Note: Descriptions are shown in the official language in which they were submitted.
92/21693 PCT/US92/04195
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
2
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
3
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
4
0 0
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
CA 02110059 2003-05-26
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
CA 02110059 2003-05-26
6
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
CA 02110059 2004-08-06
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
CA 02110059 2004-08-06
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,
CA 02110059 2004-08-06
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;
CA 02110059 2004-08-06
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;
CA 02110059 2004-08-06
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;
CA 02110059 2004-08-06
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
CA 02110059 2004-08-06
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
CA 02110059 2004-08-06
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
CA 02110059 2004-08-06
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
CA 02110059 2003-05-26
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
7
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
8
?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.
WO 92/21693 PCT/US92/04195
9
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
11
~ ~_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
WO 92/21693 PCT/US92/04195
12
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)
18
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;
WO 92/21693 PCT/US92/04195
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 . ;.;<., ......, . , . ,
_ _ .
WO 92/21693 PCT/US92/04195
16
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 . ., '..~: . _
WO 92/21693 PCT/LJS92/04195
17
U 0
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
18
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
19
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
21
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
CA 02110059 2003-05-26
22
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
WO 92/21693 PCF/US92/04195
23
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.
WO 92/21693 PCT/U592/04195
24
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
