Five highly informative microsatellite repeat polymorphic DNA markers

The invention relates to polymorphic markers (two tetranucleotide, one dinucleotide repeat polymorphisms, 27 markers characterized by primer pairs 1A-27A, and five markers characterized by primer pairs 1B-5B 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.

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 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.

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. The invention further relates to twenty-seven other 
polymorphic markers useful for human individualization. The invention 
still further relates to five other polymorphic markers and the five 
primer pairs useful for measuring the subtle genetic differences relating 
to the five polymorphic markers. 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, one 
dinucleotide repeat polymorphisms, twenty-seven other unique polymorphic 
markers, and five more unique polymorphic markers) that are useful for 
human individualization for a 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 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 oligonucteotide 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. 
Twenty-seven other primary pair sequences for detecting unique 
polymorphisms are sequences according to SEQ ID NO:10 through SEQ ID 
NO:63. Additionally, five other primary pair sequences for detecting 
unique polymorphisms are sequences according to SEQ ID NO:64 through SEQ 
ID NO:73. 
The described polymorphisms are useful for human sample individualization, 
because of their high PIC 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 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 
five further polymorphisms described are original. 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 set 
forth in Table 1, below. The 27 pairs are indicated in Table 1 as 1A-27A, 
respectively. Also indicated is the locus, the chromosomal location, the 
primer SEQ ID NO:, the degree of heterozygousness, the PIC value, the 
size, the repeat sequence and the number of alleles. 
The additional five markers are characterized by sets of oligonucleotide 
primers as set forth in Table 2, below. These five pairs are indicated in 
Table 2 as 1B-5B, respectively. Also indicated is the locus, the 
chromosomal location, the primer SEQ ID NO:, the degree of 
heterozygousness, the PIC value, the size, the repeat sequence and the 
number of alleles. 
TABLE 1 
__________________________________________________________________________ 
Chromosomal 
Primer SEQ 
Pair # 
Locus Location 
ID NO: Heteroz 
PIC 
Size Repeat No. of alleles 
__________________________________________________________________________ 
1A Int-2 11q13 10,11 84.6% 
0.79 
161-177 
(TG).sub.5 TC(TG).sub.16 
9 
2A PLA-AZ 12 12,13 73.3% 
0.76 
122-137 
(TTA).sub.16 
6 
3A FABP2 4q28-31 
14,15 64% 0.64 
99-117 
(TTA).sub.13 
6 
4A THROO1 15q15 16,17 60% 0.58 
165-181 
(CT).sub.14 
9 
5A CYARP450 
15Fq21.1 
18,19 91.3% 
0.67 
175-199 
(TTTA).sub.8 
5 
6A GCG 2q36-q37 
20,21 88% 0.77 
142-172 
(GA).sub.19 
11 
7A IL-9 5q 22,23 62.5% 
0.75 
127-139 
(TG).sub.20 
7 
8A CSTP1 20 24,25 61% 0.58 
123-141 
(GT).sub.15 
9A ANKYRIN 
8p11.1-21.1 
26,27 54% 0.45 
107-113 
(AC).sub.14 
4 
10A CD-19 16 28,29 40% 0.39 
79-91 
(GT).sub.17 
7 
11A c-fms 5q33.3-34 
