U.S. patent application number 10/769579 was filed with the patent office on 2004-07-15 for multiplex amplification of short tandem repeat loci.
This patent application is currently assigned to Promega Corporation. Invention is credited to Lins, Ann M., Schumm, James W., Sprecher, Cynthia J..
Application Number | 20040137504 10/769579 |
Document ID | / |
Family ID | 23229495 |
Filed Date | 2004-07-15 |
United States Patent
Application |
20040137504 |
Kind Code |
A1 |
Schumm, James W. ; et
al. |
July 15, 2004 |
Multiplex amplification of short tandem repeat loci
Abstract
The present invention is directed to the simultaneous
amplification of multiple distinct genetic loci using PCR or other
amplification systems to determine in one reaction the alleles of
each locus contained within the multiplex.
Inventors: |
Schumm, James W.; (Madison,
WI) ; Sprecher, Cynthia J.; (Madison, WI) ;
Lins, Ann M.; (Lodi, WI) |
Correspondence
Address: |
MICHAEL BEST & FRIEDRICH, LLP
ONE SOUTH PINCKNEY STREET
P O BOX 1806
MADISON
WI
53701
|
Assignee: |
Promega Corporation
Madison
WI
|
Family ID: |
23229495 |
Appl. No.: |
10/769579 |
Filed: |
January 30, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10769579 |
Jan 30, 2004 |
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09839478 |
Apr 20, 2001 |
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09839478 |
Apr 20, 2001 |
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09327229 |
Jun 7, 1999 |
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6221598 |
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09327229 |
Jun 7, 1999 |
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08316544 |
Sep 30, 1994 |
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Current U.S.
Class: |
435/6.11 ;
435/91.2 |
Current CPC
Class: |
C12Q 1/6858 20130101;
C12Q 2525/151 20130101; C12Q 2525/151 20130101; C12Q 2525/151
20130101; C12Q 2545/101 20130101; C12Q 2537/143 20130101; C12Q
1/6858 20130101; C12Q 2565/125 20130101; C12Q 1/6827 20130101; C12Q
1/6858 20130101; C12Q 2600/16 20130101; C12Q 1/6827 20130101 |
Class at
Publication: |
435/006 ;
435/091.2 |
International
Class: |
C12Q 001/68; C12P
019/34 |
Claims
What is claimed is:
1. A method of simultaneously determining the alleles present in at
least two loci from one or more Genomic DNA samples, comprising: a.
obtaining at least one Genomic DNA sample to be analyzed, wherein
the Genomic DNA sample has at least two loci which can be amplified
together; b. amplifying the short tandem repeat sequences in the
Genomic DNA sample; and c. evaluating the amplified fragments to
determine the alleles present at each amplified locus within the
Genomic DNA sample.
2. The method of claim 1 wherein at least one of the loci is
selected from the group consisting of: HUMCSFLPO, HUMTPOX,
HUMVWFA31, HUMFESFPS, HUMBFXIII (F13B), HUMLIPOL, HSAC04 (ACTBP2),
HUMCYP19, HUMAPOA2, HUMF13A01 and HUMMYOPK (Myotonic).
3. The method of claim 1 wherein at least two loci are selected
from the groups consisting of: HUMTH01 and HUMCSF1PO; HUMTH01 and
HUMCD4; HUMTH01 and HUMTPOX; HUMF13A01 and HUMFABP; HUMF13A01 and
HUMMYOPK (Myotonic); HUMF13A01 and HUMBFXIII (F13B); HUMBFXIII
(F13B) and HUMFESFPS; HUMBFXIII (F13B) and HUMLIPOL; HUMHPRTB and
HUMFESFPS; HSAC04 (ACTBP2) and HUMCYP19; and HSAC04 (ACTBP2) and
HUMFABP.
4. The method of claim 1 wherein the loci are selected from the
group consisting of: HUMTH01 and HUMCSF1PO; HUMTH01 and HUMCD4;
HUMTH01 and HUMTPOX; HUMF13A01 and HUMFABP; HUMF13A01 and HUMMYOPK
(Myotonic); HUMF13A01 and HUMBFXIII (F13B); HUMBFXIII (F13B) and
HUMFESFPS; HUMBFXIII (F13B) and HUMLIPOL; HUMHPRTB and HUMFESFPS;
HSAC04 (ACTBP2) and HUMCYP19; HUMCSF1PO, HUMTPOX, and HUMTH01;
HUMHPRTB, HUMFESFPS and HUMVWFA31; HSAC04 (ACTBP2), HUMCYP19 and
HUMPLA2A1; HSAC04 (ACTBP2) and HUMFABP; HUMAPOA2, HUMCYP19 and
HUMPLA2A1; HUMCD4, HUMCSF1PO and HUMTH01; HUMCYP-19, HUMFABP and
HUMPLA1; HUMCYP19, HUMHPRTB and HUMPLA1; HUMF13A01, HUMFABP and
HUMCD4; HUMHPRTB, HUMFESFPS and HUMLIPOL; HUMF13A01, HUMFABP and
HUMCD4; HUMHPRTB, HUMBFXIII (F13B) and HUMPLA2A1; HUMHPRTB,
HUMBFXIII (F13B) and HUMTPOX; HUMHPRTB, HUMBFXIII (F13B) and
HUMFESFPS; HUMCSFLPO, HUMTPOX and HUMCD4; HUMHPRTB, HUMFESFPS and
HUMMYOPK (Myotonic). HUMCSFLPO, HUMTH01 and HUMCD4; HUMCSF1PO,
HUMTH01 and HUMVWFA31; HUMHPRTB, HUMBFXIII (F13B) and HUMLIPOL;
HUMCSF1PO, HUMTPOX, HUMTH01 and HUMVWFA31; HUMHPRTB, HUMFESFPS,
HUMBFXIII (F13B) and HUMLIPOL; HUMCSF1PO, HUMTPOX, HUMTH01 and
HUMCD4; and HUMCSF1PO, HUMTH01, HUMTPOX and HUMCD4.
5. The method of claim 1 wherein the loci are HUMHPRTB and
HUMFESFPS.
6. The method of claim 1 wherein the loci are HUMCSF1PO, HUMTPOX,
and HUMTH01.
7. The method of claim 1 wherein the loci are HUMHPRTB, HUMFESFPS,
HUMBFXIII (F13B) and HUMLIPOL.
8. The method of claim 1 wherein the loci are HUMCSF1PO, HUMTPOX,
HUMTH01 and HUMVWFA31.
9. The method of claim 1 wherein the loci are HUMHPRTB, HUMFESFPS
and HUMVWFA31.
10. The method of claim 1 wherein the Genomic DNA in step b. is
amplified by polymerase chain reduction.
11. The method of claim 8 wherein the process of amplifying short
tandem repeat sequences requires primer pairs selected from the
group consisting of SEQ ID. NO. 1 and SEQ ID. NO. 2, SEQ ID. NO. 3
and SEQ ID. NO. 4, SEQ ID. NO. 5 and SEQ ID. NO. 6, SEQ ID. NO. 7
and SEQ ID. NO. 8, SEQ ID. NO. 9 and SEQ ID. NO. 10, SEQ ID. NO. 11
and SEQ ID. NO. 12, SEQ ID. NO. 13 and SEQ ID. NO. 14, SEQ ID. NO.
15 and SEQ ID. NO. 16, SEQ ID. NO. 17 and SEQ ID. NO. 18, SEQ ID.
NO. 19 and SEQ ID. NO. 20, SEQ ID. NO. 21 and SEQ ID. NO. 22, SEQ
ID. NO. 23 and SEQ ID. NO. 24, SEQ ID. NO. 25 and SEQ ID. NO. 26,
SEQ ID. NO. 27 and SEQ ID. NO. 28, SEQ ID. NO. 29 and SEQ ID. NO.
30, and SEQ ID. NO. 31 and SEQ ID. NO. 32.
12. The method of claim 1 further comprising adding short tandem
repeat allelic ladders containing nucleotide fragments of the same
lengths as two or more known alleles for each of the loci and
determining the allele content of the Genomic DNA sample by
comparison with the amplified short tandem repeat fragments for
each of the loci.
13. The method of claim 1 wherein the amplified short tandem repeat
sequences are compared by polyacrylamide gel electrophoresis.
14. The method of claim 1 wherein the amplified short tandem repeat
sequences are compared using silver stain analysis.
15. The method of claim 1 wherein the amplified short tandem repeat
sequences are compared by fluorescent analysis.
16. The method of claim 1 further comprising identifying an
appropriate set of loci and primers which provide non-overlapping
alleles.
17. The method of claim 1 wherein the samples to be tested are
selected from the group consisting of blood, semen, vaginal cells,
hair, saliva, urine or other tissue, placental cells or fetal cells
present in amniotic fluid and mixtures of body fluids.
18. A method of simultaneously determining the alleles present in
at least two loci from one or more Genomic DNA samples, comprising:
a. identifying an appropriate set of loci and primers which provide
non-overlapping alleles; b. obtaining at least one Genomic DNA
sample to be analyzed, wherein the Genomic DNA sample has at least
two loci which can be amplified together; c. amplifying the short
tandem repeat sequences in the Genomic DNA sample; and d.
evaluating the amplified fragments to determine the alleles present
at each amplified locus within the Genomic DNA sample.
19. A kit for simultaneously analyzing short tandem repeat
sequences in at least two loci from one or more Genomic DNA
samples, comprising: a. a container containing oligonucleotide
primer pairs for each of the specified loci; and b. instructions
for use.
20. The kit of claim 17 wherein the primer pairs are selected from
the group of loci consisting of SEQ ID. NO. 1 and SEQ ID. NO. 2,
SEQ ID. NO. 3 and SEQ ID. NO. 4, SEQ ID. NO. 5 and SEQ ID. NO. 6,
SEQ ID. NO. 7 and SEQ ID. NO. 8, SEQ ID. NO. 9 and SEQ ID. NO. 10,
SEQ ID. NO. 11 and SEQ ID. NO. 12, SEQ ID. NO. 13 and SEQ ID. NO.
14, SEQ ID. NO. 15 and SEQ ID. NO. 16, SEQ ID. NO. 17 and SEQ ID.
