U.S. patent application number 11/148996 was filed with the patent office on 2005-11-03 for multiplex amplification of short tandem repeat loci.
This patent application is currently assigned to Promega Corporation. Invention is credited to Schumm, James W., Sprecher, Cynthia J..
Application Number | 20050244879 11/148996 |
Document ID | / |
Family ID | 35966199 |
Filed Date | 2005-11-03 |
United States Patent
Application |
20050244879 |
Kind Code |
A1 |
Schumm, James W. ; et
al. |
November 3, 2005 |
Multiplex amplification of short tandem repeat loci
Abstract
Methods and materials are disclosed for use in simultaneously
amplifying at least thirteen loci of genomic DNA in a single
multiplex reaction, as are methods and materials for use in the
analysis of the products of such reactions. Included in the present
invention are materials and methods for the simultaneous
amplification of at least thirteen short tandem repeat loci,
including specific materials and methods for the analysis of
thirteen such loci specifically selected by the United States
Federal Bureau of Investigation as core loci for use in the
Combined DNA Index System (CODIS) database.
Inventors: |
Schumm, James W.;
(Alexandria, VA) ; Sprecher, Cynthia J.; (Madison,
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: |
35966199 |
Appl. No.: |
11/148996 |
Filed: |
June 9, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11148996 |
Jun 9, 2005 |
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10236577 |
Sep 6, 2002 |
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10236577 |
Sep 6, 2002 |
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09199542 |
Nov 25, 1998 |
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6479235 |
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09199542 |
Nov 25, 1998 |
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08632575 |
Apr 15, 1996 |
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5843660 |
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08632575 |
Apr 15, 1996 |
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08316544 |
Sep 30, 1994 |
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Current U.S.
Class: |
435/6.11 |
Current CPC
Class: |
C12Q 2600/156 20130101;
C12Q 2600/16 20130101; C12Q 1/6876 20130101; C12Q 2535/125
20130101; C12Q 1/686 20130101; C12Q 2563/107 20130101; C12Q
2537/143 20130101; C12Q 1/686 20130101 |
Class at
Publication: |
435/006 |
International
Class: |
C12Q 001/68 |
Claims
1. A method of simultaneously determining the alleles present in a
set of loci from one or more DNA samples, comprising: (a) obtaining
at least one DNA sample to be analyzed, (b) selecting a set of loci
of the DNA sample, comprising at least thirteen short tandem repeat
loci which can be co-amplified, (c) co-amplifying the loci in the
set in a multiplex amplification reaction, wherein the product of
the reaction is a mixture of amplified alleles from each of the
co-amplified loci in the set; and (d) evaluating the amplified
alleles in the mixture to determine the alleles present at each of
the loci analyzed in the set within the DNA sample.
2. (canceled)
3. (canceled)
4. The method of claim 1, wherein at least one of the at least
thirteen short tandem repeat loci in the set of loci selected in
step (b) is a pentanucleotide tandem repeat locus.
5. The method of claim 4, wherein the pentanucleotide tandem repeat
locus is selected from the group consisting of G475, C221, and
S159.
6. The method of claim 1, wherein the set of loci selected in step
(b) further comprises a locus which can be used to identify the
gender of at least one source of the DNA provided in step (a).
7. The method of claim 6, wherein the at least one source of the
DNA provided in step (a) is a human being, and the locus used to
identify the gender of the human being is an Amelogenin locus.
8. The method of claim 7, wherein the Amelogenin locus is
co-amplified using an oligonucleotide primer having a sequence
selected from the group consisting of SEQ ID NO:86, SEQ ID NO:87,
and SEQ ID NO:105.
9. The method of claim 1, wherein the multiplex amplification
reaction is done using at least one oligonucleotide primer having a
sequence selected from at least one of the groups of sequences
consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:80 and SEQ ID
NO:81, when one of the loci in the set is D7S820; SEQ ID NO:3, SEQ
ID NO:4, SEQ ID NO:82, and SEQ ID NO:83, when one of the loci in
the set is D13S317; SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:84, and SEQ
ID NO:85, when one of the loci in the set is D5S818; SEQ ID NO:7,
SEQ ID NO:8, and SEQ ID NO:49, when one of the loci in the set is
D3S1539; SEQ ID NO:9, SEQ ID NO:10, when one of the loci in the set
is D17S1298; SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:52, SEQ ID NO:
53, when one of the loci in the set is D20S481; SEQ ID NO:13, SEQ
ID NO:14, SEQ ID NO: 55, SEQ ID NO: 61, when one of the loci in the
set is D9S930; SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:54, when one
of the loci in the set is D10S1239; SEQ ID NO:17, SEQ ID NO:18,
when one of the loci in the set is D14S118; SEQ ID NO:19, SEQ ID
NO:20, when one of the loci in the set is D14S562; SEQ ID NO:21,
SEQ ID NO:22, when one of the loci in the set is D14S548; SEQ ID
NO:23, SEQ ID NO:24, when one of the loci in the set is D16S490;
SEQ ID NO:25, SEQ ID NO:26, when one of the loci in the set is
D16S753; SEQ ID NO:27, SEQ ID NO:28, when one of the loci in the
set is D17S1299; SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:58, SEQ ID
NO:79, and SEQ ID NO:97, when one of the loci in the set is
D16S539; SEQ ID NO:31, SEQ ID NO:32, when one of the loci in the
set is D22S683; SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:77, SEQ ID
NO:78, and SEQ ID NO:98, when one of the loci in the set is
HUMCSF1PO; SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:72 and SEQ ID
NO:73, when one of the loci in the set is HUMTPOX; SEQ ID NO:37,
SEQ ID NO:38, SEQ ID NO:66, SEQ ID NO:67, and SEQ ID NO:103, when
one of the loci in the set is HUMTH01; SEQ ID NO:39, SEQ ID NO:40,
SEQ ID NO:59, SEQ ID NO:60, and SEQ ID NO:76 when one of the loci
in the set is HUMvWFA31 SEQ ID NO:41, SEQ ID NO:42, when one of the
loci in the set is HUMF13A01; SEQ ID NO:43, SEQ ID NO:44, when one
of the loci in the set is HUMFESFPS; SEQ ID NO:45, SEQ ID NO:46,
when one of the loci in the set is HUMBFXIII; SEQ ID NO:47, SEQ ID
NO:48, when one of the loci in the set is HUMLIPOL; SEQ ID NO:50,
SEQ ID NO:51, when one of the loci in the set is D19S253; and SEQ
ID NO:56, SEQ ID NO:57, when one of the loci in the set is D4S2368.
SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:101, and SEQ ID NO:102, when
one of the loci in the set is D18S51; SEQ ID NO:64 and SEQ ID
NO:65, when one of the loci in the set is D21S11; SEQ ID NO:68, SEQ
ID NO:69, and SEQ ID NO:106, when one of the loci in the set is
D3S1538; SEQ ID NO:70, SEQ ID NO:71, and SEQ ID NO:107, when one of
the loci in the set is HUMFIBRA; SEQ ID NO:74, SEQ ID NO:75, and
SEQ ID NO:104, when one of the loci in the set is D8S1179; SEQ ID
NO:86, SEQ ID NO:87, and SEQ ID NO:105, when one of the loci in the
set is Amelogenin; SEQ ID NO:88, SEQ ID NO:89, and SEQ ID NO:94,
when one of the loci in the set is G475; SEQ ID NO:90, SEQ ID
NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:95, and SEQ ID NO:96,
when one of the loci in the set is S159; and SEQ ID NO:99 and SEQ
ID NO:100, when one of the loci in the set is C221.
10. The method of claim 1, wherein the amplified alleles are
separated prior to evaluating in step (d), using a separation means
selected from the group consisting of polyacrylamide gel
electrophoresis and capillary gel electrophoresis.
11. The method of claim 1, wherein the multiplex amplification
reaction is done using at least thirteen pairs of oligonucleotide
primers flanking the at least thirteen loci analyzed.
12. The method of claim 11, wherein the set of loci is co-amplified
using a polymerase chain reaction
13. The method of claim 11, wherein each of the loci co-amplified
in the multiplex reaction in step (b) is co-amplified using a pair
of primers which flank the locus, wherein at least one primer of
each pair has a fluorescent label covalently attached thereto.
14. The method of claim 13, wherein at least three of the labeled
primers have different fluorescent labels covalently attached
thereto.