30,31 86% 0.85 
95-127 
(GT).sub.26 
10 
12A CD 8 2p12 32,33 71% 0.66 
138-170 
(AC).sub.14 
7 
13A CYP2D7-8 
22 34,35 80% 0.78 
98-116 
(GT).sub.18 
10 
14A W 30 7q 36,37 74% 0.72 11 
15A HMG-14 21 38,39 69% 0.67 
69-93 
(GT).sub.19 
10 
16A RHO 3 40,41 72% 0.68 
145-169 5 
17A PFKL 21q22.3 
42,43 70% 0.66 
129-145 
(AC).sub.16 
7 
18A HSFLT 13q12 44,45 51% 0.49 
164-186 
(TG).sub.21 
8 
19A HSMYHO1 
14 46,47 66% 0.60 
90-102 
(GT).sub.15 
6 
20A HSATPSY1 
12p13-qter 
48,49 60% 0.54 
111-117 
(GT).sub.11 
4 
21A CFES PPS 
15q25-qter 
50,51 75% 0.70 
143-163 
(ATTT).sub.11 
6 
22A DHFRP2 6 52,53 70% 0.66 
157-173 
(AAAC).sub.7 
5 
23A CRYG1 2q34-35 
54,55 68% 0.61 
117-126 
(AAC).sub.9 
4 
24A F13A1 6p24-25 
56,57 78% 0.75 
180-230 
(AAAG).sub.7 
8 
25A TRM1 6p23-q12 
58,59 54% 0.50 
174-186 
(AAC).sub.8 
5 
26A II-D 6 60,61 81% 0.78 
185-206 
(CAG).sub.18 
7 
27A TH 11p15.5-p15 
62,63 78% 0.75 
244-260 
(TCAT).sub.9 
5 
__________________________________________________________________________ 
TABLE 2 
__________________________________________________________________________ 
Chromosomal 
Primer SEQ 
Pair # Locus Location 
ID NO: Heteroz 
PIC 
Size Repeat No. of 
__________________________________________________________________________ 
alleles 
1B ACPP 3q21-qter 
64 69 0.65 
260-280 
(AAAT).sub.11 
6 
65 
2B MSP 18q23-qter 
66 80% 0.77 
208-232 
(ATGG).sub.12, (TGGA).sub.9 
7 
67 79% 0.76 
122-142 
(ATGG).sub.12 
6 
3B IGF1 12q23 68 53% 0.52 
173-207 
(CT).sub.16 
11 
4B GABRB1 
4p12-p13 
70 72% 0.68 
91-99 
(AC).sub.19 
5 
5B MYC 8q24 72 86% 0.85 
87-125 
(AT).sub.23 
15 
73 
__________________________________________________________________________ 
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 polynucleotide having the sequence as set forth in SEQ ID NO:1 and a 
polynucleotide having a sequence as set forth in SEQ ID NO:2; 
b) a polynucleotide having the sequence as set forth in SEQ ID NO:3 and a 
polynucleotide having the sequence as set forth in SEQ ID NO:4; 
c) a polynucleotide having the sequence as set forth in SEQ ID NO:5 and a 
polynucleotide having the sequence as set forth in SEQ ID NO:6; 
d) a polynucleotide having the sequence as set forth in SEQ ID NO:10 and a 
polynucleotide having the sequence as set forth in SEQ ID NO:11; 
e) a polynucleotide having the sequence as set forth in SEQ ID NO:12 and a 
polynucleotide having the sequence as set forth in SEQ ID NO:13; 
f) a polynucleotide having the sequence as set forth in SEQ ID NO:14 and a 
polynucleotide having the sequence as set forth in SEQ ID NO:15; 
g) a polynucleotide having the sequence as set forth in SEQ ID NO:16 and a 
polynucleotide having the sequence as set forth in SEQ ID NO:17; 
h) a polynucleotide having the sequence as set forth in SEQ ID NO:18 and a 
polynucleotide having the sequence as set forth in SEQ ID NO:19; 
i) a polynucleotide having the sequence as set forth in SEQ ID NO:20 and a 
polynucleotide having the sequence as set forth in SEQ ID NO:21; 
j) a polynucleotide having the sequence as set forth in SEQ ID NO:22 and a 
polynucleotide having the sequence as set forth in SEQ ID NO:23; 
k) a polynucleotide having the sequence as set forth in SEQ ID NO:24 and a 
polynucleotide having the sequence as set forth in SEQ ID NO:25; 
l) a polynucleotide having the sequence as set forth in SEQ ID NO:26 and a 