NO. 18, SEQ ID. NO. 19 and SEQ ID. NO. 20, SEQ ID. NO. 21 and SEQ
ID. NO. 22, SEQ ID. NO. 23 and SEQ ID. NO. 24, SEQ ID. NO. 25 and
SEQ ID. NO. 26, SEQ ID. NO. 27 and SEQ ID. NO. 28, SEQ ID. NO. 29
and SEQ ID. NO. 30, and SEQ ID. NO. 31 and SEQ ID. NO. 32.
Description
FIELD OF THE INVENTION
[0001] The present invention is generally directed to the detection
of genetic markers in a genomic system. The present invention is
more specifically directed to the simultaneous amplification of
multiple distinct polymorphic genetic loci using the polymerase
chain reaction or other amplification systems to determine in one
reaction the alleles of each locus contained within the multiplex
system.
CITED REFERENCES
[0002] A full bibliographic citation of the references cited in
this application can be found in the section preceding the
claims.
DESCRIPTION OF THE PRIOR ART
[0003] In recent years, the discovery and development of
polymorphic short tandem repeats (STRs) as genetic markers has
stimulated progress in the development of linkage maps, the
identification and characterization of diseased genes, and the
simplification and precision of Genomic DNA typing.
[0004] Many loci, at least in the human genome, contain a
polymorphic STR region. STR loci consist of short, repetitive
sequence elements of 3 to 7 base pairs in length. It is estimated
that there are 2,000,000 expected trimeric and tetrameric STRs
present as frequently as once every 15 kilobases (kb) in the human
genome (Edwards et al. 1991; Beckmann and Weber 1992). Nearly half
of the STR loci studied by Edwards et al. (1991) are polymorphic,
which provides a rich source of genetic markers. Variation in the
number of repeat units at a particular locus is responsible for the
observed polymorphism reminiscent of VNTR loci (Nakamura et al.
1987) and minisatellite loci (Jeffreys et al. 1985), which contain
longer repeat units, and microsatellite or dinucleotide repeat loci
(Litt and Luty 1989, Tautz 1989, Weber and May 1989, Beckmann and
Weber 1992).
[0005] Polymorphic STR loci are extremely useful markers for human
identification, paternity testing and genetic mapping. STR loci may
be amplified via the polymerase chain reaction (PCR) by employing
specific primer sequences identified in the regions flanking the
tandem repeat.
[0006] Alleles of these loci are differentiated by the number of
copies of the repeat sequence contained within the amplified region
and are distinguished from one another following electrophoretic
separation by any suitable detection method including
radioactivity, fluorescence, silver stain, and color.
[0007] To minimize labor, materials and analysis time, it is
desirable to analyze multiple loci and/or more samples
simultaneously. One approach for reaching this goal involves
amplification of multiple loci simultaneously in a single reaction.
Such "multiplex" amplifications have been described extensively in
the literature. Multiplex amplification sets have been extensively
developed for analysis of genes related to human genetic diseases
such as Duchenne Muscular Dystrophy (Chamberlain et al. 1988,
Chamberlain et al. 1989, Beggs et al. 1990, Clemens et al. 1991,
Schwartz et al. 1992, Covone et al. 1992), Lesch-Nyhan Syndrome
(Gibbs et al. 1990), Cystic Fibrosis (Estivill et al. 1991, Fortina
et al. 1992, Ferrie et al. 1992, Morral and Estivill, 1992), and
Retinoblasma (Lohmann et al. 1992). Multiplex amplification of
polymorphic microsatellite markers (Clemens et al. 1991, Schwartz
et al. 1992, Huang et al. 1992) and even STR markers (Edwards et
al. 1992, Kimpton et al. 1993, Hammond et al. 1994) have been
described.
[0008] These amplified products are generally separated by one of
several methods of electrophoresis known to those skilled in the
art. Several well-known methods of detection of the amplified
products have also been described. While ethidium bromide staining
of amplified fragments is employed in some cases, in others it is
preferred to use methods which label only one of the two strands of
the amplified material. Examples of this include radioactive or
fluorescent labeling of one of the two primers prior to the
amplification of a locus. One of the more sophisticated approaches
to detection is the use of different fluorescent labels to allow
detection of amplified materials representing different loci, but
existing in the same space following electrophoresis. The products
of the different loci are differentiated with the use of filters,
which allow visualization of one fluorescent label at a time.
[0009] Reference is made to International Publications WO 93/18177
and WO 93/18178 to Fortina et al., which are directed to methods
and kits for diagnosing diseases such as Cystic Fibrosis and
.beta.-thalassemia, respectively, using an allele-specific
multiplex polymerase chain reaction system. According to Fortina et
al., multiplex PCR has also been used for simultaneous
amplification of multiple target sequences, permitting mutant
allele scanning using two lanes of an agarose gel.
[0010] Ballabio et al. (1991), disclose a single tube multiplex
allele specific PCR test using two different dye-tagged fluorescent
primers for detection of the AF508 cystic fibrosis mutation.
[0011] While there are multiplex amplification procedures for
specific loci, the use of multiplex amplification procedures is
greatly desired for the detection of alleles in other types of loci
such as specific STR loci.
SUMMARY OF THE INVENTION
[0012] It is therefore an object of the present invention to
provide a method for the simultaneous amplification of multiple
distinct polymorphic STR loci using PCR or other amplification
systems to determine, in one reaction, the alleles of each locus
contained within the multiplex. These combinations of specific loci
into multiplexes have not been heretofore shown.
[0013] It is also an object of the present invention to provide a
method and a kit specific for multiplex amplifications comprising
specified loci.
[0014] These and other objects are addressed by the present
invention which is directed to a method of simultaneously analyzing
or determining the alleles present at each individual locus of each
multiplex. This method comprises the steps of (1) obtaining at
least one Genomic DNA sample to be analyzed, wherein the Genomic
DNA sample has at least two loci which can be amplified together;
(2) amplifying the STR sequences in the DNA sample; and (3)
detecting the amplified materials in a fashion which reveals the
polymorphic nature of the systems employed.
[0015] The present invention is also directed to a method of
simultaneously analyzing multiple STR sequences wherein at least
one of the loci is selected from the group consisting of:
HUMCSFLPO, HUMTPOX, HUMVWFA31, HUMFESFPS, HUMBFXIII (F13B),
HUMLIPOL, HSAC04 (ACTBP2), HUMCYP19, HUMPLA2A1, HUMAPOA2, HUMCD4,
HUMF13A01 and HUMMYOPK (Myotonic).
[0016] Specifically, the present invention is directed to a method
of simultaneously analyzing multiple STR sequences in the following
groups of loci: HUMTH01 and HUMCSF1PO; HUMTH01 and HUMCD4; HUMTH01
and HUMTPOX; HUMF13A01 and HUMFABP; HUMF13A01 and HUMMYOPK
(Myotonic); HUMF13A01 and HUMBFXIII (F13B); HUMBFXIII (F13B) and
HUMFESFPS; HUMBFXIII (F13B) and HUMLIPOL; HUMHPRTB and HUMFESFPS;
HSAC04 (ACTBP2) and HUMCYP19; HUMCSF1PO, HUMTPOX and HUMTH01;
HUMHPRTB, HUMFESFPS and HUMVWFA31; HSAC04 (ACTBP2), HUMCYP19 and
HUMPLA2A1; HSAC04 (ACTBP2) and HUMFABP; HUMAPOA2, HUMCYP19 and
HUMPLA2A1; HUMCD4, HUMCSF1PO and HUMTH01; HUMCYP19, HUMFABP and
HUMPLA2A1; HUMCYP19, HUMHPRTB and HUMPLA2A1; HUMF13A01, HUMFABP and
HUMCD4; HUMHPRTB, HUMFESFPS and HUMLIPOL; HUMF13A01, HUMFABP and
HUMCD4; HUMHPRTB, HUMBFXIII (F13B) and HUMPLA2A1; HUMHPRTB,
HUMBFXIII (F13B) and HUMTPOX; HUMHPRTB, HUMBFXIII (F13B) and
HUMFESFPS; HUMCSF1PO, HUMTPOX and HUMCD4; HUMHPRTB, HUMFESFPS and
HUMMYOPK (Myotonic); HUMCSFLPO, HUMTH01 and HUMCD4; HUMCSFLPO,
HUMTH01 and HUMVWFA31; HUMHPRTB, HUMBFXIII (F13B) and HUMLIPOL;
HUMCSFLPO, HUMTPOX, HUMTH01 and HUMVWFA31; HUMHPRTB, HUMFESFPS,
HUMBFXIII (F13B) and HUMLIPOL; HUMCSF1PO, HUMTPOX, HUMTH01 and
HUMCD4; and HUMCSF1PO, HUMTH01, HUMTPOX and HUMCD4.
[0017] The present invention provides a high throughput method for
the detection and analysis of polymorphic genetic markers using
specific combinations of loci and specified conditions. By
selection of the appropriate detection method, the process can be
used in laboratories which have only a power supply and a standard
apparatus for/polyacrylamide gel electrophoresis or those which
have the latest in equipment for fluorescent gel scanning, e.g.,
FluorImager.TM.-575 (Molecular Dynamics, Sunnyvale, Calif.). Thus,
the process of the present invention is adaptable for a variety of
uses and laboratories.
[0018] The approach as specified in the present invention produces
a savings in time, labor and materials in the analysis of loci
contained within the multiplexes. The process of the present
invention includes all the requisite primers, allowing between two
and four or more loci to be amplified together in one amplification
tube instead of amplifying each locus independently.
[0019] The present invention has specific use in the field of
forensic analysis, paternity determination, monitoring of bone
marrow transplantation, linkage mapping, and detection of genetic
diseases and cancers.
[0020] These and other aspects of the present invention will become
evident upon reference to the following detailed description of the
invention and the attached drawings and photographs.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] FIG. 1 is a photograph illustrating the simultaneous
amplification of three loci: HUMCSF1PO, HUMTPOX and HUMTH01, with
the amplified products of each locus shown migrating next to the
corresponding allelic ladder for ease of interpretation in Example
1.
[0022] FIG. 2 is a computer image showing the fluorescent detection
of multiplex amplification of the loci HUMCSF1PO, HUMTPOX, HUMTH01
and HUMVWFA31 as detected with a FluorImager.TM.-575 (Molecular
Dynamics, Sunnyvale, Calif.) in Example 2.
[0023] FIG. 3 is a photograph showing the silver stain detection of
the multiplex amplification in Example 3.
[0024] FIG. 4 is a computer image showing the fluorescent detection
of multiplex amplification in Example 4.