15. The method of claim 1 wherein the at least one DNA sample to be
analyzed is prepared from human tissue, wherein the human tissue is
selected from the group of human tissue consisting of blood, semen,
vaginal cells, hair, saliva, urine, amniotic fluid containing
placental cells or fetal cells, and mixtures of any of the tissues
listed above.
16. The method of claim 1, wherein the amplified alleles are
evaluated by comparing the amplified alleles to a size standard,
wherein the size standard is selected from the group of size
standards consisting of a DNA marker and a locus-specific allelic
ladder.
17. A method of simultaneously identifying the alleles present in a
set of loci of from one or more DNA samples, comprising: (a)
obtaining at least one DNA sample to be analyzed, (b) selecting a
set of loci of the DNA sample, comprising short tandem repeat loci
D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11,
HUMCSF1PO, HUMFIBRA, HUMTH01, HUMTPOX, and HUMvWFA31; (c)
co-amplifying the loci in the set in a multiplex amplification
reaction, wherein the product of the reaction is a mixture of
amplified alleles from each of the co-amplified loci in the set;
and (d) evaluating the amplified alleles in the mixture to
determine the alleles present at each of the loci analyzed in the
set within the DNA sample.
18. The method of claim 17, wherein the multiplex amplification
reaction is done using at least one primer for at least one locus
in the set of at least thirteen loci selected in step (b), wherein
the primer has a sequence selected from one of the groups of primer
sequences consisting of: SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:101,
and SEQ ID NO:102, when one of the loci in the set is D18S51; SEQ
ID NO:64 and SEQ ID NO:65, for the locus D21S11; SEQ ID NO:37, SEQ
ID NO:38, SEQ ID NO:66, SEQ ID NO:67, and SEQ ID NO:103, for the
locus HUMTH01; SEQ ID NO:68, SEQ ID NO:69, and SEQ ID NO:106, for
the locus D3S1358; SEQ ID NO:70, SEQ ID NO:71, and SEQ ID NO:107,
for the locus HUMFIBRA; SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:72,
and SEQ ID NO:73, for the locus HUMTPOX; SEQ ID NO:74, SEQ ID
NO:75, and SEQ ID NO:104, for the locus D8S1179; SEQ ID NO:39, SEQ
ID NO:40, SEQ ID NO:59, SEQ ID NO:60, and SEQ ID NO:76, for the
locus HUMvWFA31; SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:77, SEQ ID
NO:78, and SEQ ID NO:98, for the locus HUMCSF1PO; SEQ ID NO:29, SEQ
ID NO:30, SEQ ID NO:58, SEQ ID NO:79, and SEQ ID NO:97, for the
locus D16S539; SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:80, and SEQ ID
NO:81, for the locus D7S820; SEQ ID NO:3, SEQ ID NO:4, SEQ ID
NO:82, and SEQ ID NO:83, for the locus D13S317; and SEQ ID NO:5,
SEQ ID NO:6, SEQ ID NO:84, and SEQ ID NO:85, for the locus
D5S818.
19. (canceled)
20. (canceled)
21. The method of claim 17, wherein the multiplex amplification
reaction is a polymerase chain reaction.
22. The method of claim 17, wherein the amplified alleles are
evaluated by comparing the amplified alleles to a size standard,
wherein the size standard is selected from the group of size
standards consisting of a DNA marker and a locus-specific allelic
ladder.
23. The method of claim 17 wherein the at least one DNA sample to
be analyzed is prepared from human tissue, wherein the human tissue
is selected from the group of human tissue consisting of blood,
semen, vaginal cells, hair, saliva, urine, bone, buccal sample,
amniotic fluid containing placental cells or fetal cells, and
mixtures of any of the tissues listed above.
24. A method of simultaneously identifying the alleles present in a
set of loci of from one or more human genomic DNA samples,
comprising: (a) obtaining at least one human genomic DNA sample to
be analyzed, (b) co-amplifying the loci in a set of at least
sixteen loci of the human genomic DNA sample in a multiplex
amplification reaction, the set of loci comprising D3S1358,
HUMTH01, D21S11, D18S51, G475, Amelogenin, HUMvWFA31, D8S1179,
HUMTPOX, HUMFIBRA, D5S818, D7S820, D13S317, D16S539, HUMCSF1PO, and
S159, wherein the product of the reaction is a mixture of amplified
alleles from each of the co-amplified loci in the set; and (c)
evaluating the amplified alleles in the mixture to determine the
alleles present at each of the at least sixteen loci of the human
genomic DNA sample.
25. The method of claim 24, wherein the multiplex amplification
reaction is done using at least one pair of primers flanking each
of the loci in the set of sixteen primers selected in step (b).
26. The method of claim 24, wherein the multiplex amplification
reaction is done using at least one primer for at least one locus
in the set of at least sixteen loci co-amplified in step (b),
wherein the primer has a sequence selected from one of the groups
of primer sequences consisting of: SEQ ID NO:62, SEQ ID NO:63, SEQ
ID NO:101, and SEQ ID NO:102, when one of the loci in the set is
D18S51; SEQ ID NO:64 and SEQ ID NO:65, for the locus D21S11; SEQ ID
NO:37, SEQ ID NO:38, SEQ ID NO:66, SEQ ID NO:67, and SEQ ID NO:103,
for the locus HUMTH01; SEQ ID NO:68, SEQ ID NO:69, and SEQ ID
NO:106, for the locus D3S1358; SEQ ID NO:70, SEQ ID NO:71, and SEQ
ID NO:107, for the locus HUMFIBRA; SEQ ID NO:35, SEQ ID NO:36, SEQ
ID NO:72, and SEQ ID NO:73, for the locus HUMTPOX; SEQ ID NO:74,
SEQ ID NO:75, and SEQ ID NO:104, for the locus D8S1179; SEQ ID
NO:39, SEQ ID NO:40, SEQ ID NO:59, SEQ ID NO:60, and SEQ ID NO:76,
for the locus HUMvWFA31; SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:77,
SEQ ID NO:78, and SEQ ID NO:98, for the locus HUMCSF1PO; SEQ ID
NO:29, SEQ ID NO:30, SEQ ID NO:58, SEQ ID NO:79, and SEQ ID NO:97,
for the locus D16S539; SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:80, and
SEQ ID NO:81, for the locus D7S820; SEQ ID NO:3, SEQ ID NO:4, SEQ
ID NO:82, and SEQ ID NO:83, for the locus D13S317; SEQ ID NO:5, SEQ
ID NO:6, SEQ ID NO:84, and SEQ ID NO:85, for the locus D5S818; SEQ
ID NO:88, SEQ ID NO:89, and SEQ ID NO:94, for the locus G475; SEQ
ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:95,
and SEQ ID NO:96, for the locus S159; and SEQ ID NO:86, SEQ ID
NO:87, and SEQ ID NO:105, for the Amelogenin locus.
27. A method of simultaneously identifying the alleles present in a
set of loci of from one or more human genomic DNA samples,
comprising: (a) obtaining at least one human genomic DNA sample to
be analyzed, (b) co-amplifying the loci in a set of at least
sixteen loci of the human genomic DNA sample in a multiplex
amplification reaction, the set of loci comprising D3S1358,
HUMTH01, D21S11, D18S51, S159, Amelogenin, HUMvWFA31, D8S1179,
HUMTPOX, HUMFIBRA, D5S818, D7S820, D13S317, D16S539, HUMCSF1PO, and
C221, wherein the product of the reaction is a mixture of amplified
alleles from each of the co-amplified loci in the set; and (c)
evaluating the amplified alleles in the mixture to determine the
alleles present at each of the at least sixteen loci of the human
genomic DNA sample.
28. The method of claim 27, wherein the multiplex amplification
reaction is done using at least one pair of primers flanking each
of the loci in the set of sixteen primers selected in step (b).