polynucleotide having the sequence as set forth in SEQ ID NO:27; 
m) a polynucleotide having the sequence as set forth in SEQ ID NO:28 and a 
polynucleotide having the sequence as set forth in SEQ ID NO:29; 
n) a polynucleotide having the sequence as set forth in SEQ ID NO:30 and a 
polynucleotide having the sequence as set forth in SEQ ID NO:31; 
o) a polynucleotide having the sequence as set forth in SEQ ID NO:32 and a 
polynucleotide having the sequence as set forth in SEQ ID NO:33; 
p) a polynucleotide having the sequence as set forth in SEQ ID NO:34 and a 
polynucleotide having the sequence as set forth in SEQ ID NO:35; 
q) a polynucleotide having the sequence as set forth in SEQ ID NO:36 and a 
polynucleotide having the sequence as set forth in SEQ ID NO:37; 
r) a polynucleotide having the sequence as set forth in SEQ ID NO:38 and a 
polynucleotide having the sequence as set forth in SEQ ID NO:39; 
s) a polynucleotide having the sequence as set forth in SEQ ID NO:40 and a 
polynucleotide having the sequence as set forth in SEQ ID NO:41; 
t) a polynucleotide having the sequence as set forth in SEQ ID NO:42 and a 
polynucleotide having the sequence as set forth in SEQ ID NO:43; 
u) a polynucleotide having the sequence as set forth in SEQ ID NO:44 and a 
polynucleotide having the sequence as set forth in SEQ ID NO:45; 
v) a polynucleotide having the sequence as set forth in SEQ ID NO:46 and a 
polynucleotide having the sequence as set forth in SEQ ID NO:47; 
w) a polynucleotide having the sequence as set forth in SEQ ID NO:48 and a 
polynucleotide having the sequence as set forth in SEQ ID NO:49; 
x) a polynucleotide having the sequence as set forth in SEQ ID NO:50 and a 
polynucleotide having the sequence as set forth in SEQ ID NO:51; 
y) a polynucleotide having the sequence as set forth in SEQ ID NO:52 and a 
polynucleotide having the sequence as set forth in SEQ ID NO:53; 
z) a polynucleotide having the sequence as set forth in SEQ ID NO:54 and a 
polynucleotide having the sequence as set forth in SEQ ID NO:55; 
aa) a polynucleotide having the sequence as set forth in SEQ ID NO:56 and a 
polynucleotide having the sequence as set forth in SEQ ID NO:57; 
bb) a polynucleotide having the sequence as set forth in SEQ ID NO:58 and a 
polynucleotide having the sequence as set forth in SEQ ID NO:59; 
cc) a polynucleotide sequence having the sequence as set forth in SEQ ID 
NO:60 and a polynucleotide sequence as set forth in SEQ ID NO:61; 
dd) a polynucleotide having the sequence as set forth in SEQ ID NO:62 and a 
polynucleotide having the sequence as set forth in SEQ ID NO:63. 
ee) a polynucleotide having the sequence as set forth in SEQ ID NO:64 and a 
polynucleotide having the sequence as set forth in SEQ ID NO:65; 
ff) a polynucleotide having the sequence as set forth in SEQ ID NO:66 and a 
polynucleotide having the sequence as set forth in SEQ ID NO:67; 
gg) a polynucleotide having the sequence as set forth in SEQ ID NO:68 and a 
polynucleotide having the sequence as set forth in SEQ ID NO:69; 
hh) a polynucleotide having the sequence as set forth in SEQ ID NO:70 and a 
polynucleotide having the sequence as set forth in SEQ ID NO:71; 
ii) a polynucleotide having the sequence as set forth in SEQ ID NO:72 and a 
polynucleotide having the sequence as set forth in SEQ ID NO:73. 