[0025] FIG. 5 is a photograph showing the silver stain detection of
the multiplex amplification in Example 5.
[0026] FIG. 6 is a photograph showing the silver stain detection of
the multiplex amplification in Example 6.
[0027] FIG. 7 is a photograph showing the silver stain detection of
the multiplex amplification in Example 7.
[0028] FIG. 8 is a photograph showing the silver stain detection of
the multiplex amplification in Example 8.
[0029] FIG. 9 is a photograph showing the silver stain detection of
the multiplex amplification in Example 9.
[0030] FIG. 10 is a photograph showing the silver stain detection
of the multiplex amplification in Example 10.
[0031] FIG. 11 is a photograph showing the silver stain detection
of the multiplex amplification in Example 11.
[0032] FIG. 12 is a photograph showing the silver stain detection
of the multiplex amplification in Example 12.
[0033] FIG. 13 is a photograph showing the silver stain detection
of the multiplex amplification in Example 13.
[0034] FIG. 14 is a photograph showing the silver stain detection
of the multiplex amplification in Example 14.
[0035] FIG. 1S is a photograph showing the silver stain detection
of the multiplex amplification in Example 15.
[0036] FIG. 16 is a photograph showing the silver stain detection
of the multiplex amplification in Example 16.
[0037] FIG. 17 is a photograph showing the silver stain detection
of the multiplex amplification in Example 17.
[0038] FIG. 18 is a photograph showing the silver stain detection
of the multiplex amplification in Example 18.
[0039] FIG. 19 is a photograph showing the silver stain detection
of the multiplex amplification in Example 19.
[0040] FIG. 20 is a photograph showing the silver stain detection
of the multiplex amplification in Example 20.
[0041] FIG. 21 is a photograph showing the silver stain detection
of the multiplex amplification in Example 21.
[0042] FIG. 22 is a photograph showing the silver stain detection
of the multiplex amplification in Example 22.
[0043] FIG. 23 is a photograph showing the silver stain detection
of the multiplex amplification in Example 23.
[0044] FIG. 25 is a photograph showing the silver stain detection
of the multiplex amplification in Example 25.
[0045] FIG. 26 is a photograph of a computer image showing the
fluorescent detection of the multiplex amplification in Example
26.
[0046] FIG. 27 is a photograph of a computer image showing the
fluorescent detection of the multiplex amplification in Example
27.
[0047] FIG. 28 is a photograph of a computer image showing the
fluorescent detection of the multiplex amplification in Example
28.
[0048] FIG. 29 is a photograph of a computer image showing the
fluorescent detection of the multiplex amplification in Example
29.
[0049] FIG. 30 is a photograph of a computer image showing the
fluorescent detection of the multiplex amplification in Example
30.
[0050] FIG. 31 is a photograph of a computer image showing the
fluorescent detection of the multiplex amplification in Example
31.
[0051] FIG. 32 is a photograph of a computer image showing the
fluorescent detection of the multiplex amplification in Example
32.
DETAILED DESCRIPTION OF THE INVENTION
[0052] The following definitions are intended to assist in
providing a clear and consistent understanding of the scope and
detail of the terms:
[0053] Allelic ladder: a standard size marker consisting of
amplified alleles from the locus.
[0054] Allele: a genetic variation/associated with a segment of
Genomic DNA, i.e., one of two or more alternate forms of a DNA
sequence occupying the same locus.
[0055] Biochemical nomenclature: standard biochemical nomenclature
is used herein in which the nucleotide bases are designated as
adenine (A); thymine (T); guanine (G); and cytosine (C).
Corresponding nucleotides are, for example,
deoxyguanosine-5'-triphosphate (dGTP).
[0056] Genomic DNA polymorphism: the condition in which two or more
different nucleotide sequences coexist in the same interbreeding
population in a Genomic DNA sequence.
[0057] Locus (or genetic locus): a specific position on a
chromosome. Alleles of a locus are located at identical sites on
homologous chromosomes.
[0058] Locus-specific primer: a primer that specifically hybridizes
with a portion of the stated locus or its complementary strand, at
least for one allele of the locus, and does not hybridize
efficiently with other Genomic DNA sequences under the conditions
used in the amplification method.
[0059] Polymerase chain reaction (PCR): a technique in which cycles
of denaturation, annealing with primer, and extension with Genomic
DNA polymerase are used to amplify the number of copies of a target
Genomic DNA sequence by >10.sup.16 times. The polymerase chain
reaction process for amplifying nucleic acid is covered by U.S.
Pat. Nos. 4,683,195 and 4,683,202, which are incorporated herein by
reference for a description of the process.
[0060] Polymorphism information content (PIC): a measure of the
amount of polymorphism present at a locus (Botstein et al., 1980).
PIC values range from 0 to 1.0, with higher values indicating
greater degrees of polymorphism. This measure generally displays
smaller values than the other commonly used measure, i.e.,
heterozygosity. For markers that are highly informative
(heterozygosities exceeding about 70%), the difference between
heterozygosity and PIC is slight.
[0061] Primary reaction: initial reaction using the purified human
genomic DNA as template for the PCR.
[0062] Primers: two single-stranded oligonucleotides or DNA
fragments which hybridize with opposing strands of a locus such
that the 3' termini of the primers are in closest proximity.
[0063] Primer pair: two primers including primer 1 that hybridizes
to a single strand at one end of the Genomic DNA sequence to be
amplified and primer 2 that hybridizes with the other end on the
complementary strand of the Genomic DNA sequence to be
amplified.
[0064] Primer site: the area of the target Genomic DNA to which a
primer hybridizes.
[0065] Secondary reaction: reamplification with the same or
different primer pair using a dilution of the primary reaction as
template for the PCR.
[0066] Construction of the Multiplex System
[0067] Prior to constructing the multiplex system, an appropriate
set of loci, primers, and amplification protocols must be selected
such that amplification generates fragments such that alleles of
the various loci do not overlap in size or, when such overlap
occurs, fragments representing different loci are detectable by
separate means. In addition, the selected loci must be compatible
for use with a single amplification-protocol. The specific
combinations of loci described herein are unique in this
application. Combinations of loci may be rejected for either of
these reasons, or because, in combination, one or more of the loci
do not produce adequate product yield, or fragments which do not
represent authentic alleles are produced in this reaction.
[0068] Successful combinations are generated by trial and error of
locus combinations and by adjustment of primer concentrations to
identify an equilibrium in which all included loci may be
amplified.
[0069] Of particular importance in the multiplex system is the size
range of amplified alleles produced from the individual loci which
will be analyzed together. For ease of analysis with current
technologies, systems which can be detected by amplification of
fragments smaller than 500 bases were preferably selected.
[0070] The following multiplex combinations have been developed and
are considered ideal combinations for use in the present
system:
[0071] 1. HUMTH01 and HUMCSFLPO;
[0072] 2. HUMTH01 and HUMCD4;
[0073] 3. HUMTH01 and HUMTPOX;
[0074] 4. HUMF13A01 and HUMFABP;
[0075] 5. HUMF13A01 and HUMMYOPK (Myotonic);
[0076] 6. HUMF13A01 and HUMBFXIII (F13B);
[0077] 7. HUMBFXIII (F13B) and HUMFESFPS;
[0078] 8. HUMBFXIII (F13B) and HUMLIPOL;
[0079] 9. HUMHPRTB and HUMFESFPS;
[0080] 10. HSAC04 (ACTBP2) and HUMCYP19;
[0081] 11. HSAC04 (ACTBP2) and HUMFABP;
[0082] 12. HUMCSFLPO, HUMTPOX and HUMTH01;
[0083] 13. HUMHPRTB, HUMFESFPS and HUMVWFA31;
[0084] 14. HSAC04 (ACTBP2), HUMCYP19 and HUMPLA2A1;
[0085] 15. HUMAPOA2, HUMCYP19 and HUMPLA2A1;
[0086] 16. HUMCD4, HUMCSF1PO and HUMTH01;
[0087] 17. HUMCYP19, HUMFABP and HUMPLA2A1;
[0088] 18. HUMCYP19, HUMHPRTB and HUMPLA2A1;
[0089] 19. HUMF13A01, HUMFABP and HUMCD4;
[0090] 20. HUMHPRTB, HUMFESFPS and HUMLIPOL;
[0091] 21. HUMF13A01, HUMFABP and HUMCD4;
[0092] 22. HUMHPRTB, HUMBFXIII (F13B) and HUMPLA2A1;
[0093] 23. HUMHPRTB, HUMBFXIII (F13B) and HUMTPOX;
[0094] 24. HUMHPRTB, HUMBFXIII (F13B) and HUMFESFPS;
[0095] 25. HUMCSFLPO, HUMTPOX and HUMCD4;
[0096] 26. HUMHPRTB, HUMFESFPS and HUMMYOPK (Myotonic);
[0097] 27. HUMCSF1PO, HUMTH01 and HUMCD4;
[0098] 28. HUMCSF1PO, HUMTH01 and HUMVWFA31;
[0099] 29. HUMHPRTB, HUMBFXIII (F13B) and HUMLIPOL;
[0100] 30. HUMCSF1PO, HUMTPOX, HUMTH01 and HUMVWFA31;
[0101] 31. HUMHPRTB, HUMFESFPS, HUMBFXIII (F13B) and HUMLIPOL;
[0102] 32. HUMCSF1PO, HUMTPOX, HUMTH01 and HUMCD4; and
[0103] 33. HUMCSF1PO, HUMTH01, HUMTPOX and HUMCD4.
[0104] The primers must also be designed so that the size of the
resulting amplification products differ in length, thereby
facilitating assignment of alleles to individual loci during
detection. Inappropriate selection of primers can produce several
undesirable effects such as lack of amplification, amplification at
multiple sites, primer dimer formation, undesirable interaction of
primer sequences from different loci, production of alleles from
one locus which overlap with alleles from another, or requirement
for amplification conditions or protocols for the different loci
which are incompatible in a multiplex. The synthesis of the primers
is conducted by procedures known to those skilled in the art.
[0105] Using Multiplexes of Two Loci to Develop Multiplexes Using
More than Two Loci
[0106] Once a multiplex containing two loci is developed, it may be
used as a core to create multiplexes containing more than two loci.