29. The method of claim 27, wherein the multiplex amplification
reaction is done using at least one primer for at least one locus
in the set of at least sixteen loci co-amplified in step (b),
wherein the primer has a sequence selected from one of the groups
of primer sequences consisting of: SEQ ID NO:62, SEQ ID NO:63, SEQ
ID NO:101, and SEQ ID NO:102, for the locus D18S51; SEQ ID NO:64
and SEQ ID NO:65, for the locus D21S11; SEQ ID NO:37, SEQ ID NO:38,
SEQ ID NO:66, SEQ ID NO:67, and SEQ ID NO:103, for the locus
HUMTH01; SEQ ID NO:68, SEQ ID NO:69, and SEQ ID NO:106, for the
locus D3S1358; SEQ ID NO:70, SEQ ID NO:71, and SEQ ID NO:107, for
the locus HUMFIBRA; SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:72, and
SEQ ID NO:73, for the locus HUMTPOX; SEQ ID NO:74, SEQ ID NO:75,
and SEQ ID NO:104, for the locus D8S1179; SEQ ID NO:39, SEQ ID
NO:40, SEQ ID NO:59, SEQ ID NO:60, and SEQ ID NO:76, for the locus
HUMvWFA31; SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:77, SEQ ID NO:78,
and SEQ ID NO:98, for the locus HUMCSF1PO; SEQ ID NO:29, SEQ ID
NO:30, SEQ ID NO:58, SEQ ID NO:79, and SEQ ID NO:97, for the locus
D16S539; SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:80, and SEQ ID NO:81,
for the locus D7S820; SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:82, and
SEQ ID NO:83, for the locus D13S317; SEQ ID NO:5, SEQ ID NO:6, SEQ
ID NO:84, and SEQ ID NO:85, for the locus D5S818; SEQ ID NO:90, SEQ
ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:95, and SEQ ID
NO:96, for the locus S159; SEQ ID NO:99 and SEQ ID NO:100, for the
locus C221; and SEQ ID NO:86, SEQ ID NO:87, and SEQ ID NO:105, for
the Amelogenin locus.
30.-40. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S. patent
application Ser. No. 08/632,575, filed Apr. 15, 1996, now U.S. Pat.
No. 5,843,660, issued Dec. 1, 1998, which is a continuation-in-part
of U.S. patent application Ser. No. 08/316,544, filed Sep. 30,
1994. The entire disclosure of those applications is incorporated
by reference herein.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] Not applicable.
FIELD OF THE INVENTION
[0003] 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.
BACKGROUND OF THE INVENTION
[0004] DNA typing is commonly used to identify the parentage of
human children, and to confirm the lineage of horses, dogs, other
animals, and agricultural crops. DNA typing is also commonly
employed to identify the source of blood, saliva, semen, and other
tissue found at a crime scenes or other sites requiring
identification of human remains. DNA typing is also employed in
clinical settings to determine success or failure of bone marrow
transplantation and presence of particular cancerous tissues. DNA
typing involves the analysis of alleles of genomic DNA with
characteristics of interest, commonly referred to as "markers".
Most typing methods in use today are specifically designed to
detect and analyze differences in the length and/or sequence of one
or more regions of DNA markers known to appear in at least two
different forms in a population. Such length and/or sequence
variation is referred to as "polymorphism." Any region (i.e.
"locus") of DNA in which such a variation occurs is referred to as
a "polymorphic locus." The methods and materials of the present
invention are designed for use in the detection of multiple loci of
DNA, some or all of which are polymorphic loci.
[0005] Genetic markers which are sufficiently polymorphic with
respect to length or sequence have long been sought for use in
identity applications, such as paternity testing and identification
of tissue samples collected for forensic analysis. The discovery
and development of such markers and methods for analyzing such
markers have gone through several phases of development over the
last several years.
[0006] The first identified DNA variant markers were simple base
substitutions, i.e. simple sequence polymorphisms, which were most
often detected by Southern hybridization assays. For examples of
references describing the identification of such markers, designed
to be used to analyze restriction endonuclease-digested DNA with
radioactive probes, see: Southern, E. M. (1975), J. Mol. Biol.
98(3):503-507; Schumm, et al. (1988), American Journal of Human
Genetics 42:143-159; and Wyman, A. and White, R. (1980) Proc. Natl.
Acad. Sci, U.S.A. 77:6754-6758.
[0007] The next generation of markers were size variants, i.e.
length polymorphisms, specifically "variable number of tandem
repeat" (VNTR) markers (Nakamura Y., et al. (1987), Science 235:
1616-1622; and U.S. Pat. No. 4,963,663 issued to White et al.
(1990); U.S. Pat. No. 5,411,859 continuation of U.S. Pat. No.
4,963,663 issued to White et al. (1995)) and "minisatellite"
markers (Jeffreys et al. (1985a), Nature 314:67-73; Jeffreys et al.
(1985b) Nature 316:76-79., U.S. Pat. No. 5,175,082 for an invention
by Jeffreys). Both VNTR and minisatellite markers, contain regions
of nearly identical sequences repeated in tandem fashion. The core
repeat sequence is 10 to 70 bases in length, with shorter core
repeat sequences referred to as "minisatellite" repeats and longer
repeats referred to as VNTRs. Different individuals in a human
population contain different numbers of the repeats. The VNTR
markers are generally more highly polymorphic than base
substitution polymorphisms, sometimes displaying up to forty or
more alleles at a single genetic locus. However, the tedious
process of restriction enzyme digestion and subsequent Southern
hybridization analysis are still required to detect and analyze
most such markers.
[0008] The next advance involved the joining of the polymerase
chain reaction (PCR) (U.S. Pat. No. 4,683,202 by Mullis, K. B.)
technology with the analysis of VNTR loci (Kasai, K. et al. (1990)
Journal Forensic Science 35(5):1196-1200). Amplifiable VNTR loci
were discovered, which could be detected without the need for
Southern transfer. The amplified products are separated through
agarose or polyacrylamide gels and detected by incorporation of
radioactivity during the amplification or by post-staining with
silver or ethidium bromide. However, PCR can only be used to
amplify relatively small DNA segments reliably, i.e. only reliably
amplifying DNA segments under 3,000 bases in length Ponce, M &
Micol, L. (1992) NAR 20(3):623; Decorte R, et al. (1990) DNA Cell
Biol. 9(6):461-469). Consequently, very few amplifiable VNTRs have
been developed.
[0009] 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 DNA typing. Specifically, with the
discovery and development of polymorphic markers containing
dinucleotide repeats (Litt and Luty (1989) Am J. Hum Genet
3(4):599-605; Tautz, D (1989) NAR 17:6463-6471; Weber and May
(1989) Am J Hum Genet 44:388-396; German Pat. No. DE 38 34 636 C2,
inventor Tautz, D; U.S. Pat. No. 5,582,979 filed by Weber, L.),
STRs with repeat units of three to four nucleotides (Edwards, A.,
et al. (1991) Am. J. Hum. Genet. 49: 746-756.; Hammond, H. A., et
al. (1994) Am. J. Hum. Genet. 55: 175-189; Fregeau, C. J.; and
Fourney, R. M. (1993) BioTechniques 15(1): 100-119.; Schumm, J. W.
et al. (1994) in The Fourth International Symposium on Human
Identification 1993, pp.177-187 (pub. by Promega Corp., 1994); and
U.S. Pat. No. 5,364,759 by Caskey et al.; German Pat. No. DE 38 34
636 C2 by Tautz, D.) and STRs with repeat units of five to seven
bases (See, e.g. Edwards et al. (1991) Nucleic Acids Res. 19:4791;
Chen et al. (1993) Genomics 15(3): 621-5; Harada et al. (1994) Am.
J. Hum. Genet. 55: 175-189; Comings et al. (1995), Genomics
29(2):390-6; and Utah Marker Development Group (1995), Am. J.
Genet. 57:619-628; and Jurka and Pethiyagoda (1995) J. Mol. Evol.
40:120-126)), many of the deficiencies of previous methods have
been overcome. STR markers are generally shorter than VNTR markers,
making them better substrates for amplification than most VNTR
markers.
[0010] STR loci are similar to amplifiable VNTR loci in that the
amplified alleles at each such locus may be differentiated based on
length variation. Generally speaking STR loci are less polymorphic
at each individual locus than VNTR loci;. Thus, it is desirable to
amplify and detect multiple STR systems in a single amplification
reaction and separation to provide information for several loci
simultaneously. Systems containing several loci are called
multiplex systems and many such systems containing up to 11
separate STR loci have been described. See, e.g., Proceedings:
American Academy of Forensic Sciences (Feb. 9-14, 1998), Schumm,
James W. et al., p. 53, B88; Id., Gibson, Sandra D. et al., p. 53,
B89; Id., Lazaruk, Katherine et al., p. 51, B83; Sparkes, R. et
al., Int J Legal Med (1996) 109:186-194; AmpFlSTR Profiler.TM. PCR
Amplification Kit User's Manual (1997), pub by Perkin-Elmer Corp,
i-viii and 1-1 to 1-10; AmpFlSTR Profiler Plus.TM. PCR
Amplification Kit User's Manual (1997), pub by Perkin-Elmer Corp.,
i viii and 1-1 to 1-10; AmpFlSTR COfiler.TM. PCR Amplification Kit
User Bulletin (1998), pub by Perkin-Elmer Corp. i-iii and 1-1 to
1-10; 9th International Symposium on Human Identification (Oct.
