forth in SEQ ID NO:12 and a nucleotide sequence as set forth in SEQ ID NO:13;
f) a nucleotide sequence having the sequence as set forth in SEQ ID NO:14 and a nucleotide sequence as set forth in SEQ ID NO:15;
g) a nucleotide sequence having the sequence as set forth in SEQ ID NO:16 and a nucleotide sequence as set forth in SEQ ID NO:17;

WO 92/21693 PCA'/1JS92/04195 2 -L J_0 0 h) a nucleotide sequence having the sequence as set forth in SEQ ID N0:18 and a nucleotide sequence as set forth in SEQ ID NO:19;
i) a nucleotide sequence having the sequence as .5 set forth in SEQ ID NO:20 and a nucleotide sequence as set forth in SEQ ID NO:21;
j) a nucleotide sequence having the sequence as set forth in SEQ ID NO:22 and a nucleotide sequence as set forth in SEQ ID N :23;
k) a nucleotide sequence having the sequence as set forth in SEQ ID NO:24 and a nucleotide sequence as set forth in SEQ ID NO:25;
aõr' =
1) a nucleotide seauence having the sequence as set forth in SEQ ID NO:26 and a nucleotide sequence as set forth in SEQ ID NO:27;
m) a nucleotide sequence having the sequence as set forth in SEQ ID NO:28 and a nucleotide sequence as set forth in SEQ ID NO:29;
n) a nucleotide sequence having the sequence as set forth in SEQ ID NO:30 and a nucleotide sequence as set forth in SEQ ID NO:31;
o) 'a nucleotide seauence having the sequence as set forth in SEQ ID NO:32 and a nucleotide sequence as set forth in SEQ ID NO:33;
p) a nucleotide seauence having the sequence as set forth in SEQ ID NO:34 and a nucleotide sequence as set forth in SEQ ID NO:35;
q) a nucleotide sequence having the sequence as set forth in SEQ ID NO:36 and a nucleotide sequence as set forth in SEQ ID TVOs37;
r) a nucleotide seauence having the sequence as set forth in SEQ ID NO:38 and a nucleotide sequence as set forth in SEQ ID NO:39;