New combinations are created including the first two loci. For
example, the core multiplex containing loci HUMTH01 and HUMCSF1PO
was used to generate derivative multiplexes of HUMTH01, HUMCSFLPO,
and HUMTPOX; HUMTH01, HUMCSF1PO, and HUMCD4; HUMTH01, HUMCSF1PO,
and HUMVWFA31; HUMTH01, HUMCSF1PO, HUMVWFA31, and HUMTPOX; and
HUMTH01, HUMCSFLPO, HUMCD4, and HUMTPOX. Many other derivative
multiplexes can be generated based upon a working multiplex. The
derivative multiplexes are, in some sense, routine extensions of
the core multiplex.
[0107] Preparation of Genomic DNA
[0108] All methods of Genomic DNA preparation which are compatible
with the amplification process for a single locus should be
appropriate for multiplex amplification. Many examples of
preparation methods have been described in the literature (Patel et
al. 1984, Gill et al. 1985). Genomic DNA concentrations are
measured fluorometrically (Brunk et al. 1979).
[0109] Amplification of Genomic DNA
[0110] Human genomic DNA samples are subjected to PCR amplification
using primers and thermocycling conditions specific for each locus.
Reference is made to Table 1 for details of the primer sequences.
The amplification protocol specific to each multiplex is listed in
the specific examples.
1 Sequence Designation Primer sequences ID Number HSAC04 primer 1:
ACA TCT CCC CTA CCG CTA TA 1 (ACTBP2) primer 2: AAT CTG GGC GAC AAG
AGT GA 2 HUMAPOA2 primer 1: GGA GCA GTC CTA GGG CCG CGC CGT 3
(APOCIII) primer 2: GTG ACA GAG GGA GAC TCC ATT AAA 4 HUMCSF1PO
primer 1: AAC CTG AGT CTG CCA AGG ACT AGC 5 primer 2: TTC CAC ACA
CCA CTG GCC ATC TTC 6 HUMCYP19 primer 1: GCA GGT ACT TAG TTA GCT AC
7 (CYARP450) primer 2: TTA CAG TGA GCC AAG GTC GT 8 HUMCD4 primer
1: CCA GGA AGT TGA GGC TGC AGT GAA 9 primer 2: TTG GAG TCG CAA GCT
GAA CTA GCG 10 HUMF13A01 primer 1: GAG GTT GCA CTC CAG CCT TTG CAA
11 primer 2: TTC CTG AAT CAT CCC AGA GCC ACA 12 HUMBFXIII primer 1:
TGA GGT GGT GTA CTA CCA TA 13 (F13B) primer 2: GAT CAT GCC ATT GCA
CTC TA 14 HUMFABP primer 1: GTA GTA TCA GTT TCA TAG GGT CAC C 15
primer 2: CAG TTC GTT TCC ATT GTC TGT CCG 16 HUMFESFPS primer 1:
GCT GTT AAT TCA TGT AGG GAA GGC 17 primer 2: GTA GTC CCA GCT ACT
TGG CTA CTC 18 HUMHPRTB primer 1: ATG CCA CAG ATA ATA CAC ATC CCC
19 (HPRT-1) primer 2: CTC TCC AGA ATA GTT AGA TGT AGG 20 HUMNYOPK
primer 1: GCT CGA AGG GTC CTT GTA GCC GGG 21 Myotonic primer 2: GAT
AGG TGG GGG TGC GTG GAG GAT 22 HUMLIPOL primer 1: CTG ACC AAG GAT
AGT GGG ATA TAG 23 primer 2: GGT AAC TGA GCG AGA CTC TGT CT 24
HUMPLA2A1 primer 1: GGT TGT AAG CTC CAT GAG GTT AGA 25 (PLA-AZ)
primer 2: TTG AGC ACT TAC TAT GTG CCA GGC T 26 HUMTH01 primer 1:
GTG GGC TGA AAA GCT CCC GAT TAT 27 primer 2: ATT CAA AGG GTA TCT
GGG CTC TGG 28 HUMTPOX primer 1: ACT GGC ACA GAA CAG GCA CTT AGG 29
primer 2: GGA GGA ACT GGG AAC CAC ACA GGT 30 HUMVWFA31 primer 1: GA
AAG CCC TAG TGG ATG AGA ATA ATC 31 primer 2: GGA CAG ATG ATA AAT
ACA TAG GAT GGA TGG 32
[0111] Reference is made to the examples below for additional
details of the specific procedure relating to each multiplex. The
locus-specific primers include a number of nucleotides which, under
the conditions used in the hybridization, are sufficient to
hybridize with an allele of the locus to be amplified and to be
essentially free from amplification of alleles of other loci.
Reference is made to U.S. Pat. No. 5,192,659 to Simons, which is
incorporated herein by reference for a more detailed description of
locus-specific primers.
[0112] Separation and Detection of Genomic DNA Fragments
[0113] Following amplification, products are then separated by
electrophoresis, e.g., denaturing polyacrylamide gel
electrophoresis (Sambrook et al., 1989). Preferred gel preparation
and electrophoresis procedures are conducted as described in
Example 1. Fragment separation occurs based on size and charge of
the sample.
[0114] The Genomic DNA is then detected by, e.g., silver staining
(Bassam et al. 1991). Alternatively, if radioactively-labeled or
fluorescently-labeled primers were used for each locus, the
products are detected by means available to detect these reporters
as known to those skilled in the art. Amplified materials may be
detected using any of a number of reporters including, e.g., silver
staining, radioisotopes, fluorescers, chemiluminescers and enzymes
in combination with detectable substrates.
[0115] Individual Genomic DNA samples containing amplified alleles
are preferably compared with a size standard such as a Genomic DNA
marker or locus-specific allelic ladder to determine the alleles
present at each locus within the sample. The preferred size marker
for evaluation of a multiplex amplification containing two or more
polymorphic STR loci consists of a combination of allelic ladders
for the loci being evaluated.
[0116] The preferred size marker for evaluation of a multiplex
amplification containing two or more polymorphic STR loci which are
generated using fluorescently-labeled primers for each locus
consists of a combination of fluorescently-labeled allelic ladders
for the loci being evaluated.
[0117] Following the construction of allelic ladders for individual
loci, they may be mixed and loaded for gel electrophoresis at the
same time as the loading of amplified samples occurs. Each allelic
ladder co-migrates with alleles in the sample from the
corresponding locus.
[0118] A permanent record of the data can be generated with the use
of electrophoresis duplicating film (STR systems manual #TMD004,
Promega Corporation, Madison, Wis.).
[0119] Advantage of Fluorescent Detection
[0120] With the advent of automated fluorescent imaging, faster
detection and analysis of multiplex amplification products can be
achieved. For fluorescent analyses, one fluoresceinated primer can
be included in the amplification of each locus. Separation of the
amplified fragments is achieved in precisely the same manner as
with the silver stain detection method. The resulting gel is loaded
onto a FluorImager.RTM. 575 (Molecular Dynamics, Sunnyvale, Calif.)
which scans the gel and digitizes the data in three minutes. The
FluorImager.RTM. contains an argon laser emitting 488 nm light
which sweeps through the gel using a galvanometer-controlled
mirror. The light activates fluorescent molecules in its path and
they, in turn, emit light of higher wavelength. A filter prohibits
passage of the original light, but allows collection of the emitted
light by a fiber optic collector. A second filter selected by the
user may be inserted between the fiber optic collector and the
photomultiplier, allowing detection of specific wavelength bands
(or colors) with each scan.
[0121] The image has an overall cleaner appearance than that
obtained with the silver stain for three reasons. First, only one
of the two PCR product strands is labeled with primer, simplifying
the two band per allele images of the silver stain. Second, in the
silver stain reaction, the entire gel is exposed to silver and
prone to silver deposition causing a significant general
background. With the fluorescent reporter, only the primer is
labeled and the unincorporated primers migrate out of the bottom of
the gel prior to detection. Third, some artifact bands of the PCR
reaction are plentiful, but contain very little primer.
[0122] Because this fluorescent method detects only products with
one particular primer, some of these artifacts which appear in
silver stain of multiplex amplifications are not detected. In fact,
this characteristic has allowed development of the more complex
quadriplex as shown in FIG. 2 in place of the triplex shown in FIG.
1.
Kit
[0123] The present invention is also directed to kits that utilize
the process described. A basic kit includes a container having a
locus-specific primer pair (or alternately separate containers
containing each primer of a primer pair) for each locus. The kit
also includes instructions for use.
[0124] Other ingredients may include an allelic ladder directed to
each of the specified loci, a sufficient quantity of enzyme for
amplification, amplification buffer to facilitate the
amplification, loading solution for preparation of the amplified
material for gel electrophoresis, human genomic DNA as a control to
test that the system is working well, a size marker to insure that
materials migrate as anticipated in the gel, and a protocol and
manual to educate the user and to limit error in use. The amounts
of the various reagents in the kits can be varied depending upon a
number of factors, such as the optimum sensitivity of the process.
The instructions for use are suitable to enable any analyst to
carry out the desired test. It is within the scope of this
invention to provide test kits for use in manual applications or
test kits for use with automated detectors or analyzers.
EXAMPLES
[0125] The following examples are presented to illustrate the
advantages of the present invention and to assist one of ordinary
skill in making and using the same. The examples are not intended
in any way to otherwise limit the scope of the disclosure or
protection granted by the patent.
[0126] Genomic DNA isolation and quantitation were performed
essentially as described by Puers et al., 1993. These methods are
generally known to those skilled in the art and are preferred, but
not required, for application of the invention.
[0127] Amplification products were separated by electrophoresis
through a 0.4 mm thick 4% denaturing polyacrylamide gel (19:1 ratio
of acrylamide to bis-acrylamide) which contained 7 M urea (Sambrook
et al., 1989) and was chemically cross-linked to one glass plate
(Kobayashi, 1988). Genomic DNA samples were mixed with 3 .mu.l
loading solution (10 mM NaOH, 95% formamide, 0.05% bromophenol
blue, 0.05% xylene cyanol), denatured at 95.degree. C. for 2 min.,
and chilled on ice prior to loading.
[0128] Electrophoresis was performed at 60 W in 0.5.times.TBE for
1-2 hrs. The Genomic DNA was detected by silver staining (Bassam et
al., 1991). Permanent images were obtained by exposure to
Electrophoresis Duplicating Films (EDF, Kodak, Cat. No. 809 6232).
Alternatively, detection can be performed by fluorescent scanning
(Schumm et al., 1994) or radioactive detection (Hammond et al.,
1994).