7-10, 1998), pub. by Promega Corp., Staub, Rick W. et al., Poster
Abstract 15; Id., Willard, Jeanne M. et al., Poster Abstract 73;
and Id., Walsh, P. Sean, et al., Speaker Abstract for
8:50am-9:20am, Thursday, Oct. 8, 1998.
[0011] Amplification protocols with STR loci can be designed to
produce small products, generally from 60 to 500 base pairs (bp) in
length, and alleles from each locus are often contained within a
range of less than 100 bp. This allows simultaneous electrophoretic
analysis of several systems on the same gel or capillary
electrophoresis by careful design of PCR primers such that all
potential amplification products from an individual system do not
overlap the range of alleles of other systems. Design of these
systems is limited, in part, by the difficulty in separating
multiple loci in a single gel or capillary. This occurs because
there is spacial compression of fragments of different sizes,
especially longer fragments in gels or capillaries, i.e., commonly
used means for separation of DNA fragments by those skilled in the
art.
[0012] The United States Federal Bureau of Investigation ("FBI")
has established and maintains a Combined DNA Index System
("CODIS"), a database of DNA typing information. Local, state, and
national law enforcement agencies use the CODIS system to match
forensic DNA evidence collected at crime scenes with DNA
information in the database. CODIS and other national database
systems have proven to be an effective tool for such agencies to
use in solving violent-crimes. (See, e.g. Niezgoda, Stephen, in
Cambridge Healthtech Institute's Second Annual Conference on DNA
Forensics: Science, Evidence, and Future Prospects (Nov. 17-18,
1998), pp. 1-21.; Niezgoda, Stephen in Proceedings From The Eighth
International Symposium on Human Identification 1997, pub. by
Promega Corporation (1998), pp 48-49; Frazier, Rachel R. E. et al.
Id., pp. 56-60; Niezgoda, S. J. Profiles in DNA 1(3): 12-13;
Werrett, D. J. and Sparkes, R. in Speaker Abstracts: 9th
International Symposium on Human Identification (Oct. 7-10, 1998)
pp. 5-6). Until recently, only restriction fragment length
polymorphism ("RFLP") data obtained from the analysis of particular
VNTR loci was considered a core component in the database. The FBI
has recently identified thirteen polymorphic STR loci for inclusion
in the CODIS database. The thirteen CODIS STR loci are HUMCSF1PO,
D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11,
HUMFIBRA, HUMTH01, HUMTPOX, and HUMvWFA31. (Budowle, Bruce and
Moretti, Tamyra in Speaker Abstracts: 9th International Symposium
on Human Identification (Oct. 7-10, 1998) pp. 7-8). Both VNTR and
STR marker data are currently maintained in the CODIS database.
(See, e.g. Niezgoda, Stephen in Second Annual Conference on DNA
Forensics, supra). Until the present invention, the number of loci
which could be co-amplified in a single reaction, and analyzed
thereafter was limited. Specifically, no materials or methods had
been developed for use in multiplex amplification of thirteen or
more STR loci, much less the thirteen polymorphic STR loci
identified for use in the CODIS database.
[0013] The materials and methods of the present method are designed
for use in multiplex analysis of particular polymorphic loci of DNA
of various types, including single-stranded and double-stranded DNA
from a variety of different sources. The present invention
represents a significant improvement over existing technology,
bringing increased power of discrimination, precision, and
throughput to DNA profiling for linkage analysis, criminal justice,
paternity testing, and other forensic, medical, and genetic
identification applications.
SUMMARY OF THE INVENTION
[0014] It is, therefore, an object of the present invention to
provide a method and materials for the simultaneous amplification
of sets of loci, which include multiple distinct polymorphic short
tandem repeat (STR) loci, in a single multiplex reaction, using PCR
or other amplification systems in combination with gel
electrophoresis, capillary electrophoresis or other separation and
detection methods to analyze and compare the relative lengths of
the alleles of each locus amplified in the multiplex reaction.
Multiplex analysis of the sets of loci disclosed herein has not
been previously described in the prior art. There has also not been
any previous description of the sequences for many of the primers
disclosed herein below, all of which are shown to be useful in
multiplex amplification-of the sets of loci disclosed.
[0015] It is also an object of the present invention to provide a
method, a kit, and primers specific for multiplex amplifications
comprising specified loci.
[0016] These and other objects are addressed by the present
invention which is directed to a method and materials for
simultaneously analyzing or determining the alleles present at each
individual locus of each multiplex. In general, the method of this
invention comprises the steps of (a) obtaining at least one DNA
sample to be analyzed, wherein the DNA sample has at least thirteen
loci which can be co-amplified; (b) co-amplifying the at least
thirteen loci of the DNA sample; and (c) detecting the amplified
materials in a fashion which reveals the polymorphic nature of the
systems employed.
[0017] In one embodiment, the present invention is a method of
simultaneously determining the alleles present in a set of loci
from one or more DNA samples, comprising the steps of:
[0018] (a) obtaining at least one DNA sample to be analyzed;
[0019] (b) selecting a set of loci of the DNA sample, comprising at
least thirteen short tandem repeat loci which can be
co-amplified;
[0020] (c) co-amplifying the loci in the set in a multiplex
amplification reaction, wherein the product of the reaction is a
mixture of amplified alleles from each of the co-amplified loci in
the set; and
[0021] (d) evaluating the amplified alleles in the mixture to
determine the alleles present at each of the loci analyzed in the
set within the DNA sample.
[0022] At least four of the at least thirteen short tandem repeat
loci are preferably selected from the group of loci consisting
of:
[0023] D3S1539, D4S2368, D5S818, D7S820, D9S930, D10S1239, D13S317,
D14S118, D14S548, D14S562, D16S490, D16S539, D16S753, D17S1298,
D17S1299, D19S253, D20S481, D22S683, HUMCSF1PO, HUMTPOX, HUMTH01,
HUMF13A01, HUMBFXIII, HUMLIPOL, HUMvWFA31.
[0024] In another embodiment of the invention, the set of loci
selected in step (b) of
[0025] In another embodiment of the invention, the set of loci
selected in step (b) of the method comprises thirteen CODIS STR
loci (i.e., D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539,
D18S51, D21S11, HUMCSF1PO, HUMFIBRA, HUMTH01, HUMTPOX, and
HUMvWFA31) which can be co-amplified and analyzed by themselves, or
with additional loci using methods of the present invention.
[0026] In a further aspect, this invention is a kit for
simultaneously analyzing a set of loci of genomic DNA, comprising
oligonucleotide primers for co-amplifying a set of loci of the
genomic DNA to be analyzed, wherein the set of loci comprises at
least thirteen short tandem repeat loci which can be co-amplified
in the same multiplex reaction, and wherein the primers are in one
or more containers. More preferably, the kit comprises
oligonucleotide primer pairs for co-amplifying a set of at least
thirteen loci of human genomic DNA, the set of loci comprising
D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11,
HUMCSF1PO, HUMFIBRA, HUMTH01, HUMTPOX, and HUMvWFA31.
[0027] In yet a further aspect, the invention is primer sequences
and primer pairs for amplifying specific loci of human DNA. Use of
the primers and primer pairs of this invention for multiplex
analysis of human DNA is demonstrated herein, below. The primers of
this invention are suitable for use in the method of this
invention, wherein they can be used in labeled form, as noted
below, to assist the evaluation step of the method.
[0028] The approaches specified in the present invention produce
savings of time, labor, and materials in the analysis of loci
contained within the multiplexes. The method of the present
invention allows thirteen or more, even as many as sixteen or more,
loci to be co-amplified in one tube using a single amplification
reaction, instead of amplifying each locus independently in
separate tubes or in smaller groups of loci.
[0029] 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. By allowing thirteen methods of the present
invention significantly increase the certainty with which one can
match DNA prepared from different samples from the same individual.
The need to match or distinguish accurately between samples
containing very small amounts of DNA is particularly acute in
forensics applications, where many convictions (and acquittals)
turn on DNA typing analysis.