WO 92/21693 PC'T/US92l04195 s) a nucleotide sequence having the sequence as set forth in SEQ ID NO:40 and a nucleotide sequence as set forth in SEQ ID NO:41;
t) a nucleotide sequence having the sequence as 5 set forth in SEQ ID NO:42 and a nucleotide sequence as set forth in SEQ ID NO:43;
u) a nucleotide sequence having the sequence as set forth in SEQ ID NO:44 and a nucleotide sequence as set forth in SEQ ID NO:45;
10 v) a nucleotide sequence having the sequence as set forth in SEQ ID NO:46 and a nucleotide sequence as set forth in SEQ ID NO:47;
w) .a nucleotide sequence having the sequence as set forth in SEQ ID NO:48 and a nucleotide sequence as 15 set forth in SEQ ID NO:49;
x) a nucleotide sequence having the sequence as set forth in SEQ ID NO:50 and a nucleotide sequence as set forth in SEQ ID NO:51;
y) a nucleotide sequence having the sequence as 20 set forth in SEQ ID NO:52 and a nucleotide sequence as set forth in SEQ ID NO:53;
z) a nucleotide sequence having the sequence as set forth in SEQ ID NO:54 and a nucleotide seauence as set forth in SEQ ID NO:55;
aa) a nucleotide sequence having the sequence as set forth in SEQ ID NO:56 and a nucleotide sequence as set forth in SEQ ID NO:57;
bb) a nucleotide sequence having the sequence as set forth in SEQ ID NO:58 and a nucleotide sequence as set forth in SEQ ID NO:59;
cc) a nucleotide sequence having the sequence as set forth in SEQ ID NO:60 and a nucleotide sequence as set forth in SEQ ID NO:61; and WO 92/21693 PCT/tJS92/04195 dd) a nucleotide sequence having the sequence as set forth in SEQ ID NO:62 and a nucleotide sequence as set forth in SEQ ID NO:63;
wherein said nucleotide is 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.
Also, the polymorphism corresponding to 1A-27A as described above and characterizes by their 27 primer pairs according to SEQ ID N0:10-SEQ N z63 are usefdll for human sample individualization evaluation because of their high PIC values.
Further, the assayaccording 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 mol.ecular, 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 microsatel.lites described in the present invention.
The general PCR technique step is conducted generally as desc:ribed in U.S. Patent No. 4,683,195 to Mullis et al and U.S. Patent No. 4,683,202 to Mul:lis et al. Further, electrical motility enhancing DNA analogs can optionally be used duririg 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 polymo:rphism DNA segment according to the present invention yields excellent results. Further use of primer sequences corresponding to SEQ ID NO:10 through SEQ ID NO:63 to detect the polymorphism 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 0 0.r.' 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:
Examnle 1 The samples are of DNA are prepared as follows.
60ng of genomic DNA are used as template for PCR
with 80ng of each oligonucleotide primer, 0.6 units of Taq Polymerase 50mM KCL, 10mM Tris (pH 8.3), 1.5mM
MgC12, 0.01% gelatin, 200uk1 of each dGTP, dATP, dTTP, 2.5uM dCTP and 10 microcuries of alpha P32 dCTP., in a finalreaction volume of 15 microliters. The samples are overlaid with 15 micro].iters of mineral oil to prevent evaporation.
Example 2 PCR is performed for each of the samples a~nd primers described in Example 1, above.
PCR is perf ornned in a Techne MW-1 microplate thermocycler under the following conditions denaturation of 94 degrees C for 1.4 mi.n., annealing at 55 degrees C for 2 min., and extension at 72 degrees C
for 2 rnin. 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 electrovhoresis 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 3a16 hours.
The foregoing descriiption of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without departing from the generic concept and therefore such adaptations are intended to be comprehended within the meaning and range of equivalents of a disclosed embodiment. it is to be understood that the phraseology or terminology employed herein is for the purposes of description only and not of limitation. *

1 '4

Claims (18)

1. An isolated 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, and a sequence according to SEQ ID NO:6.
2. The isolated nucleotide sequence according to claim 1, wherein the sequence is a sequence according to SEQ ID NO:1.
3. The isolated nucleotide sequence according to claim 1, wherein the sequence is a sequence according to SEQ ID NO:2.
4. The isolated nucleotide sequence according to claim 1, wherein the sequence is a sequence according to SEQ ID NO:3.
5. The isolated nucleotide sequence according to claim 1, wherein the sequence is a sequence according to SEQ ID NO:4.
6. The isolated nucleotide sequence according to claim 1, wherein the sequence is a sequence according to SEQ ID NO:5.
7. The isolated nucleotide sequence according to claim 1, wherein the sequence is a sequence according to SEQ ID NO:6.
8. 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 polymorphism information content of about 0.9, the method comprising the steps of:

i) obtaining a DNA fragment comprising the repeat polymorphisms in an amount sufficient for amplification by polymerase chain reaction;