EXAMPLE 1
Silver Stain Detection of Multiplex Amplification of Loci
HUMCSFLPO, HUMTPOX, and HUMTH01
[0129] In this example, a Genomic DNA template (three Genomic DNA
samples) was amplified at the individual loci HUMCSFLPO, HUMTPOX,
and HUMTH01 simultaneously in a single reaction vessel. The PCR
amplifications were performed in 50 .mu.l volumes using 25 ng
template, 0.03 U Taq Genomic DNA Polymerase/.mu.l 1.times.STR
Buffer (50 mM KCl, 10 mM Tris-HCl (pH 9.0 at 25.degree. C.), 0.1%
Triton X-100, 1.5 mM MgCl2 and 200 .mu.M each of dATP, dCTP, dGTP
and dTTP), and using a Thermal Cycler 480 (Perkin Elmer Cetus).
Amplification protocol 1 (96.degree. C. for 2 min., then 10 cycles
of 94.degree. C. for 1 min., 64.degree. C. for 1 min., and
70.degree. C. for 1.5 min., followed by 20 cycles of 90.degree. C.
for 1 min., 64.degree. C. for 1 min., 70.degree. C. for 1.5 min.)
was employed.
[0130] Six amplification primers were used in combination,
including 0.2 .mu.M each HUMCSF1PO primers 1 [SEQ. ID. 5] and 2
[SEQ. ID. 6], 0.2 .mu.M each HUMTPOX primers 1 [SEQ. ID. 29] and 2
[SEQ. ID. 30], and 0.6 .mu.M each HUMTH01 primers 1 [SEQ. ID. 27]
and 2 [SEQ. ID. 28].
[0131] Amplified products were separated by denaturing acrylamide
gel electrophoresis on a 40 cm gel for 60-90 min. at 60 W and
products were visualized by silver stain analysis according the
protocol of Bassam et al. (1991).
[0132] Reference is made to FIG. 1 which reveals the silver stain
detection of the multiplex amplification. Lanes 2, 3, and 5 contain
Genomic DNA samples simultaneously co-amplified for the loci
HUMCSFLPO, HUMTPOX, and HUMTH01. Lanes 1, 4, and 7 contain allelic
ladders for the three loci and lane 6 displays a sample without
Genomic DNA subjected to the same procedures, i.e., a negative
control.
EXAMPLE 2
Fluorescent Detection of Multiplex Amplification of Loci HUMCSFLPO,
HUMTPOX, HUMTH01, and HUMVWFA31
[0133] In this example, a Genomic DNA template was amplified at the
individual loci HUMCSFLPO, HUMTPOX, HUMTH01, and HUMVWFA31
simultaneously in a single reaction vessel. The PCR amplifications
were performed in 25 .mu.l volumes using 25 ng template, 0.04 U Taq
Genomic DNA Polymerase/.mu.l, 1.times.STR Buffer (50 mM KCl, 10 mM
Tris-HCl (pH 9.0 at 25.degree. C.), 0.1% Triton X-100, 1.5 mM MgCl2
and 200 .mu.M each of DATP, dCTP, dGTP and dTTP), and using a
Thermal Cycler 480 (Perkin Elmer Cetus). Amplification protocol 1,
as described in Example 1, was employed. Eight amplification
primers were used in combination, including 1 .mu.M each HUMCSF1PO
primer 2 [SEQ. ID. 5] and fluorescein-labeled primer 1 [SEQ. ID.
5], 0.15 .mu.M each HUMTPOX primer 1 [SEQ. ID. 29] and
fluorescein-labeled primer 2 [SEQ. ID. 30], 0.2 .mu.M each HUMTH01
primer 2 [SEQ. ID. 28] and fluorescein-labeled primer 1 [SEQ. ID.
271, and 1M each HUMVWFA31 primer 1 [SEQ. ID. 31] and
fluorescein-labeled primer 2 [SEQ. ID. 32].
[0134] Amplified products were separated by denaturing acrylamide
gel electrophoresis on a 32 cm gel for 45 minutes at 40 watts.
Detection of the fluorescent signal was achieved using the
FluorImager.TM.575 (Molecular Dynamics, Sunnyvale, Calif.).
Reference is made to FIG. 2 which is a computer image of a
FluorImager scan. Lanes 2-7 contain Genomic DNA samples
simultaneously co-amplified for the loci HUMCSF1PO, HUMTPOX,
HUMTH01, and HUMVWFA31. Lane 1 contains allelic ladders for the 4
loci.
EXAMPLE 3
Multiplex Amplification of Loci HUMHPRTB. HUMFESFPS, and
HUMVWFA31
[0135] In this example, a Genomic DNA template was amplified at the
loci HUMHPRTB, HUMFESFPS, and HUMVWFA31 simultaneously in a single
reaction vessel. The PCR amplifications were performed in 25 .mu.l
volumes using 25 ng template, 0.03 U Taq Genomic DNA
Polymerase/.mu.l, 1.times.STR Buffer (described in example 1), and
a Thermal Cycler 480 (Perkin Elmer Cetus). Amplification protocol 2
(96.degree. C. for 2 min., then 10 cycles of 94.degree. C. for 1
min., 60.degree. C. for 1 min., and 70.degree. C. for 1.5 min.,
followed by 20 cycles of 90.degree. C. for 1 min., 64.degree. C.
for 1 min., 70.degree. C. for 1.5 min.) was employed. Amplified
products were separated by denaturing acrylamide gel
electrophoresis on a 32 cm gel for 45 min. at 40 W and products
were visualized by silver stain analysis according the protocol of
Bassam et al. (supra.). Six primers were used in combination
including 0.2 .mu.M each HUMHPRTB primers 1 [SEQ. ID. 19] and 2
[SEQ. ID. 20], 1.5 .mu.M each HUMFESFPS primers 1 [SEQ. ID. 17] and
2 [SEQ. ID. 18], and 1 .mu.M each HUMVWFA31 primers 1 [SEQ. ID. 31]
and 2 [SEQ. ID. 32].
[0136] Reference is made to FIG. 3 which reveals the silver stain
detection of the multiplex amplification. Lanes 2-6 contain Genomic
DNA samples simultaneously co-amplified for the loci HUMHPRTB,
HUMFESFPS, and HUMVWFA31. Lanes 1 and 7 contain allelic ladders for
the 3 loci.
EXAMPLE 4
Fluorescent Detection of Multiplex Amplification of Loci HUMHPRTB.
HUMFESFPS. HUMBFXIII (F13B), and HUMLIPOL
[0137] In this example, a Genomic DNA template was amplified at the
loci HUMHPRTB, HUMFESFPS, HUMBFXIII (F13B), and HUMLIPOL
simultaneously in a single reaction vessel. The PCR amplifications
and other manipulations were performed as described in Example 2
using amplification protocol 2, as described in Example 3.
[0138] Eight primers were used in combination, including 1M each
HUMHPRTB primer 2 [SEQ. ID. 20] and fluorescein-labeled primer 1
[SEQ. ID. 19], 2.5 .mu.M each HUMFESFPS primer 2 [SEQ. ID. 18] and
fluorescein-labeled primer 1 [SEQ. ID. 17], 1 .mu.M each HUMBFXIII
(F13B) primer 2 [SEQ. ID. 14] and fluorescein-labeled primer 1
[SEQ. ID. 13], and 0.5 .mu.M each HUMLIPOL primer 2 [SEQ. ID. 24]
and fluorescein-labeled primer 1 [SEQ. ID. 23].
[0139] Reference is made to FIG. 4 which is a computer image of a
FluorImager scan. Lanes 2-7 contain Genomic DNA samples
simultaneously co-amplified for the loci HUMHPRTB, HUMFESFPS,
HUMBFXIII (F13B), and HUMLIPOL. Lane 1 contains allelic ladders for
the 4 loci.
EXAMPLE 5
Multiplex Amplification of Loci HSAC04 (ACTBP2) and HUMCYP19
[0140] In this example, a Genomic DNA template was amplified at the
individual loci HSAC04 and HUMCYP19 simultaneously in a single
reaction vessel. The PCR amplifications were performed in 15 .mu.l
volumes with 25 ng template, 0.01 U Taq Genomic DNA
Polymerase/.mu.l, 1.times.Taq Genomic DNA Polymerase Buffer (50 mM
KCl, 10 mM Tris-HCl (pH 9.0 at 25.degree. C.), 0.1% Triton X-100
and 1.5 mM MgCl2) and 200 .mu.M each of dATP, dCTP, dGTP and dTTP
using a Thermal Cycler 480 (Perkin Elmer Cetus). Amplification
protocol 2, as described in Example 3, was employed. Amplified
products were separated and detected per example 1. Four primers
were used in combination, including 1 .mu.M each HSAC04 (ACTBP2)
primers 1 [SEQ. ID. 1] and 2 [SEQ. ID. 2], and 1 .mu.M each
HUMCYP19 primers 1 [SEQ. ID. 7] and 2 [SEQ. ID. 8].
[0141] Reference is made to FIG. 5 which reveals the silver stain
detection of the multiplex amplification. Lanes 1-3 contain Genomic
DNA samples simultaneously co-amplified for the loci HSAC04
(ACTBP2) and HUMCYP19. Lane 4 displays a sample without Genomic DNA
subjected to the same procedures, i.e., a negative control.
EXAMPLE 6
Multiplex Amplification of Loci HSAC04 (ACTBP2), HUMCYP19, and
HUMPLA2A1
[0142] In this example, a Genomic DNA template was amplified at the
loci HSAC04 (ACTBP2), HUMCYP19, and HUMPLA2A1 simultaneously in a
single reaction vessel. The PCR amplifications were performed in 15
.mu.l volumes with 25 ng template, 0.02 U Taq DNA Polymerase/.mu.l,
1.times.Taq Genomic DNA Polymerase Buffer (50 mM KCl, 10 mM
Tris-HCl (pH 9.0 at 25.degree. C.), 0.1% Triton X-100 and 1.5 mM
MgCl2) and 200 .mu.M each of DATP, dCTP, dGTP and dTTP using a
Thermal Cycler 480 (Perkin Elmer Cetus). Amplification protocol 2,
as described in Example 3, was employed. Amplified products were
separated and detected per example 1. Six primers were used in
combination, including 1 .mu.M each HSAC04 (ACTBP2) primers 1 [SEQ.
ID. 1] and 2 [SEQ. ID. 2], 1 .mu.M each HUMPLA2A1 primers 1 [SEQ.