[0030] Scientists, particularly forensic scientists, have long
appreciated the need to analyze multiple polymorphic loci of DNA in
order to ensure that a match between two samples of DNA is
statistically significant. (Presley, L. A. et al., in The Third
International Symposium on Human Identification 1992, pp. 245-269
(pub. by Promega Corp., 1993); Bever, R. A., et al., in The Second
International Symposium on Human Identification 1991, pp. 103-128.
(pub. by Promega Corp., 1992)) However, until this invention, one
could not simultaneously analyze thirteen or more STR loci in a
single reaction. To realize the importance of such multiplexing
capabilities, it helps to understand some of the mathematics behind
DNA typing analysis.
[0031] For purposes of illustration, suppose every STR locus has a
genotype (i.e., pattern of two alleles) frequency of one in ten. In
other words, suppose that the chance of two randomly selected
individuals have a matching type for a single STR is {fraction
(1/10)}. However, if two different STR loci are analyzed, the
chance of a random match with both systems is {fraction (1/100)}.
If three STR loci are analyzed, the chances of a random match with
each of the three systems is {fraction (1/1,000)} and so on.
Consequently, it is easy to see how increasing the number of STR
loci analyzed reduces the likelihood of random matches within the
general population, thereby increasing the chance one can
accurately identify a suspect's presence at a crime scene by
comparing the individual's type with crime scene evidence. Similar
reasoning can be used to conclude that the method of this invention
also would increase the likelihood of accurately identifying a
suspected father in a paternity case, of correctly matching bone
marrow tissue, of developing significant results from linkage
mapping studies, and of detecting genetic diseases and cancers.
[0032] Further objects, features, and advantages of the invention
will be apparent from the following best mode for carrying out the
invention and the illustrative drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0033] FIG. 1A is a plot of the output of three-color fluorescent
detection of the products of simultaneous amplification of the loci
D3S1358, D5S818, D7S820, D8S1179, D 13S317, D16S539, D18S51,
D21S11, HUMCSF1 PO, HUMFIBRA, HUMTH01, HUMTPOX, and HUMvWFA31 of a
sample of human genomic DNA, as detected with the ABI PRISM.RTM.
310 Genetic Analyzer in Example 1.
[0034] FIG. 1B is a plot of the output of three-color fluorescent
detection of a control sample processed the same way as FIG. 1A,
with no genomic DNA in the amplification reaction.
[0035] FIG. 2A is a plot of the output of three-color fluorescent
detection of the products of simultaneous amplification of the loci
D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21
S11, HUMCSF1 PO, HUMFIBRA, HUMTH01, HUMTPOX, HUMvWFA31, G475, S159,
and Amelogenin of a sample of human genomic DNA, as detected with
the ABI PRISM.RTM. 310 Genetic Analyzer in Example 2.
[0036] FIG. 2B is a plot of the output of three-color fluorescent
detection of a control sample processed the same way as FIG. 2A,
with no genomic DNA substrate in the amplification reaction.
[0037] FIG. 3A is a plot of the output of three-color fluorescent
detection of the products of simultaneous amplification of the loci
D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11,
HUMCSF1 PO, HUMFIBRA, HUMTH01, HUMTPOX, HUMvWFA31, G475, S159, and
Amelogenin of a sample of human genomic DNA, as detected with an
ABI PRISM.RTM. 377 DNA Sequencer in Example 3.
[0038] FIG. 3B is a plot of the output of three-color fluorescent
detection of a control sample processed the same way as FIG. 3A,
with no genomic DNA substrate in the amplification reaction.
[0039] FIGS. 4A and 4B are laser printed images of the results of
fluorescent detection of the products of simultaneous amplification
of the loci D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539,
D18S51, D21S11, HUMCSF1PO, HUMFIBRA, HUMTH01, HUMTPOX, HUMvWFA31,
G475, S159, and Amelogenin as detected using the fluorescein
channel (FIG. 4A) and carboxy-tetramethylrhodamine channel (FIG.
4B) of a Hitachi FMBIO.RTM. II Fluorescent Scanner, as described in
Example 4.
[0040] FIGS. 5A and 5B are laser printed images of the results of
fluorescent detection of the products of simultaneous amplification
of the loci D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539,
D18S51, D21S11, HUMCSF1PO, HUMFIBRA, HUMTH01, HUMTPOX, HUMvWFA31,
G475, S159, and Amelogenin as detected using the fluorescein
channel (FIG. 5A) and carboxy-tetramethylrhodamine channel (FIG.
5B) of a Hitachi FMBIO.RTM. II Fluorescent Scanner, as described in
Example 5.
[0041] FIG. 6A and 6B are laser printed images of the results of
fluorescent detection of the products of simultaneous amplification
of the loci D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539,
D18S51, D21S11, HUMCSF1PO, HUMFIBRA, HUMTH01, HUMTPOX, HUMvWFA31,
C221, S159, and Amelogenin as detected using the fluorescein
channel (FIG. 6A) and carboxy-tetramethylrhodamine channel (FIG.
6B) of a Hitachi FMBIO.RTM. II Fluorescent Scanner, as described in
Example 6.
DETAILED DESCRIPTION OF THE INVENTION
[0042] A. Definitions
[0043] The following definitions are intended to assist in
providing a clear and consistent understanding of the scope and
detail of the following terms, as used to describe and define the
present invention:
[0044] "Allelic ladder": a standard size marker consisting of
amplified alleles from the locus.
[0045] "Allele": a genetic variation associated with a segment of
DNA, i.e., one of two or more alternate forms of a DNA sequence
occupying the same locus.
[0046] "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).
[0047] "DNA polymorphism": the condition in which two or more
different nucleotide sequences in a DNA sequence coexist in the
same interbreeding population.
[0048] "Locus" or "genetic locus": a specific position on a
chromosome. Alleles of a locus are located at identical sites on
homologous chromosomes.
[0049] "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 DNA sequences under the conditions
used in the amplification method.
[0050] "Pentanucleotide tandem repeat": a subclass of the STR
polymorphisms defined below. Unless specified otherwise, the term
"pentanucleotide tandem repeat" encompasses perfect STRs wherein
the repeat unit is a five base sequence, and imperfect STRs wherein
at least one repeat unit is a five base repeat.
[0051] "Polymerase chain reaction" or "PCR": a technique in which
cycles of denaturation, annealing with primer, and extension with
DNA polymerase are used to amplify the number of copies of a target
DNA sequence by approximately 10.sup.6 times or more. 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.
[0052] "Polymorphic short tandem repeat loci": STR loci, defined
below, in which the number of repetitive sequence elements (and net
length of sequence) in a particular region of genomic DNA varies
from allele to allele, and from individual to individual.
[0053] "Polymorphism information content" or "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.
[0054] "Primer": a single-stranded oligonucleotide or DNA fragment
which hybridizes with a DNA strand of a locus in such a manner that
the 3' terminus of the primer may act as a site of polymerization
using a DNA polymerase enzyme.
[0055] "Primer pair": two primers including, primer 1 that
hybridizes to a single strand at one end of the DNA sequence to be
amplified and primer 2 that hybridizes with the other end on the
complementary strand of the DNA sequence to be amplified.
[0056] "Primer site": the area of the target DNA to which a primer
hybridizes.
[0057] "Short tandem repeat loci" or "STR loci": regions of genomic
DNA which contain short, repetitive sequence elements of 3 to 7
base pairs in length. The term STR also encompasses a region of
genomic DNA wherein more than a single three to seven base sequence
is repeated in tandem or with intervening bases, provided that at
least one of the sequences is repeated at least two times in
tandem. Each sequence repeated at least once within an STR is
referred to herein as a "repeat unit."
[0058] The sequences of the STR loci analyzed using the materials
and methods of the present invention can be divided into two
general categories, perfect and imperfect. The term "perfect" STR,
as used herein, refers to a region of double-stranded DNA
containing a single three to seven base repeat unit repeated in
tandem at least two times, e.g. (AAAAT).sub.2. The term "imperfect"
STR, as used herein, refers to a region of DNA containing at least
two tandem repeats of a perfect repeat unit and at least one repeat
of an imperfect repeat unit, wherein the imperfect repeat unit
consists of a DNA sequence which could result from one, two, three,
or four base insertions, deletions, or substitutions in the
sequence of the perfect repeat unit, e.g.