ii) amplifying the DNA fragment by polymerase chain reaction using a pair of oligonucleotide primers comprising at least one DNA fragment according to claim 1, wherein said pair of oligonucleotide primers is 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 to produce amplification; and iii) determining the length of the amplification products of step (ii) in order to detect the added or omitted set of dinucleotide or tetranucleotide repeat polymorphisms.
9. 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 polymorphism 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 said repeat polymorphisms in an amount sufficient for amplification by polymerase chain reaction, b. amplifying said DNA fragment by a polymerase chain reaction procedure using a pair of oligonucleotide primers which amplify said DNA fragment, c. resolving the amplified DNA fragment using polyacrylamide gel electrophoresis, and d. comparing the resolved DNA fragment by autoradiography to observe the differences in migration patterns due to length variation.
10. An assay according to claim 9, wherein said pair of oligonucleotide primers is 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.
11. 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 polymorphism information content of about 0.9 comprising an amount of a pair of oligonucleotide primers sufficient for amplification by polymerase chain reaction comprising at least one nucleotide sequence having a sequence according to claim 1, wherein said pair of oligonucleotide primers is 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.
12. 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 beta-actin related pseudogene, wherein said method results in a polymorphism information content of about 0.9, and wherein said genetic material comprises a DNA fragment comprising a nucleotide 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 method comprises a. obtaining a DNA fragment comprising said repeat polymorphisms in an amount sufficient for amplification by polymerase chain reaction, b. amplifying said DNA fragment by a polymerase chain reaction procedure using a pair of oligonucleotide primers which amplify said DNA fragment, c. resolving the amplified DNA fragment using polyacrylamide gel electrophoresis, and d. comparing the resolved DNA fragment by autoradiography to observe the differences in migration patterns due to length variation.
13. A method according to claim 12, wherein said pair of oligonucleotide primers is 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 as set forth in SEQ ID NO:5, and a sequence according to SEQ ID NO:6, wherein said method results in.
a polymorphism information content of about 0.9.
14. An isolated nucleotide sequence selected from the group consisting of a sequence according to any one of SEQ ID NOS: 10 to 63.
15. An assay for measuring the subtle differences in genetic material regarding an added or omitted set of dinucleotide or tetranucleotide repeat polymorphisms wherein said genetic material comprises a sequence amplifiable by one of primer pairs 1A-27A, which comprises a. obtaining nucleotide segments comprising said repeat polymorphisms in an amount sufficient for amplification by polymerase chain reaction, b. amplifying said segments by a PCR procedure using a pair of oligonucleotide primers capable of amplifying said polymorphisms containing segments, c. resolving the amplified segments using polyacrylamide gel electrophoresis, and d. comparing the resolved segments by autoradiography to observe the differences in migration patterns due to length variation.
16. The assay according to claim 15, wherein said pair of oligonucleotide primers of step (b) is selected from the group of primer pairs consisting of d) a nucleotide sequence having the sequence as set forth in SEQ ID NO:10 and a nucleotide sequence as set forth in SEQ ID NO:11;
e) a nucleotide sequence having the sequence as set forth in SEQ ID NO:12 and a nucleotide sequence as set forth in SEQ ID NO:13;

f) a nucleotide sequence having the sequence as set forth in SEQ ID NO:14 and a nucleotide sequence as set forth in SEQ ID NO:15;

g) a nucleotide sequence having the sequence as set forth in SEQ ID NO:16 and a nucleotide sequence as set forth in SEQ ID NO:17;

h) a nucleotide sequence having the sequence as set forth in SEQ ID NO:18 and a nucleotide sequence as set forth in SEQ ID NO:19;

i) a nucleotide sequence having the sequence as set forth in SEQ ID NO:20 and a nucleotide sequence as set forth in SEQ ID NO:21;

j) a nucleotide sequence having the sequence as set forth in SEQ ID NO:22 and a nucleotide sequence as set forth in SEQ ID NO:23;

k) a nucleotide sequence having the sequence as set forth in SEQ ID NO:24 and a nucleotide sequence as set forth in SEQ ID NO:25;

l) a nucleotide sequence having the sequence as set forth in SEQ ID NO:26 and a nucleotide sequence as set forth in SEQ ID NO;27;

m) a nucleotide sequence having the sequence as set forth in SEQ ID NO:28 and a nucleotide sequence as set forth in SEQ ID NO:29;

n) a nucleotide sequence having the sequence as set forth in SEQ ID NO:30 and a nucleotide sequence as set forth in SEQ ID NO:31;

o) a nucleotide sequence having the sequence as set forth in SEQ ID NO:32 and a nucleotide sequence as set forth in SEQ ID NO:33;