ID. 25] and 2 [SEQ. ID. 26], and 1 .mu.M each HUMCYP19 primers 1
[SEQ. ID. 7] and 2 [SEQ. ID. 8].
[0143] Reference is made to FIG. 6 which reveals the silver stain
detection of the multiplex amplification. Lanes 1-3 contain Genomic
DNA samples simultaneously co-amplified for the loci HSAC04
(ACTBP2), HUMCYP19, and HUMPLA2A1. Lane 4 displays a sample without
Genomic DNA subjected to the same procedures, i.e., a negative
control.
EXAMPLE 7
[0144] Multiplex Amplification of Loci HSAC04 (ACTBP2) and
HUMFABP
[0145] In this example, a Genomic DNA template was amplified at the
loci HSAC04 (ACTBP2) and HUMFABP simultaneously in a single
reaction vessel. The PCR amplifications and other manipulations
were performed as described in Example 5 using amplification
protocol 2, as described in Example 3. Four primers were used in
combination, 1 .mu.M each HSAC04 (ACTBP2) primers 1 [SEQ. ID. 1]
and 2 [SEQ. ID. 2], and 1 .mu.M each HUMFABP primers 1 [SEQ. ID.
15] and 2 [SEQ. ID. 16].
[0146] Reference is made to FIG. 7 which reveals the silver stain
detection of the multiplex amplification. Lanes 1-3 contain Genomic
DNA samples simultaneously co-amplified for the loci HSAC04
(ACTBP2) and HUMFABP. Lane 4 displays a sample without Genomic DNA
subjected to the same procedures, i.e., a negative control.
EXAMPLE 8
Multiplex Amplification of Loci HUMAPOA2, HUMCYP19, and
HUMPLA2A1
[0147] In this example, a Genomic DNA template was amplified at the
loci HUMAPOA2, HUMCYP19, and HUMPLA2A simultaneously in a single
reaction vessel. The PCR amplifications and other manipulations
were performed as described in Example 6 using amplification
protocol 2, as described in Example 3. Six primers were used in
combination, including 1 .mu.M each HUMAPOA2 primers 1 [SEQ. ID. 3]
and 2 [SEQ. ID. 4], 1 .mu.M each HUMCYP19 primers 1 [SEQ. ID. 7]
and 2 [SEQ. ID. 8], and 1M each HUMPLA2A1 primers 1 [SEQ. ID. 25]
and 2 [SEQ. ID. 26]. Reference is made to FIG. 8 which reveals the
silver stain detection of the multiplex amplification. Lanes 1 and
3 contain Genomic DNA samples simultaneously co-amplified for the
loci HUMAPOA2, HUMCYP19, and HUMPLA2A1. Lane 2 contains a Genomic
DNA sample which failed to amplify and lane 4 displays a sample
without Genomic DNA subjected to the same procedures, i.e., a
negative control.
EXAMPLE 9
Multiplex Amplification of Loci HUMCD4. HUMCSFLPO, and HUMTH01
[0148] In this example, a Genomic DNA template was amplified at the
loci HUMCD4, HUMCSF1PO, and HUMTH01 simultaneously in a single
reaction vessel. The PCR amplifications were performed in 50 .mu.l
volumes with 25 ng template, 0.02 U Taq Genomic DNA
Polymerase/.mu.l, 1.times.Taq Genomic DNA Polymerase Buffer (50 mM
KCl, 10 mM Tris-HCl (pH 9.0 at 25.degree. C.), 0.1% Triton X-100
and 1.5 mM MgCl2) and 200 .mu.M each of DATP, dCTP, dGTP and dTTP
using a Thermal Cycler 480 (Perkin Elmer Cetus). Amplification
protocol 1, as described in Example 1, was employed. Amplified
products were separated and detected as described in Example 1. Six
primers were used in combination, including 1 .mu.M each HUMCD4
primers 1 [SEQ. ID. 9] and 2 [SEQ. ID. 10], 1 .mu.M each HUMCSF1PO
primers 1 [SEQ. ID. 5] and 2 [SEQ. ID. 6], and 1 .mu.M each HUMTH01
primers 1 [SEQ. ID. 27] and 2 [SEQ. ID. 28].
[0149] Reference is made to FIG. 9 which reveals the silver stain
detection of the multiplex amplification. Lanes 1-3 contain Genomic
DNA samples simultaneously co-amplified for the loci HUMCD4,
HUMCSF1PO, and HUMTH01. Lane 4 displays a sample without Genomic
DNA subjected to the same procedures, i.e., a negative control.
EXAMPLE 10
Multiplex Amplification of Loci HUMCYP19. HUMFABP, and
HUMPLA2A1
[0150] In this example, a Genomic DNA template was amplified at the
loci HUMCYP19, HUMFABP, and HUMPLA2A1 simultaneously in a single
reaction vessel. The PCR amplifications and other manipulations
were performed as described in Example 6 using amplification
protocol 2, as described in Example 3. Six primers were used in
combination, including 1 .mu.M each HUMCYP19 primers 1 [SEQ. ID. 7]
and 2 [SEQ. ID. 8], 1 .mu.M each HUMFABP primers 1 [SEQ. ID. 15]
and 2 [SEQ. ID. 16] and 1 .mu.M each HUMPLA1 primers 1 [SEQ. ID.
25] and 2 [SEQ. ID. 26].
[0151] Reference is made to FIG. 10 which reveals the silver stain
detection of the multiplex amplification. Lanes 1-3 contain Genomic
DNA samples simultaneously co-amplified for the loci HUMCYP19,
HUMFABP, and HUMPLA2A1. Lane 4 displays a sample without Genomic
DNA subjected to the same procedures, i.e., a negative control.
EXAMPLE 11
Multiplex Amplification of Loci HUMCYP19. HUMHPRTB, and
HUMPLA2A1
[0152] In this example, a Genomic DNA template was amplified at the
loci HUMCYP19, HUMHPRTB, and HUMPLA2A1 simultaneously in a single
reaction vessel. The PCR amplifications and other manipulations
were performed as described in Example 9 using amplification
protocol 2, as described in Example 3. Six primers were used in
combination, including 1 .mu.M each HUMCYP19 primers 1 [SEQ. ID. 7]
and 2 [SEQ. ID. 8], 1 .mu.M each HUMHPRTB primers 1 [SEQ. ID. 19]
and 2 [SEQ. ID. 20], and 1 .mu.M each HUMPLA2A1 primers 1 [SEQ. ID.
25] and 2 [SEQ. ID. 26].
[0153] Reference is made to FIG. 11 which reveals the silver stain
detection of the multiplex amplification. Lanes 1-3 contain Genomic
DNA samples simultaneously co-amplified for the loci HUMCYP19,
HUMHPRTB, and HUMPLA2A1. Lane 4 displays a sample without Genomic
DNA subjected to the same procedures, i.e., a negative control.
EXAMPLE 12
Multiplex Amplification of Loci HUMF13A01 and HUMFABP
[0154] In this example, a Genomic DNA template was amplified at the
loci HUMF13A01 and HUMFABP simultaneously in a single reaction
vessel. The PCR amplifications and other manipulations were
performed as described in Example 5 using amplification protocol 1,
as described in Example 1. Four primers were used in combination,
including 1 .mu.M each HUMF13A01 primers 1 [SEQ. ID. 11] and 2
[SEQ. ID. 12], and 1 .mu.M each HUMFABP primers 1 [SEQ. ID. 15] and
2 [SEQ. ID. 16].
[0155] Reference is made to FIG. 12 which reveals the silver stain
detection of the multiplex amplification. Lanes 1-3 contain Genomic
DNA samples simultaneously co-amplified for the loci HUMF13A01 and
HUMFABP. Lane 4 displays a sample without DNA subjected to the same
procedures, i.e., a negative control.
EXAMPLE 13
Multiplex Amplification of Loci HUMBFXIII (F13B) and HUMFESFPS
[0156] In this example, a Genomic DNA template was amplified at the
loci HUMBFXIII (F13B) and HUMFESFPS simultaneously in a single
reaction vessel. The PCR amplifications and other manipulations
were performed as described in Example 6 using amplification
protocol 1, as described in Example 1. Four primers were used in
combination, including 1 .mu.M each HUMBFXIII (F13B) primers 1
[SEQ. ID. 13] and 2 [SEQ. ID. 14], and 1 .mu.M each HUMFESFPS
primers 1 [SEQ. ID. 17] and 2 [SEQ. ID. 18].
[0157] Reference is made to FIG. 13 which reveals the silver stain
detection of the multiplex amplification. Lanes 1-3 contain Genomic
DNA samples simultaneously co-amplified for the loci HUMBFXIII
(F13B) and HUMFESFPS. Lane 4 displays a sample without Genomic DNA
subjected to the same procedures, i.e., a negative control.
EXAMPLE 14
Multiplex Amplification of Loci HUMBFXIII (F13B). HUMHPRTB, and
HUMPLA2A1
[0158] In this example, a Genomic DNA template was amplified at the
loci HUMBFXIII (F13B), HUMHPRTB, and HUMPLA2A1 simultaneously in a
single reaction vessel. The PCR amplifications and other
manipulations were performed as described in Example 6 using
amplification protocol 2, as described in Example 3. Six primers
were used in combination, including 1 .mu.M each HUMBFXIII (F13B)
primers 1 [SEQ. ID. 13] and 2 [SEQ. ID. 14], 1M each HUMHPRTB
primers 1 [SEQ. ID. 19] and 2 [SEQ. ID. 20], and 1M each HUMPLA2A1
primers 1 [SEQ. ID. 25] and 2 [SEQ. ID. 26].
[0159] Reference is made to FIG. 14 which reveals the silver stain
detection of the multiplex amplification. Lanes 1-3 contain Genomic
DNA samples simultaneously co-amplified for the loci HUMBFXIII
(F13B), HUMHPRTB, and HUMPLA2A1. Lane 4 displays a sample without
Genomic DNA subjected to the same procedures, i.e., a negative
control.
EXAMPLE 15
Multiplex Amplification of Loci HUMF13A01, HUMFABP, and HUMCD4
[0160] In this example, a Genomic DNA template was amplified at the
loci HUMF13A01, HUMFABP, and HUMCD4 simultaneously in a single
reaction vessel. The PCR amplifications and other manipulations
were performed as described in Example 5 using amplification
protocol 1, as described in Example 1. Six primers were used in
combination, including 1 .mu.M each HUMF13A01 primers 1 [SEQ. ID.