(AAAAT).sub.12(AAAAAT).sub.- 5AAT(AAATT).sub.4. Every imperfect STR
sequence contains at least one perfect STR sequence. Specifically,
every STR sequence, whether perfect or imperfect, includes at least
one repeat unit sequence appearing at least two times in tandem, a
repeat unit sequence which can be represented by formula (I):
(A.sub.wG.sub.xT.sub.yC.sub.z) (I)
[0059] wherein A, G, T, and C represent the nucleotides which can
be in any order; w, x, y and z represent the number of each
nucleotide in the sequence and range from 0 to 7 with the sum of
w+x+y+z ranging between 3 and 7; and n represents the number of
times the sequence is tandemly repeated and is at least 2.
[0060] B. Selection of Multiplex Reaction Components
[0061] The method of the present invention contemplates selecting
an appropriate set of loci, primers, and amplification protocols to
generate amplified alleles from multiple co-amplified loci which
preferably do not overlap in size or, more preferably, which are
labeled in a way which enables one to differentiate between the
alleles from different loci which overlap in size. In addition,
this method contemplates the selection of short tandem repeat loci
which are 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 the above two 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.
[0062] Successful combinations in addition to those disclosed
herein can be generated by trial and error of locus combinations,
by selection of primer pair sequences, and by adjustment of primer
concentrations to identify an equilibrium in which all included
loci may be amplified. Once the method and materials of this
invention are disclosed, various methods of selecting loci, primer
pairs, and amplification techniques for use in the method and kit
of this invention are likely to be suggested to one skilled in the
art. All such methods are intended to be within the scope of the
appended claims.
[0063] Of particular importance in the practice of the method of
this invention is the size range of amplified alleles produced from
the individual loci which are co-amplified in the multiplex
amplification reaction step. For ease of analysis with current
technologies, systems which can be detected by amplification of
fragments smaller than 500 bases are most preferable.
[0064] Practice of the method of the present invention begins with
selection of a set of loci comprising at least thirteen STR loci,
which can be co-amplified in a single multiplex amplification
reaction. Selection of loci and oligonucleotide primers used to
amplify the loci in the multiplex amplification reaction of the
present method is described herein below, and illustrated in the
Examples below.
[0065] C. Use of Multiplexes of Three Loci to Develop Multiplexes
Using More than Three Loci
[0066] Any one of a number of different techniques can be used to
select a set of loci for use in the present invention. One
preferred technique for developing useful sets of loci for use in
this method of analysis is described below. Once a multiplex
containing three STR loci is developed, it may be used as a core to
create multiplexes containing more than three loci. New
combinations of more than three loci can, thus, be created which
include the first three loci. For example, the core multiplex
containing loci D7S820, D13S317, and D5S818 was used to generate
derivative multiplexes of:
[0067] D16S539, D7S820, D13S317, and D5S818;
[0068] HUMCSF1PO, HUMTPOX, D16S539, D7S820, D13S317, and
D5S818;
[0069] HUMCSF1PO, HUMTPOX, HUMTH01, D16S539, D7S820, D13S317, and
D5S818;
[0070] HUMCSF1PO, HUMTPOX, HUMTH01, HUMvWFA31, D16S539, D7S820,
D13S317, and D5S818;
[0071] D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51,
D21S11, HUMCSF1PO, HUMFIBRA, HUMTH01, HUMTPOX, and HUMvWA31;
[0072] S159, HUMCSF1PO, D16S539, D7S820, D13S317, and D5S818;
[0073] D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51,
D21S11, HUMCSF1PO, HUMFIBRA, HUMTH01, HUMTPOX, and HUMvWFA31;
and
[0074] D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51,
D21S11, HUMCSF1PO, HUMFIBRA, HUMTH01, HUMTPOX, HUMvWFA31, G475,
S159, and Amelogenin.
[0075] It is contemplated that core sets of loci can be used to
generate other appropriate derivative sets of STR loci for
multiplex analysis using the method of this invention. Regardless
of what method is used to select the loci analyzed using the method
of the present invention, all the loci selected for multiplex
analysis share the following characteristics: (1) they produce
sufficient amplification product to allow evaluation; (2) they
generate few if any artifacts due to the addition (or lack of
addition) of a base to the amplified alleles during the multiplex
amplification step; (3) they generate few, if any, artifacts due to
premature termination of amplification reactions by a polymerase;
and (4) they produce little or no "trailing" bands of smaller
molecular weight from consecutive single base deletions below a
given authentic amplified allele. See, e.g., Schumm et al., Fourth
International Symposium on Human Identification 1993, pp. 177-187
(pub. by Promega Corp., 1994).
[0076] The same technique used to identify the set of at least
three loci, described above, can be applied to select thirteen or
more loci of human genomic DNA or multiplex analysis, according to
a preferred embodiment of the method of analysis of the present
invention. Any set of loci identified as described above is
suitable for multiplex analysis in accordance with the present
invention, provided the set of loci comprises at least thirteen STR
loci. More preferably, at least four of the at least thirteen STR
loci analyzed according to the present invention are selected from
the group of loci consisting of:
[0077] D3S1539, D4S2368, D5S818, D7S820, D9S930, D10S1239, D13S317,
D14S118, D14S548, D14S562, D16S490, D16S539, D16S753, D17S1298,
D17S1299, D19S253, D20S481, D22S683, HUMCSF1PO, HUMTPOX, HUMTH01,
HUMF13A01, HUMBFXIII, HUMLIPOL, and HUMvWFA31
[0078] Even more preferably, the set of loci analyzed according to
the present invention includes all thirteen CODIS loci, i.e.
D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11,
HUMCSF1PO, HUMFIBRA, HUMTH01, HUMTPOX, and HUMvWFA31.
[0079] At least one of the loci selected for co-amplification in
the present multiplex reaction is preferably an STR focus with a
repeat unit of five to seven bases or base pairs in length, more
preferably an STR locus with a pentanucleotide repeat. As is
demonstrated in U.S. patent application Ser. No. 09/018,584, which
is incorporated by reference herein, loci with such intermediate
length repeats can be amplified with minimal incidence of
artifacts, e.g. due to repeat slippage. Three such loci with
pentanucleotide repeats, G475, C221 and S159, are included in the
sets of loci identified immediately above. The terms "G475",
"C221", and "S159", as used herein, refer to names assigned to
pentanucleotide repeat loci identified, as described in U.S. patent
application Ser. No. 09/018,584, incorporated by reference above.
Each name corresponds to a clone from which each pentanucleotide
locus was identified. The sequence of the G475 clone, described
therein as SEQ ID NO:34, is identified herein as SEQ ID NO:108. The
sequence of the C221 clone, described therein as SEQ ID NO:2, is
identified herein as SEQ ID NO:109. The sequence of the S159 clone,
described therein as SEQ ID NO: 26, is identified herein as SEQ ID
NO:110. Individual primers and primer pairs identified for use in
amplifying G475, C221, and S159 therein can also be used to amplify
the same loci in the sets of at least thirteen loci co-amplified
and analyzed according to the present invention.
[0080] The set of loci selected for co-amplification and analysis
according to the invention preferably further comprises at least
one locus in addition to the at least thirteen STR loci. The
additional locus preferably includes a sequence polymorphism, or
another feature which identifies a particular characteristic which
separates the DNA of an individual from the DNA of other
individuals in the population. The additional locus more preferably
is a locus which identifies the gender of the source of the DNA
sample analyzed. When the DNA sample is human genomic DNA, a gender
identifying locus such as the Amelogenin locus is preferably
selected for co-amplification and analysis according to the present
method. The Amelogenin locus is identified by GenBank as HUMAMELY
(when used to identify a locus on the Y chromosome contained in
male DNA) or as HUMAMELX (when used to identify a locus on the X
chromosome in male or female DNA). When the Amelogenin locus is
co-amplified in the same multiplex amplification reaction as the
set of at least thirteen short tandem repeat loci, the sequence of
at least one of the primers used to amplify this particular locus
in the multiplex amplification reaction preferably has a sequence
selected from: SEQ ID NO:86, SEQ ID NO:105, and SEQ ID NO:87.
[0081] D. Selection of Primers
[0082] Once a set of loci for co-amplification in a single
multiplex reaction is identified, one can determine primers
suitable for co-amplifying each locus in the set. Care should be
used in selecting the sequence of primers used in the multiplex
reaction. 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 the need for
amplification conditions or protocols for the different loci which
are incompatible in a multiplex. Primers used in the present method
or included in the present kits of the invention are preferably
selected according to the following selection process.