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 no 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 and 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 polynucleotides selected from the 
group of polynucleotide pairs consisting of 
a) a polynucleotide having the sequence as set forth in SEQ ID NO:1 and a 
polynucleotide having the sequence as set forth in SEQ ID NO:2; 
b) a polynucleotide having the sequence as set forth in SEQ ID NO:3 and a 
polynucleotide having the sequence as set forth in SEQ ID NO:4; 
c) a polynucleotide having the sequence as set forth in SEQ ID NO:5 and a 
polynucleotide having the sequence as set forth in SEQ ID NO:6, 
d) a polynucleotide having the sequence as set forth in SEQ ID NO:10 and a 
polynucleotide having the sequence as set forth in SEQ ID NO:11; 
e) a polynucleotide having the sequence as set forth in SEQ ID NO:12 and a 
polynucleotide having the sequence as set forth in SEQ ID NO:13; 
f) a polynucleotide having the sequence as set forth in SEQ ID NO:14 and a 
polynucleotide having the sequence as set forth in SEQ ID NO:15; 
g) a polynucleotide having the sequence as set forth in SEQ ID NO:16 and a 
polynucleotide having the sequence as set forth in SEQ ID NO:17; 
h) a polynucleotide having the sequence as set forth in SEQ ID NO:18 and a 
polynucleotide having the sequence as set forth in SEQ ID NO:19; 
i) a polynucleotide having the sequence as set forth in SEQ ID NO:20 and a 
polynucleotide having the sequence as set forth in SEQ ID NO:21; 
j) a polynucleotide having the sequence as set forth in SEQ ID NO:22 and a 
polynucleotide having the sequence as set forth in SEQ ID NO:23; 
k) a polynucleotide having the sequence as set forth in SEQ ID NO:24 and a 
polynucleotide having the sequence as set forth in SEQ ID NO:25; 
l) a polynucleotide having the sequence as set forth in SEQ ID NO:26 and a 
polynucleotide having the sequence as set forth in SEQ ID NO:27; 
m) a polynucleotide having the sequence as set forth in SEQ ID NO:28 and a 
polynucleotide having the sequence as set forth in SEQ ID NO:29; 
n) a polynucleotide having the sequence as set forth in SEQ ID NO:30 and a 
polynucleotide having the sequence as set forth in SEQ ID NO:31; 
o) a polynucleotide having the sequence as set forth in SEQ ID NO:32 and a 
polynucleotide having the sequence as set forth in SEQ ID NO:33; 
p) a polynucleotide having the sequence as set forth in SEQ ID NO:34 and a 
polynucleotide having the sequence as set forth in SEQ ID NO:35; 
q) a polynucleotide having the sequence as set forth in SEQ ID NO:36 and a 
polynucleotide having the sequence as set forth in SEQ ID NO:37; 
r) a polynucleotide having the sequence as set forth in SEQ ID NO:38 and a 
polynucleotide having the sequence as set forth in SEQ ID NO:39; 
s) a polynucleotide having the sequence as set forth in SEQ ID NO:40 and a 
polynucleotide having the sequence as set forth in SEQ ID NO:41; 
t) a polynucleotide having the sequence as set forth in SEQ ID NO:42 and a 
polynucleotide having the sequence as set forth in SEQ ID NO:43; 
u) a polynucleotide having the sequence as set forth in SEQ ID NO:44 and a 
polynucleotide having the sequence as set forth in SEQ ID NO:45; 
v) a polynucleotide having the sequence as set forth in SEQ ID NO:46 and a 
polynucleotide having the sequence as set forth in SEQ ID NO:47; 
w) a polynucleotide having the sequence as set forth in SEQ ID NO:48 and a 
polynucleotide having the sequence as set forth in SEQ ID NO:49; 
x) a polynucleotide having the sequence as set forth in SEQ ID NO:50 and a 
polynucleotide having the sequence as set forth in SEQ ID NO:51; 
y) a polynucleotide having the sequence as set forth in SEQ ID NO:52 and a 
polynucleotide having the sequence as set forth in SEQ ID NO:53; 
z) a polynucleotide having the sequence as set forth in SEQ ID NO:54 and a 
polynucleotide having the sequence as set forth in SEQ ID NO:55; 