11] and 2 [SEQ. ID. 12], 1M each HUMFABP primers 1 [SEQ. ID. 15]
and 2 [SEQ. ID. 16], and 1M each HUMCD4 primers 1 [SEQ. ID. 9] and
2 [SEQ. ID. 101.
[0161] Reference is made to FIG. 15 which reveals the silver stain
detection of the multiplex amplification. Lanes 1-3 contain Genomic
DNA samples simultaneously co-amplified for the loci HUMF13A01,
HUMFABP, and HUMCD4. Lane 4 displays a sample without Genomic DNA
subjected to the same procedures, i.e., a negative control.
EXAMPLE 16
Multiplex Amplification of Loci HUMHPRTB and HUMFESFPS
[0162] In this example, a Genomic DNA template was amplified at the
loci HUMHPRTB and HUMFESFPS simultaneously in a single reaction
vessel. The PCR amplifications and other manipulations were
performed as described in Example 1 using 500-0.5 ng template, 0.02
U Taq Genomic DNA Polymerase/A1 and amplification protocol 2, as
described in Example 3. Four primers were used in combination,
including 0.2 .mu.M each HUMHPRTB primers 1 [SEQ. ID. 19] and 2
[SEQ. ID. 20] and 1.5 .mu.M each HUMFESFPS primers 1 [SEQ. ID. 171
and 2 [SEQ. ID.
[0163] 18).
[0164] Reference is made to FIG. 16 which reveals the silver stain
detection of the multiplex amplification. Lanes 1-6 contain Genomic
DNA samples simultaneously co-amplified for the loci HUMHPRTB and
HUMFESFPS-using 500, 50, 25, 5, 1 and 0.5 ng DNA template. Lane 7
displays a sample without Genomic DNA subjected to the same
procedures, i.e., a negative control.
EXAMPLE 17
Multiplex Amplification of Loci HUMHPRTB. HUMFESFPS, and
HUMLIPOL
[0165] In this example, a Genomic DNA template was amplified at the
loci HUMHPRTB, HUMFESFPS, and HUMLIPOL simultaneously in a single
reaction vessel. The PCR amplifications and other manipulations
were performed as described in Example 1 using amplification
protocol 2, as described in Example 3. Six primers were used in
combination, including 0.4 .mu.M each HUMHPRTB primers 1 [SEQ. ID.
19] and 2 [SEQ. ID. 20], 3 .mu.M each HUMFESFPS primers 1 [SEQ. ID.
17] and 2 [SEQ. ID. 18], and 2 .mu.M each HUMLIPOL primers 1 [SEQ.
ID. 23] and 2 [SEQ. ID. 24].
[0166] Reference is made to FIG. 17 which reveals the silver stain
detection of the multiplex amplification. Lanes 1-3 contain Genomic
DNA samples simultaneously co-amplified for the loci HUMHPRTB,
HUMFESFPS and HUMLIPOL. Lane 4 displays a sample without Genomic
DNA subjected to the same procedures, i.e., a negative control.
EXAMPLE 18
Multiplex Amplification of Loci HUMBFXIII (F13B) and HUMLIPOL
[0167] In this example, a Genomic DNA template was amplified at the
loci HUMBFXIII (F13B) and HUMLIPOL Simultaneously in a single
reaction vessel. The PCR amplifications and other manipulations
were performed as described in Example 1 using 0.02 U Taq Genomic
DNA Polymerase/pl and amplification protocol 2, as described in
Example 3. Four primers were used in combination, including 1 .mu.M
each HUMBFXIII (F13B) primers 1 [SEQ. ID. 13] and 2 [SEQ. ID. 14]
and 1 .mu.M each HUMLIPOL primers 1 [SEQ. ID. 23] and 2 [SEQ. ID.
24].
[0168] Reference is made to FIG. 18 which reveals the silver stain
detection of the multiplex amplification. Lanes 1-3 contain Genomic
DNA samples simultaneously co-amplified for the loci HUMBFXIII
(F13B) and HUMLIPOL.
EXAMPLE 19
Multiplex Amplification of Loci HUMHPRTB. HUMTPOX, and HUMBFXIII
(F13B)
[0169] In this example, a Genomic DNA template was amplified at the
loci HUMHPRTB, HUMTPOX, and HUMBFXIII (F13B) simultaneously in a
single reaction vessel. The PCR amplifications and other
manipulations were performed as described in Example 1 using
amplification protocol 2, as described in Example 3. Six primers
were used in combination, including 1M each HUMHPRTB primers 1.
[SEQ. ID. 19] and 2 [SEQ. ID. 20], 0.2 .mu.M each HUMTPOX primers 1
[SEQ. ID. 29] and 2 [SEQ. ID. 30], and 2 .mu.M each HUMBFXIII
(F13B) primers 1 [SEQ. ID. 13] and 2 [SEQ. ID. 14].
[0170] Reference is made to FIG. 19 which reveals the silver stain
detection of the multiplex amplification. Lanes 1-3 contain Genomic
DNA samples simultaneously co-amplified for the loci HUMHPRTB,
HUMTPOX, and HUMBFXIII (F13B).
EXAMPLE 20
Multiplex Amplification of Loci HUMHPRTB. HUMFESFPS, and HUMBFXIII
(F13B)
[0171] In this example, a Genomic DNA template was amplified at the
loci HUMHPRTB, HUMFESFPS, and HUMBFXIII (F13B) simultaneously in a
single reaction vessel. The PCR amplifications and other
manipulations were performed as described in Example 1 using
amplification protocol 2, as described in Example 3. Six primers
were used in combination, including 1 .mu.M each HUMHPRTB primers 1
[SEQ. ID. 19] and 2 [SEQ. ID. 20], 2 .mu.M each HUMFESFPS primers 1
[SEQ. ID. 17] and 2 [SEQ. ID. 18], and 2 .mu.M each HUMBFXIII
(F13B) primers 1 [SEQ. ID. 13] and 2 [SEQ. ID. 14].
[0172] Reference is made to FIG. 20 which reveals the silver stain
detection of the multiplex amplification. Lanes 1-3 contain Genomic
DNA samples simultaneously co-amplified for the loci HUMHPRTB,
HUMFESFPS, and HUMBFXIII (F13B).
EXAMPLE 21
Multiplex Amplification of Loci HUMCSFLPO. HUMTPOX, and HUMCD4
[0173] In this example, a Genomic DNA template was amplified at the
loci HUMCSF1PO, HUMTPOX, and HUMCD4 simultaneously in a single
reaction vessel. The PCR amplifications and other manipulations
were performed as described in Example 1 using amplification
protocol 1, as described in Example 1. Six primers were used in
combination, including 1 .mu.M each HUMCSFLPO primers 1 [SEQ. ID.
5] and 2 (SEQ. ID. 61], 1 .mu.M each HUMTPOX primers 1 [SEQ. ID.
29] and 2 [SEQ. ID. 30], and 1 .mu.M each HUMCD4 primers 1 [SEQ.
ID. 9] and 2 [SEQ. ID. 10].
[0174] Reference is made to FIG. 21 which reveals the silver stain
detection of the multiplex amplification. Lanes 1-3 contain Genomic
DNA samples simultaneously co-amplified for the loci HUMCSF1PO,
HUMTPOX, and HUMCD4.
EXAMPLE 22
Multiplex Amplification of Loci HUMHPRTB, HUMFESFPS, and HUMMYOPK
(Myotonic)
[0175] In this example, a Genomic DNA template was amplified at the
loci HUMHPRTB, HUMFESFPS, and HUMMYOPK simultaneously in a single
reaction vessel. The PCR amplifications and other manipulations
were performed as described in Example 1 using amplification
protocol 2, as described in Example 3. Six primers were used in
combination, including 1 .mu.M each HUMHPRTB primers 1 [SEQ. ID.
19] and 2 (SEQ. ID. 20], 1M each HUMFESFPS primers 1 [SEQ. ID. 17]
and 2 [SEQ. ID. 18], and 1 .mu.M each HUMMYOPK (Myotonic) primers 1
[SEQ. ID. 21] and 2 [SEQ. ID. 22].
[0176] Reference is made to FIG. 22 which reveals the silver stain
detection of the multiplex amplification. Lanes 1-3 contain Genomic
DNA samples simultaneously co-amplified for the loci HUMHPRTB,
HUMFESFPS, and HUMMYOPK (Myotonic).
EXAMPLE 23
Multiplex Amplification of Loci HUMCSF1PO. HUMTPOX. HUMTH01, and
HUMCD4
[0177] In this example, a Genomic DNA template was amplified at the
loci HUMCSFLPO, HUMTPOX, HUMTH01, and HUMCD4 simultaneously in a
single reaction vessel. The PCR amplifications and other
manipulations were performed as described in Example 1 using 0.04 U
Taq Genomic DNA Polymerase/A1 and amplification protocol 1, as
described in Example 1. Eight primers were, used in combination,
including 1 .mu.M each HUMCSF1PO primers 1 (SEQ. ID. 5] and 2 (SEQ.
ID. 6], 1 .mu.M each HUMTPOX primers 1 [SEQ. ID. 291 and 2 [SEQ.
ID. 30], 1 .mu.M each HUMTH01 primers 1 (SEQ. ID. 27] and 2 [SEQ.
ID. 28], and 1 .mu.M each HUMCD4 primers 1 [SEQ. ID. 9] and 2 [SEQ.
ID. 10].
[0178] Reference is made to FIG. 23 which reveals the silver stain
detection of the multiplex amplification.
[0179] Lanes 1-3 contain Genomic DNA samples simultaneously
co-amplified for the loci HUMCSF1PO, HUMTPOX, HUMTH01, and
HUMCD4.
EXAMPLE 24
Multiplex Amplification of Loci HUMF13A01 and HUMMYOPK
(Myotonic)
[0180] In this example, a Genomic DNA template was amplified at the
loci HUMF13A01 and HUMMYOPK (Myotonic) simultaneously in a single
reaction vessel. The PCR amplifications and other manipulations
were performed as described in Example 1 using 0.04 U Taq Genomic
DNA Polymerase/A1 and amplification protocol 1, as described in
Example 1. Four primers were used in combination, including 0.1
.mu.M each HUMF13A01 primers 1 [SEQ. ID. 11] and 2 [SEQ. ID. 12]
and 1 .mu.M each HUMMYOPK (Myotonic) primers 1 [SEQ. ID. 21] and 2
[SEQ. ID. 22].