[0083] Primers are preferably developed and selected for use in the
multiplex systems of the invention by employing a re-iterative
process of selecting primer sequences, mixing the primers for
co-amplification of the selected loci, co-amplifying the loci, then
separating and detecting the amplified products. Initially, this
process often produces the amplified alleles in an imbalanced
fashion (i.e., higher product yield for some loci than for others)
and may also generate amplification products which do not represent
the alleles themselves. These extra fragments may result from any
number of causes described above.
[0084] To eliminate such extra fragments from the multiplex
systems, individual primers from the total set are used with
primers from the same or other loci to identify which primers
contribute to the amplification of the extra fragments. Once two
primers which generate one or more of the fragments are identified,
one or both contributors are modified and retested, either in a
pair alone or in the multiplex system (or a subset of the multiplex
system). This process is repeated until evaluation of the products
yields amplified alleles with no or an acceptable level of extra
fragments in the multiplex system.
[0085] On occasion, extra fragments can be eliminated by labeling
the opposite primer in a primer pair. This change reveals the
products of the opposing primer in the detection step. This newly
labeled primer may amplify the true alleles with greater fidelity
than the previously labeled primer generating the true alleles as a
greater proportion of the total amplification product.
[0086] The determination of primer concentration may be performed
either before or after selection of the final primer sequences, but
is preferably performed after that selection. Generally, increasing
primer concentration for any particular locus increases the amount
of product generated for that locus. However, this is also a
re-iterative process because increasing yield for one locus may
decrease it for one or more other loci. Furthermore, primers may
interact directly affecting yield of the other loci. Linear
increases in primer concentration do not necessarily produce linear
increases in product yield for the corresponding locus.
[0087] Locus to locus balance is also affected by a number of
parameters of the amplification protocol such as the amount of
template used, the number of cycles of amplification, the annealing
temperature of the thermal cycling protocol, and the inclusion or
exclusion of an extra extension step at the end of the cycling
process. Absolutely even balance across all alleles and loci is
generally not achieved.
[0088] The process of multiplex system development may also be a
re-iterative process in another sense described, above. That is, it
is possible, first, to develop a multiplex system for a small
number of loci, this system being free or nearly free of extra
fragments from amplification. Primers of this system may be
combined with primers for one or more additional loci. This
expanded primer combination may or may not produce extra fragments
from amplification. In turn, new primers may be introduced and
evaluated.
[0089] One or more of the re-iterative selection processes
described above are repeated until a complete set of primers is
identified which can be used to co-amplify the at least thirteen
loci selected for co-amplification as described above. It is
understood that many different sets of primers may be developed to
amplify a particular set of loci.
[0090] Synthesis of the primers used in the present method can be
conducted using any standard procedure for oligonucleotide
synthesis known to those skilled in the art. At least one primer
for each locus is preferably covalently attached to a dye label, as
described in Section F, below.
[0091] Table 1, below, provides a list sequences of primers which
have been determined to be suitable for use in amplifying the
corresponding polymorphic STR loci listed therein. At least one
primer listed in Table 1 is preferably used to amplify at least one
of the loci selected for co-amplification and analysis as described
above. It is understood that other primers could be identified
which are suitable for simultaneous amplification of the loci
listed below.
1 TABLE 1 Locus Primer SEQ ID NO: 's D7S820 1, 2, 80 and 81 D13S317
3, 4, 82 and 83 D5S818 5, 6, 84 and 85 D3S1539 7, 8 and 49 D17S1298
9 and 10 D20S481 11, 12, 52 and 53 D9S930 13, 14, 55 and 61
D10S1239 15, 16 and 54 D14S118 17 and 18 D14S562 19 and 20 D14S548
21 and 22 D16S490 23 and 24 D16S753 25 and 26 D17S1299 27 and 28
D16S539 29, 30, 58, 79 and 97 D22S683 31 and 32 HUMCSF1PO 33, 34,
77, 78 and 98 HUMTPOX 35, 36, 72 and 73 HUMTH01 37, 38, 66, 67 and
103 HUMvWFA31 39, 40, 59, 60 and 76 HUMF13A01 41 and 42 HUMFESFPS
43 and 44 HUMBFXIII 45 and 46 HUMLIPOL 47 and 48 D19S253 50 and 51
D4S2368 56 and 57 D18S51 62, 63, 101 and 102 D21S11 64 and 65
D3S1538 68, 69 and 106 HUMFIBRA 70, 71 and 107 D8S1179 74, 75 and
104 G475 88, 89 and 94 S159 90, 91, 92, 93, 95 and 96 C221 99 and
100
[0092] E. Preparation of DNA Samples
[0093] Samples of genomic DNA can be prepared for use in the method
of this invention using any method of DNA preparation which is
compatible with the amplification of DNA. Many such methods are
known by those skilled in the art. Examples include, but are not
limited to DNA purification by phenol extraction (Sambrook, J., et
al. (1989) Molecular Cloning: A Laboratory Manual, Second Edition,
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp.
9.14-9.19), and partial purification by salt precipitation (Miller,
S. et al. (1988) Nucl. Acids Res. 16:1215) or chelex (Walsh et al.,
(1991) BioTechniques 10:506-513, Comey, et al., (1994) Forensic
Sci. 39:1 254) and the release of unpurified material using
untreated blood (Burckhardt, J. (1994) PCR Methods and Applications
3:239-243, McCabe, Edward R. B.,(1991) PCR Methods and Applications
1:99-106, Nordv.ang.g, Bj.o slashed.rn-Yngvar (1992) BioTechniques
12:4 pp. 490-492).
[0094] When the at least one DNA sample to be analyzed using the
method of this invention is human genomic DNA, the DNA is
preferably prepared from tissue, selected from the group consisting
of blood, semen, vaginal cells, hair, saliva, urine, bone, buccal
samples, amniotic fluid containing placental cells or fetal cells,
chorionic villus, and mixtures of any of the tissues listed
above.
[0095] Optionally, DNA concentrations can be measured prior to use
in the method of the present invention, using any standard method
of DNA quantification known to those skilled in the art. In such
cases, the DNA concentration is preferably determined by
spectrophotometric measurement as described by Sambrook, J., et al.
(1989), supra, Appendix E.5, or fluorometrically using a
measurement technique such as that described by Brunk C. F., et al.
(1979), Anal Biochem 92: 497-500. The DNA concentration is more
preferably measured by comparison of the amount of hybridization of
DNA standards with a human-specific probe such as that described by
Waye, J. S., et al. (1991) "Sensitive and specific quantification
of human genomic deoxyribonucleic acid (DNA) in forensic science
specimens: casework examples," J. Forensic Sci., 36:1198-1203. Use
of too much template DNA in the amplification reactions can produce
artifacts which appear as extra bands which do not represent true
alleles.
[0096] F. Amplification of DNA
[0097] Once a sample of genomic DNA is prepared, the targeted loci
can be co-amplified in the multiplex amplification step of the
present method. Any one of a number of different amplification
methods can be used to amplify the loci, including, but not limited
to, polymerase chain reaction (PCR) (Saiki, R. K., et al. (1985),
Science 230: 1350-1354), transcription based amplification (Kwoh,
D. Y., and Kwoh, T. J. (1990), American Biotechnology Laboratory,
October, 1990) and strand displacement amplification (SDA) (Walker,
G. T., et al. (1992) Proc. Natl. Acad. Sci., U.S.A. 89: 392-396).
Preferably, the DNA sample is subjected to PCR amplification using
primer pairs specific to each locus in the set. Reference is made
to the Sequence Listing at the end of this specification for
details of the primer sequences used in the Examples below, some of
which sequences are alternative embodiments of this invention.
[0098] At least one primer for each locus is preferably covalently
attached to a dye label, more preferably a fluorescent dye label.
The primers and dyes attached thereto are preferably selected for
the multiplex amplification reaction, such that alleles amplified
using primers for each locus labeled with one color do not overlap
the alleles of the other loci in the set co-amplified therein using
primers labeled with the same color, when the alleles are
separated, preferably, by gel or capillary electrophoresis.
[0099] In a particularly preferred embodiment of the method of the
present invention, at least one primer for each locus co-amplified
in the multiplex reaction is labeled with a fluorescent label prior
to use in the reaction. Fluorescent labels suitable for attachment
to primers for use in the present invention are commercially
available. See, e.g. fluorescein and carboxy-tetramethylrhodamine
labels and their chemical derivatives from PE Biosystems and
Molecular Probes. Most preferably, at least three different labels
are used to label the different primers used in the multiplex
amplification reaction. When a size marker is included to evaluate
the multiplex reaction, the primers used to prepare the size marker
are preferably labeled with a different label from the primers used
to amplify the loci of interest in the reaction.