aa) a polynucleotide having the sequence as set forth in SEQ ID NO:56 and a 
polynucleotide having the sequence as set forth in SEQ ID NO:57; 
bb) a polynucleotide having the sequence as set forth in SEQ ID NO:58 and a 
polynucleotide having the sequence as set forth in SEQ ID NO:59; 
cc) a polynucleotide having the sequence as set forth in SEQ ID NO:60 and a 
polynucleotide having the sequence as set forth in SEQ ID NO:61; 
dd) a polynucleotide having the sequence as set forth in SEQ ID NO:62 and a 
polynucleotide having the sequence as set forth in SEQ ID NO:63; 
ee) a polynucleotide having the sequence as set forth in SEQ ID NO:64 and a 
polynucleotide having the sequence as set forth in SEQ ID NO:65; 
ff) a polynucleotide having the sequence as set forth in SEQ ID NO:66 and a 
polynucleotide having the sequence as set forth in SEQ ID NO:67; 
gg) a polynucleotide having the sequence as set forth in SEQ ID NO:68 and a 
polynucleotide having the sequence as set forth in SEQ ID NO:69; 
hh) a polynucleotide having the sequence as set forth in SEQ ID NO:70 and a 
polynucleotide having the sequence as set forth in SEQ ID NO:71; and 
ii) a polynucleotide having the sequence as set forth in SEQ ID NO:72 and a 
polynucleotide having the sequence as set forth in SEQ ID NO:73; 
wherein said polynucleotide 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 NO:10-SEQ NO:63 
are useful for human sample individualization evaluation because of their 
high PIC values. Additionally, the polymorphisms corresponding to 1B-5B as 
described above and characterizes by their five primer pairs according to 
SEQ ID NO:64-SEQ ID NO:73 are useful for human sample individualization 
evaluation because of their high PIC values. 
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. Further use of primer sequences corresponding to SEQ ID 
NO:10 through SEQ ID NO:63 or SEQ ID NO:64 through SEQ ID NO:73 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 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 overlaid 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. 
The foregoing description 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. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 73 
(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: 
AATCTGGGC GACAAGAGTGA20 
(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) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
ACATCTCCCCT ACCGCTATA20 
(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: 
TCCAGCCTCGGAGA CAGAAT20 
(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: 
AGTCCTTTCTCCAGAGC AGGT21 
(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: 
GCCAGTGATGCTAAAGGTTG 20 
(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: 
AACATACGTGGCTCTATGCA 20 
(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: 
AATCTGGGCGACAAGAGTGAAACTC CGTCAAAAGAAAGAAAGAAAGAGACAAAGAGAGTT60 
AGAAAGAAAGAAAGAGAGAGAGAGAGAAAGGAAGGAAGGAAGAAAAAGAAAGAAAAAGAA120 
AGAAAGAGAAAGAAAGAAAGAGAAAGAAAGAAAGAAAGAAAGAAAGAAAGAAAGAAAGAA180 
AGAAAGAA AAAGAAAGAAAGAAAGAAAGAAAGAAAGAAAGAAAGAAAGAAAGAAAGAAAG240 
AAAGAAAGGAAGGAAAGAAAGAGCAAGTTACTATAGCGGTAGGGGAGATGT291 
(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: 
GCCAGTGATGCTAAAGGTTGTATTGCATATATACATATATATATATATATATATATATAT60 
ATATATATATATATATATATATATATATATTTTAATTTGATAGTATTGTGCATAGAGCC A120 
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: 
TCCAGCCTCGGAGACAGAATGAGACTCCATCAAAAACAAGAAAGAAAGAAAGACAAAGAG60 
AGAAAGAAAGAAAGAAAGAAAGAAAGAAAGAGAGAGAGAGAGAGAGAGAGAGAAAGAAAG120 
AAAGAAAGAAAGAAAGAAAGAAAGAAAGAAAGAAAGAAAGAAA GAAAGAAGGAAAGAAAG180 
AAAGGAAACTAAAATAACTAAATAACTGAGTAGCACCACACCACCTGCTCTGGAGAAAGG240 
ACT243 
(2) INFORMATION FOR SEQ ID NO:10: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 19 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: 
TTTCTGGGTGTGTCTGAAT19 
(2) INFORMATION FOR SEQ ID NO:11: 
(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:11: 
ACACAGTTGCTCTAAAGGGT20 
(2) INFORMATION FOR SEQ ID NO:12: 
(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:12: 