p) a nucleotide sequence having the sequence as set forth in SEQ ID NO:34 and a nucleotide sequence as set forth in SEQ ID NO:35;

q) a nucleotide sequence having the sequence as set forth in SEQ ID NO:36 and a nucleotide sequence as set forth in SEQ ID NO:37;

r) a nucleotide sequence having the sequence as set forth in SEQ ID NO:38 and a nucleotide sequence as set forth in SEQ ID NO:39;

s) a nucleotide sequence having the sequence as set forth in SEQ ID NO:40 and a nucleotide sequence as set forth in SEQ ID NO:41;

t) a nucleotide sequence having the sequence as set forth in SEQ ID NO:42 and a nucleotide sequence as set forth in SEQ ID NO:43;

u) a nucleotide sequence having the sequence as set forth in SEQ ID NO:44 and a nucleotide sequence as set forth in SEQ ID NO:45;

v) a nucleotide sequence having the sequence as set forth in SEQ ID NO:46 and a nucleotide sequence as set forth in SEQ ID NO:47;

w) a nucleotide sequence having the sequence as set forth in SEQ ID NO:48 and a nucleotide sequence as set forth in SEQ ID NO:49;
x) a nucleotide sequence having the sequence as set forth in SEQ ID NO:50 and a nucleotide sequence as set forth in SEQ ID NO:51;
y) a nucleotide sequence having the sequence as set forth in SEQ ID NO:52 and a nucleotide sequence as set forth in SEQ ID NO:53;
z) a nucleotide sequence having the sequence as set forth in SEQ ID NO:54 and a nucleotide sequence as set forth in SEQ ID NO:55;
aa) a nucleotide sequence having the sequence as set forth in SEQ ID NO:56 and a nucleotide sequence as set forth in SEQ ID NO:57;
bb) a nucleotide sequence having the sequence as set forth in SEQ ID NO:58 and a nucleotide sequence as set forth in SEQ ID NO:59;
cc) a nucleotide sequence having the sequence as set forth in SEQ ID NO:60 and a nucleotide sequence as set forth in SEQ ID NO:61; and dd) a nucleotide sequence having the sequence as set forth in SEQ ID NO:62 and a nucleotide sequence as set forth in SEQ ID NO:63.
17. An assay kit for conducting a PCR procedure to detect an added or omitted set of dinucleotide or tetranucleotide repeat polymorphisms, comprising an amount sufficient for amplification by polymerase chain reaction of at least one nucleotide sequence having a sequence according to claim 14, wherein the nucleotide sequence is part of a primer pair selected from the group of primer pairs consisting of d) a nucleotide sequence having the sequence as set forth in SEQ ID NO:10 and a nucleotide sequence as set forth in SEQ ID NO:11;
e) a nucleotide sequence having the sequence as set forth in SEQ ID NO:12 and a nucleotide sequence as set forth in SEQ ID NO:13;
f) a nucleotide sequence having the sequence as set forth in SEQ ID NO:14 and a nucleotide sequence as set forth in SEQ ID NO:15;
g) a nucleotide sequence having the sequence as set forth in SEQ ID NO:16 and a nucleotide sequence as set forth in SEQ ID NO:17;
h) a nucleotide sequence having the sequence as set forth in SEQ ID NO:18 and a nucleotide sequence as set forth in SEQ ID NO:19;
i) a nucleotide sequence having the sequence as set forth in SEQ ID NO:20 and a nucleotide sequence as set forth in SEQ ID NO:21;
j) a nucleotide sequence having the sequence as set forth in SEQ ID NO:22 and a nucleotide sequence as set forth in SEQ ID NO:23;
k) a nucleotide sequence having the sequence as set forth in SEQ ID NO:24 and a nucleotide sequence as set forth in SEQ ID NO:25;