[0181] Reference is made to FIG. 24 which reveals the silver stain
detection of the multiplex amplification. Lanes 1-3 contain Genomic
DNA samples simultaneously co-amplified for the loci HUMF13A01 and
HUMMYOPK (Myotonic).
EXAMPLE 25
Multiplex Amplification of Loci HUMF13A01 and HUMBFXIII (F13B)
[0182] In this example, a Genomic DNA template was amplified at the
loci HUMF13A01 and HUMBFXIII (F13B) simultaneously in a single
reaction vessel. The PCR amplifications and other manipulations
were performed as described in Example 1 using 0.03 U Taq Genomic
DNA Polymerase/A1 and amplification protocol 2, as described in
Example 3. Four primers were used in combination, including 0.1
.mu.M each HUMF13A01 primers 1 [SEQ. ID. 11] and 2-[SEQ. ID. 12]
and 0.5 .mu.M each HUMBFXIII (F13B) primers 1 [SEQ. ID. 13] and 2
[SEQ. ID. 14].
[0183] Reference is made to FIG. 25 which reveals the silver stain
detection of the multiplex amplification. Lanes 1-3 contain Genomic
DNA samples simultaneously co-amplified for the loci HUMF13A01 and
HUMBFXIII (F13B).
EXAMPLE 26
Fluorescent Detection of Multiplex Amplification of Loci HUMCSFLPO.
HUMTPOX. HUMTH01, and HUMCD4
[0184] In this example, a Genomic DNA template was amplified at the
individual loci HUMCSF1PO, HUMTPOX, HUMTH01, and HUMCD4
simultaneously in a single reaction vessel. The PCR amplifications
were performed as described in Example 1 using 0.04 U Taq Genomic
DNA Polymerase/A1 and amplification protocol 1, as described in
Example 1. Eight amplification primers were used in combination,
including 2 .mu.M each HUMCSFLPO primer 2 [SEQ. ID. 6] and
fluorescein-labeled primer 1 [SEQ. ID. 5], 0.5 .mu.M each HUMTPOX
primer 1 [SEQ. ID. 29] and fluorescein-labeled primer 2 [SEQ. ID.
30], 0.5 .mu.M each HUMTH01 primer 2 [SEQ. ID. 28] and
fluorescein-labeled primer 1 [SEQ. ID. 27] and 0.5 .mu.M each
HUMCD4 primer 1 [SEQ. ID. 9] and fluorescein-labeled primer 2 [SEQ.
ID. 10].
[0185] Amplified products were detected as in Example 2. Reference
is made to FIG. 26 which is a photograph of a computer image of a
FluorImager scan. Lanes 1-3 contain Genomic DNA samples
simultaneously co-amplified for the loci HUMCSF1PO, HUMTPOX,
HUMTH01, and HUMCD4.
EXAMPLE 27
Fluorescent Detection of Multiplex Amplification of Loci HUMCSFLPO.
HUMTH01, and HUMCD4
[0186] In this example, a Genomic DNA template was amplified at the
individual loci HUMCSF1PO, HUMTH01, and HUMCD4 simultaneously in a
single reaction vessel. The PCR amplifications were performed as
described in Example 1 using 0.02 U Taq Genomic DNA Polymerase/A1
and amplification protocol 1, as described in Example 1. Six
amplification primers were used in combination, including 1 .mu.M
each HUMCSF1PO primer 2 [SEQ. ID. 6] and fluorescein-labeled primer
1 [SEQ. ID. 5], 1 .mu.M each HUMTH01 primer 2 [SEQ. ID. 281 and
fluorescein-labeled primer 1 [SEQ. ID. 27] and 1 .mu.M each HUMCD4
primer 1 [SEQ. ID. 9] and fluorescein-labeled primer 2 [SEQ. ID.
10].
[0187] Amplified products were detected as in Example 2. Reference
is made to FIG. 27 which is a photograph of a computer image of a
FluorImager scan. Lanes 1 and 2 contain DNA samples simultaneously
co-amplified for the loci HUMCSFLPO, HUMTH01, and HUMCD4.
EXAMPLE 28
Fluorescent Detection of Multiplex Amplification of Loci HUMCSF1PO.
HUMTH01, and HUMVWFA31
[0188] In this example, a Genomic DNA template was amplified at the
individual loci HUMCSFLPO, HUMTH01, and HUMVWFA31 simultaneously in
a single reaction vessel. The PCR amplifications were performed as
described in Example 1 using 0.02 U Taq Genomic DNA Polymerase/A1
and amplification protocol 1, as described in Example 1. Six
amplification primers were used in combination, including 1M each
HUMCSF1PO primer 2 [SEQ. ID. 6] and fluorescein-labeled primer 1
[SEQ. ID. 5], 1 .mu.M each HUMTH01 primer 2 [SEQ. ID. 28] and
fluorescein-labeled primer 1 [SEQ. ID. 27], and 1 .mu.M each
HUMVWFA31 primer 1 [SEQ. ID. 31] and fluorescein-labeled primer 2
[SEQ. ID. 32].
[0189] Amplified products were detected as in Example 2. Reference
is made to FIG. 28 which is a photograph of a computer image of a
FluorImager scan. Lanes 1 and 2 contain DNA samples simultaneously
co-amplified for the loci HUMCSF1PO, HUMTH01, and HUMVWFA31.
EXAMPLE 29
Fluorescent Detection of Multiplex Amplification of Loci HUMHPRTB.
HUMBFXIII (F13B), and HUMLIPOL
[0190] In this example, a Genomic DNA template was amplified at the
individual loci HUMHPRTB, HUMBFXIII (F13B), and HUMLIPOL
simultaneously in a single reaction vessel. The PCR amplifications
were performed as described in Example 1 using 0.03 U Taq Genomic
DNA Polymerase/A1 and amplification protocol 2, as described in
Example 3. Six amplification primers were used in combination,
including 1 .mu.M each HUMHPRTB primer 2 (SEQ. ID. 20] and
fluorescein-labeled primer 1 [SEQ. ID. 19], 1M each HUMBFXIII
(F13B) primer 2 [SEQ. ID. 14] and fluorescein-labeled primer 1
[SEQ. ID. 13], and 1 .mu.M each HUMLIPOL primer 2 [SEQ. ID. 24] and
fluorescein-labeled primer 1 [SEQ. ID. 23].
[0191] Amplified products were detected as in Example 2. Reference
is made to FIG. 29 which is a photograph of a computer image of a
FluorImager scan. Lanes 1-3 contain Genomic DNA samples
simultaneously co-amplified for the loci HUMHPRTB, HUMBFXIII
(F13B), and HUMLIPOL.
EXAMPLE 30
Fluorescent Detection of Multiplex Amplification of Loci HUMCSFLPO
and HUMTH01
[0192] In this example, a Genomic DNA template was amplified at the
individual loci HUMCSF1PO and HUMTH01 simultaneously in a single
reaction vessel. The PCR amplifications were performed as described
in Example 1 using 0.02 U Taq Genomic DNA Polymerase/A1 and
amplification protocol 1, as described in Example 1. Four
amplification primers were used in combination, including 2 .mu.M
each HUMCSF1PO primer 2 [SEQ. ID. 6] and fluorescein-labeled primer
1 [SEQ. ID. 5] and 1 .mu.M each HUMTH01 primer 2 [SEQ. ID. 28] and
fluorescein-labeled primer 1 [SEQ. ID. 27].
[0193] Amplified products were detected as in Example 2. Reference
is made to FIG. 30 which is a photograph of a computer image of a
FluorImager scan. Lanes 1-3 contain Genomic DNA samples
simultaneously co-amplified for the loci HUMCSFLPO and HUMTH01.
EXAMPLE 31
Fluorescent Detection of Multiplex Amplification of Loci HUMTH01
and HUMCD4
[0194] In this example, a Genomic DNA template was amplified at the
individual loci HUMTH01 and HUMCD4 simultaneously in a single
reaction vessel. The PCR amplifications were performed as described
in Example 1 using 0.02 U Taq Genomic DNA Polymerase/A1 and
amplification protocol 1, as described in Example 1. Four
amplification primers were used in combination, including 1 .mu.M
each HUMTH01 primer 2 [SEQ. ID. 28] and fluorescein-labeled primer
1 [SEQ. ID. 27] and 1 .mu.M each HUMCD4 primer 1 [SEQ. ID. 9] and
fluorescein-labeled primer 2 [SEQ. ID. 10].
[0195] Amplified products were detected as in Example 2. Reference
is made to FIG. 31 which is a photograph of a computer image of a
FluorImager scan. Lanes 1-3 contain Genomic DNA samples
simultaneously co-amplified for the loci HUMTH01 and HUMCD4.
EXAMPLE 32
Fluorescent Detection of Multiplex Amplification of Loci HUMTH01
and HUMTPOX
[0196] In this example, a Genomic DNA template was amplified at the
individual loci HUMTH01 and HUMTPOX simultaneously in a single
reaction vessel. The PCR amplifications were performed as described
in Example 1 using 0.02 U Taq Genomic DNA Polymerase/A1 and
amplification protocol 1, as described in Example 1. Four
amplification primers were used in combination, including 1 .mu.M
each HUMTH01 primer 2 [SEQ. ID. 28] and fluorescein-labeled primer
1 [SEQ. ID. 27] and 1 .mu.M each HUMTPOX primer 2 [SEQ. ID. 30] and
fluorescein-labeled primer 1 [SEQ. ID. 29].
[0197] Amplified products were detected as in Example 2. Reference
is made to FIG. 32 which is a photograph of a computer image of a
FluorImager scan. Lanes 1-3 contain Genomic DNA samples
simultaneously co-amplified for the loci HUMTH01 and HUMTPOX.
[0198] It is understood that the invention is not confined to the
particular construction and arrangements herein illustrated and
described, but embraces such modified forms thereof and come within
the scope of the claims following the bibliography.
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953-958.
[0229] Sambrook J. et al. (1989) In "Molecular cloning--A
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[0230] Schumm, J. W. et al. (1994) "Development of nonisotopic
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[0231] Schwartz, J. S., et al. (1992) "Fluorescent multiple linkage
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[0232] Tautz, D., et al. (1986) "Cryptic simplicity in Genomic DNA
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[0233] Weber, J. L. and May, P. E. (1989) "Abundant class of human
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Sequence CWU 1
1
* * * * *