CTAGGTTGTAAGCTCCATGA20 
(2) INFORMATION FOR SEQ ID NO:13: 
(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:13: 
TTGAGCACTTACTCTGTGCC20 
(2) INFORMATION FOR SEQ ID NO:14: 
(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:14: 
AACTCAGAACAGTGCCTGAC20 
(2) INFORMATION FOR SEQ ID NO:15: 
(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:15: 
ATTTCCCTCAAGGCTCCAGGT21 
(2) INFORMATION FOR SEQ ID NO:16: 
(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:16: 
CTGATCTTGCTCACCTTCGA20 
(2) INFORMATION FOR SEQ ID NO:17: 
(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:17: 
GCGTTTGCTGAAATGAAGGA20 
(2) INFORMATION FOR SEQ ID NO:18: 
(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:18: 
GCAGGTACTTAGTTAGCTAC20 
(2) INFORMATION FOR SEQ ID NO:19: 
(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:19: 
TTACAGTGAGCCAAGGTCGT20 
(2) INFORMATION FOR SEQ ID NO:20: 
(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:20: 
TTTGTCTGGATAGACTGGAG20 
(2) INFORMATION FOR SEQ ID NO:21: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 19 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: 
CCATCTTCCTGTGGCTGTA19 
(2) INFORMATION FOR SEQ ID NO:22: 
(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:22: 
CTAATGCAGAGATTTAGGGC20 
(2) INFORMATION FOR SEQ ID NO:23: 
(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:23: 
GTGGTGTAAAGACTGCATAG20 
(2) INFORMATION FOR SEQ ID NO:24: 
(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:24: 
ATGTGACTGATGTGGGTCAG20 
(2) INFORMATION FOR SEQ ID NO:25: 
(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:25: 
CATCTGCACTCATGCTCCAT20 
(2) INFORMATION FOR SEQ ID NO:26: 
(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:26: 
TCCCAGATCGCTCTACATGA20 
(2) INFORMATION FOR SEQ ID NO:27: 
(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:27: 
CACAGCTTCAGAAGTCACAG20 
(2) INFORMATION FOR SEQ ID NO:28: 
(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:28: 
GAGCAATGTTGCTTAGGATG20 
(2) INFORMATION FOR SEQ ID NO:29: 
(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:29: 
TGGAAGTGTCACTGGCATGT20 
(2) INFORMATION FOR SEQ ID NO:30: 
(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:30: 
TGT GTCCAGCCTTAGTGTGCA21 
(2) INFORMATION FOR SEQ ID NO:31: 
(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:31: 
TCATCACT TCCAGAATGTGC20 
(2) INFORMATION FOR SEQ ID NO:32: 
(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:32: 
ACTGCCTCATCC AGTTTCAG20 
(2) INFORMATION FOR SEQ ID NO:33: 
(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:33: 
GAGCAGGCACTTGTTAG ATG20 
(2) INFORMATION FOR SEQ ID NO:34: 
(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:34: 
CCTCTTGGCTCTAACAGCAA 20 
(2) INFORMATION FOR SEQ ID NO:35: 
(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:35: 
AGCAAGACCCTGTCTCAAGA 20 
(2) INFORMATION FOR SEQ ID NO:36: 
(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:36: 
CAAGGCCCATCTTCAGTAGA 20 
(2) INFORMATION FOR SEQ ID NO:37: 
(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:37: 
CCTTCTCACTCCTTTACTAG 20 
(2) INFORMATION FOR SEQ ID NO:38: 
(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:38: 
GAAGACTGAGGAGGTCAGAA 20 
(2) INFORMATION FOR SEQ ID NO:39: 
(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:39: 
CTACTGTTCAGAGTCAAAGC 20 
(2) INFORMATION FOR SEQ ID NO:40: 
(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:40: 
TGCCCCACATTAGGATGCAT 20 
(2) INFORMATION FOR SEQ ID NO:41: 
(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:41: 
AGGGACACGAATCAGATCAG 20 
(2) INFORMATION FOR SEQ ID NO:42: 
(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:42: 
GTGGTACCTCATTGTGGCTA 20 
(2) INFORMATION FOR SEQ ID NO:43: 
(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:43: 
AGGCATCCTTGTGCTGACAT20 
(2) INFORMATION FOR SEQ ID NO:44: 
(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:44: 
TTTGGCCGACAGTGGTGTAA20 
(2 ) INFORMATION FOR SEQ ID NO:45: 