1) a nucleotide sequence having the sequence as set forth in SEQ ID NO:26 and a nucleotide sequence as set forth in SEQ ID NO;27;

m) a nucleotide sequence having the sequence as set forth in SEQ ID NO:28 and a nucleotide sequence as set forth in SEQ ID NO:29;
n) a nucleotide sequence having the sequence as set forth in SEQ ID NO:30 and a nucleotide sequence as set forth in SEQ ID NO:31;
o) a nucleotide sequence having the sequence as set forth in SEQ ID NO:32 and a nucleotide sequence as set forth in SEQ ID NO:33;
p) a nucleotide sequence having the sequence as set forth in SEQ ID NO:34 and a nucleotide sequence as set forth in SEQ ID NO:35;
q) a nucleotide sequence having the sequence as set forth in SEQ ID NO:36 and a nucleotide sequence as set forth in SEQ ID NO:37;
r) a nucleotide sequence having the sequence as set forth in SEQ ID NO:38 and a nucleotide sequence as set forth in SEQ ID NO:39;
s) a nucleotide sequence having the sequence as set forth in SEQ ID NO:40 and a nucleotide sequence as set forth in SEQ ID NO:41;
t) a nucleotide sequence having the sequence as set forth in SEQ ID NO:42 and a nucleotide sequence as set forth in SEQ ID NO:43;
u) a nucleotide sequence having the sequence as set forth in SEQ ID NO:44 and a nucleotide sequence as set forth in SEQ ID NO:45;
v) a nucleotide sequence having the sequence as set forth in SEQ ID NO:46 and a nucleotide sequence as set forth in SEQ ID NO:47;

w) a nucleotide sequence having the sequence as set forth in SEQ ID NO:48 and a nucleotide sequence as set forth in SEQ ID NO:49;
x) a nucleotide sequence having the sequence as set forth in SEQ ID NO:50 and a nucleotide sequence as set forth in SEQ ID NO:51;

y) a nucleotide sequence having the sequence as set forth in SEQ ID NO:52 and a nucleotide sequence as set forth in SEQ ID NO:53;
z) a nucleotide sequence having the sequence as set forth in SEQ ID NO:54 and a nucleotide sequence as set forth in SEQ ID NO:55;
aa) a nucleotide sequence having the sequence as set forth in SEQ ID NO:56 and a nucleotide sequence as set forth in SEQ ID NO:57;
bb) a nucleotide sequence having the sequence as set forth in SEQ ID NO:58 and a nucleotide sequence as set forth in SEQ ID NO:59;
cc) a nucleotide sequence having the sequence as set forth in SEQ ID NO:60 and a nucleotide sequence as set forth in SEQ ID NO:61; and dd) a nucleotide sequence having the sequence as set forth in SEQ ID NO:62 and a nucleotide sequence as set forth in SEQ ID NO:63;
wherein said amount of said nucleotide sequence is in combination with an effective amount of ancillary PCR
reagents.
18. A method for conducting a PCR procedure to detect an added or omitted set of dinucleotide or tetranucleotide repeat polymorphisms, comprising using an amount sufficient for amplification by polymerase chain reaction of at least one nucleotide sequence having a sequence according to claim 14, wherein the nucleotide sequence is part of a primer pair selected from the group of primer pairs consisting of d) a nucleotide sequence having the sequence as set forth in SEQ ID NO:10 and a nucleotide sequence as set forth in SEQ ID NO:11;
e) a nucleotide sequence having the sequence as set forth in SEQ ID NO:12 and a nucleotide sequence as set forth in SEQ ID NO:13;