(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:45: 
AGGACCAAACCATGTCTGTC20 
(2) INFORMATION FOR SEQ ID NO:46: 
(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:46: 
CTGCATCTGAGCATATGGGA20 
(2) INFORMATION FOR SEQ ID NO:47: 
(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:47: 
CATTCAGACTATGCAGGCTT20 
(2) INFORMATION FOR SEQ ID NO:48: 
( i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 19 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:48: 
CTGGGACTACTGGCACATG19 
(2) INFORMATION FOR SEQ ID NO:49: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 19 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:49: 
GGCAACGTGGTGAAACCTT19 
(2) INFORMATION FOR SEQ ID NO:50: 
(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:50: 
GGAAGATGGAGTGGCTGTTA20 
(2) INFORMATION FOR SEQ ID NO:51: 
(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:51: 
CTCCAGCCTGGCGAAAGAAT20 
(2) INFORMATION FOR SEQ ID NO:52: 
(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:52: 
GTAAGACTTTTGGAGCCATT20 
(2) INFORMATION FOR SEQ ID NO:53: 
(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:53: 
TTCAGGGAGAATGAGATGGG20 
(2) INFORMATION FOR SEQ ID NO:54: 
(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:54: 
GACAGAGTGAGACTCCATCT20 
(2) INFORMATION FOR SEQ ID NO:55: 
(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:55: 
GATCCTATCTTCTCAGGAGG20 
(2) INFORMATION FOR SEQ ID NO:56: 
(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:56: 
GAGGTTGCACTCCAGCCTTT20 
(2) INFORMATION FOR SEQ ID NO:57: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 19 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:57: 
ATGCCATGCAGATTAGAAA19 
(2) INFORMATION FOR SEQ ID NO:58: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 19 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:58: 
GGAAAGAAACAGTGAAAGA19 
(2) INFORMATION FOR SEQ ID NO:59: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:59: 
ATCCATCGACCTCTGGGTTA20 
(2) INFORMATION FOR SEQ ID NO:60: 
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(A) LENGTH: 20 
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:60: 
GACCCCACAGCCTATTCAGA20 
(2) INFORMATION FOR SEQ ID NO:61: 
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(A) LENGTH: 20 
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:61: 
TTGACTGCTGAACGGCTGCA20 
(2) INFORMATION FOR SEQ ID NO:62: 
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(A) LENGTH: 19 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:62: 
CAGCTGCCCTAGTCAGCAC19 
(2) INFORMATION FOR SEQ ID NO:63: 
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(A) LENGTH: 20 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:63: 
GCTTCCGAGTGCAGGTCACA20 
(2) INFORMATION FOR SEQ ID NO:64: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:64: 
GGGCAACATGGTGAAACCTT20 
(2) INFORMATION FOR SEQ ID NO:65: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:65: 
CCTAGCCTATACTTCCTTTC20 
(2) INFORMATION FOR SEQ ID NO:66: 
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(A) LENGTH: 20 
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:66: 
GGACCTCGTGAATTACAATC20 
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(A) LENGTH: 20 
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:67: 
ATTTACCTACCTGTTCATCC20 
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:68: 
TTGTGTCAACTGCTGATATG20 
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:69: 
AACCAAAACATCATTCCCTA20 
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:70: 
CGT AAGCGTGCACTATACCCT21 
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(A) LENGTH: 19 
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:71: 
CTGAGGAT TCATCCACCTG19 
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(A) LENGTH: 20 
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:72: 
CCTGAGTAGCTG TTAAGGGA20 
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(A) LENGTH: 20 
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:73: 
GCACATGTACCCTAGAA CTT20