f) a nucleotide sequence having the sequence as set forth in SEQ ID NO:14 and a nucleotide sequence as set forth in SEQ ID NO:15;
g) a nucleotide sequence having the sequence as set forth in SEQ ID NO:16 and a nucleotide sequence as set forth in SEQ ID NO:17;
h) a nucleotide sequence having the sequence as set forth in SEQ ID NO:18 and a nucleotide sequence as set forth in SEQ ID NO:19;
i) a nucleotide sequence having the sequence as set forth in SEQ ID NO:20 and a nucleotide sequence as set forth in SEQ ID NO:21;
j) a nucleotide sequence having the sequence as set forth in SEQ ID NO:22 and a nucleotide sequence as set forth in SEQ ID NO:23;
k) a nucleotide sequence having the sequence as set forth in SEQ ID NO:24 and a nucleotide sequence as set forth in SEQ ID NO:25;
l) a nucleotide sequence having the sequence as set forth in SEQ ID NO:26 and a nucleotide sequence as set forth in SEQ ID NO:27;

m) a nucleotide sequence having the sequence as set forth in SEQ ID NO:28 and a nucleotide sequence as set forth in SEQ ID NO:29;

n) a nucleotide sequence having the sequence as set forth in SEQ ID NO:30 and a nucleotide sequence as set forth in SEQ ID NO:31;
o) a nucleotide sequence having the sequence as set forth in SEQ ID NO:32 and a nucleotide sequence as set forth in SEQ ID NO:33;

p) a nucleotide sequence having the sequence as set forth in SEQ ID NO:34 and a nucleotide sequence as set forth in SEQ ID NO:35;

q) a nucleotide sequence having the sequence as set forth in SEQ ID NO:36 and a nucleotide sequence as set forth in SEQ ID NO:37;

r) a nucleotide sequence having the sequence as set forth in SEQ ID. NO:38 and a nucleotide sequence as set forth in SEQ ID NO:39;

s) a nucleotide sequence having the sequence as set forth in SEQ ID NO:40 and a nucleotide sequence as set forth in SEQ ID NO:41;

t) a nucleotide sequence having the sequence as set forth in SEQ ID NO:42 and a nucleotide sequence as set forth in SEQ ID NO:43;

u) a nucleotide sequence having the sequence as set forth in SEQ ID NO:44 and a nucleotide sequence as set forth in SEQ ID NO:45;

v) a nucleotide sequence having the sequence as set forth in SEQ ID NO:46 and a nucleotide sequence as set forth in SEQ ID NO:47;

w) a nucleotide sequence having the sequence as set forth in SEQ ID NO:48 and a nucleotide sequence as set forth in SEQ ID NO:49;

x) a nucleotide sequence having the sequence as set forth in SEQ ID NO:50 and a nucleotide sequence as set forth in SEQ ID NO:51;

y) a nucleotide sequence having the sequence as set forth in SEQ ID NO:52 and a nucleotide sequence as set forth in SEQ ID NO:53;

z) a nucleotide sequence having the sequence as set forth in SEQ ID NO:54 and a nucleotide sequence as set forth in SEQ ID NO:55;

aa) a nucleotide sequence having the sequence as set forth in SEQ ID NO:56 and a nucleotide sequence as set forth in SEQ ID NO:57;

bb) a nucleotide sequence having the sequence as set forth in SEQ ID NO:58 and a nucleotide sequence as set forth in SEQ ID NO:59;

cc) a nucleotide sequence having the sequence as set forth in SEQ ID NO:60 and a nucleotide sequence as set forth in SEQ ID NO:61; and dd) a nucleotide sequence having the sequence as set forth in SEQ ID NO:62 and a nucleotide sequence as set forth in SEQ ID NO:63.
CA002110059A 1991-05-29 1992-05-27 Three highly informative microsatellite repeat polymorphic dna markers Expired - Lifetime CA2110059C (en)

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US70750191A 1991-05-29 1991-05-29
US707,501 1991-05-29
US07/799,828 US5378602A (en) 1991-05-29 1991-11-27 Highly informative microsatellite repeat polymorphic DNA markers twenty-[seven]six
US799,828 1991-11-27
PCT/US1992/004195 WO1992021693A1 (en) 1991-05-29 1992-05-27 Three highly informative microsatellite repeat polymorphic dna markers

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CA2110059C true CA2110059C (en) 2007-08-14

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