[0100] Details of the most preferred amplification protocol for
each of the most preferred combinations of loci for use in the
method of this invention are given in the Examples below. Reference
is also made to the Examples for additional details of the specific
procedure relating to each multiplex. The sequences of the
locus-specific primers used in the Examples 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, the teaching of which is incorporated herein by reference
for a more detailed description of locus-specific primers.
[0101] G. Separation and Detection of DNA Fragments
[0102] Once a set of amplified alleles is produced from the
multiplex amplification step of the present method, the amplified
alleles are evaluated. The evaluation step of this method can be
accomplished by any one of a number of different means, the most
preferred of which are described below.
[0103] Electrophoresis is preferably used to separate the products
of the multiplex amplification reaction, more preferably capillary
electrophoresis (see, e.g., Buel, Eric et al. (1998), Journal of
Forensic Sciences; 43:(1) pp. 164-170) or denaturing polyacrylamide
gel electrophoresis (see, e.g., Sambrook, J. et al. (1989) In
Molecular Cloning--A Laboratory Manual, 2nd edition, Cold Spring
Harbor Laboratory Press, pp. 13.45-13.57). Gel preparation and
electrophoresis procedures and conditions for suitable for use in
the evaluating step of the method of this invention are illustrated
in the Examples, below. Separation of DNA fragments in a denaturing
polyacrylamide gel and in capillary electrophoresis occurs based
primarily on fragment size.
[0104] Once the amplified alleles are separated, the alleles and
any other DNA in the gel or capillary (e.g., DNA size markers or an
allelic ladder) can then be visualized and analyzed. Visualization
of the DNA in the gel can be accomplished using any one of a number
of prior art techniques, including silver staining or reporters
such as radioisotopes, fluorescers, chemiluminescers and enzymes in
combination with detectable substrates. However, the preferred
method for detection of multiplexes containing thirteen or more
loci is fluorescence (see, e.g., Schumm, J. W. et al. in
Proceedings from the Eighth International Symposium on Human
Identification, (pub. 1998 by Promega Corporation), pp. 78-84;
Buel, Eric et al. (1998), supra.), wherein primers for each locus
in the multiplexing reaction is followed by detection of the
labeled products employing a fluorometric detector. The references
cited above, which describe prior art methods of visualizing
alleles, are incorporated by reference herein.
[0105] The alleles present in the DNA sample are preferably
determined by comparison to a size standard such as a DNA marker or
a locus-specific allelic ladder to determine the alleles present at
each locus within the sample. The most preferred size marker for
evaluation of a multiplex amplification containing two or more
polymorphic STR loci consists of a combination of allelic ladders
for each of the loci being evaluated. See, e.g., Puers, Christoph
et al., (1993) Am J. Hum Genet. 53:953-958, Puers, Christoph, et
al. (1994) Genomics 23:260-264. See also, U.S. Pat. Nos. 5,599,666;
5,674,686; and 5,783,406 for descriptions of allelic ladders
suitable for use in the detection of STR loci, and methods of
ladder construction disclosed therein.
[0106] Following the construction of allelic ladders for individual
loci, these 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.
[0107] The products of the multiplex reactions of the present
invention can be evaluated using an internal lane standard, a
specialized type of size marker configured to run in the same lane
of a polyacrylamide gel or same capillary. The internal lane
standard preferably consists of a series of fragments of known
length. The internal lane standard more preferably is labeled with
a fluorescent dye which is distinguishable from other dyes in the
amplification reaction.
[0108] Following construction of the internal lane standard, this
standard can also be mixed with amplified sample or allelic ladders
and loaded for electrophoresis for comparison of migration in
different lanes of gel electrophoresis or different capillaries of
capillary electrophoresis. Variation in the migration of the
internal lane standard indicates variation in the performance of
the separation medium. Quantitation of this difference and
correlation with the allelic ladders allows correction in the size
determination of alleles in unknown samples.
[0109] H. Preferred Detection Technique: Fluorescent Detection
[0110] In one of the most preferred embodiments of the method of
this invention, fluorescent detection is used to evaluate the
amplified alleles in the mixture produced by the multiplex
amplification reaction. Below is a brief summary of how that method
of detection preferably is practiced.
[0111] With the advent of automated fluorescent imaging, faster
detection and analysis of multiplex amplification products can be
achieved. For fluorescent analysis, one fluorescent labeled primer
can be included in the amplification of each locus. Fluorescent
labeled primers preferably suited for use in the present invention
include the fluorescein-labeled (FL-),
carboxy-tetramethylrhodamine-labeled (TMR-), and
5,6-carboxyrhodamine 6G-labeled (R6G) primers, such as are
illustrated in the Examples, below. Separation of the amplified
fragments produced using such labeled primers is achieved
preferably by slab gel electrophoresis or capillary
electrophoresis. The resulting separated fragments can be analyzed
using fluorescence detection equipment such as an ABI PRISM.RTM.
310 Genetic Analyzer, an ABI PRISM.RTM. 377 DNA Sequencer (Applied
Biosystems Division, Perkin Elmer, Foster City, Calif.), or a
Hitachi FMBIO.RTM. II Fluorescent Scanner (Hitachi Software
Engineering America, Ltd. South San Francisco, Calif.).
[0112] In summary, the method of this invention is most preferably
practiced using fluorescent detection as the detection step. In
this preferred method of detection, one or both of each pair of
primers used in the multiplex amplification reaction has a
fluorescent label attached thereto, and as a result, the amplified
alleles produced from the amplification reaction are fluorescently
labeled. In this most preferred embodiment of the invention, the
amplified alleles are subsequently separated by capillary
electrophoresis and the separated alleles visualized and analyzed
using a fluorescent image analyzer.
[0113] Fluorescent detection is preferred over radioactive methods
of labeling and detection, because it does not require the use of
radioactive materials, and all the regulatory and safety problems
which accompany the use of such materials.
[0114] Fluorescent detection employing labeled primers is also
preferred over other non-radioactive methods of detection, such as
silver staining, because fluorescent methods of detection generally
reveal fewer amplification artifacts than silver staining. The
smaller number of artifacts are due, in part, to the fact that only
amplified strands of DNA with labels attached are detected in
fluorescent detection, while both strands of every amplified allele
of DNA produced from the multiplex amplification reaction is
stained and detected using the silver staining method of
detection.
[0115] I. Kit
[0116] The present invention is also directed to kits that utilize
the process described above. A basic kit comprises a container
having one or more locus-specific primers. Instructions for use
optionally may be included.
[0117] Other optional kit components 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 electrophoresis, genomic DNA as a template control, a
size marker to insure that materials migrate as anticipated in the
separation medium, and a protocol and manual to educate the user
and to limit error in use. The amounts of the various reagents in
the kits also can be varied depending upon a number of factors,
such as the optimum sensitivity of the process. 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
[0118] 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 intended to be
illustrative, and are not intended in any way to otherwise limit
the scope of the claims or protection granted by the patent.
[0119] The human genomic DNA samples assayed in the Example below
were prepared from blood or tissue culture cells, using a standard
procedure described by Miller and Dykes in (Miller, S. et al.
(1988) Nucl. Acids Res. 16:1215). The isolation and quantification
methods described therein are generally known to those skilled in
the art and are preferred, but not required, for application of the
invention.
[0120] Each Example below is an example of the use of the method of
this invention, to determine simultaneously the alleles present in
at least thirteen loci from one or more DNA samples of human
genomic DNA. Each set of loci co-amplified below includes the
thirteen short tandem repeat loci identified for use in the CODIS
system (i.e., D3S1358, HUMTHO1, D21S11, D18S51, HUMvWFA31, D8S1179,
HUMTPOX, HUMFIBRA, D5S818, D13S317, D7S820, D16S539, and
HUMCSF1PO). Some sets of loci co-amplified below also include one
or more additional short tandem repeat loci, such as loci with
pentanucleotide repeats (e.g., G475, S159, or C221), and a non-STR
locus, Amelogenin.
[0121] Table 2 summarizes which set of loci was co-amplified in the
multiplex amplification reaction described in each Example below.
The table also indicates which primer pair was used to amplify each
such locus in each such multiplex reaction. One primer of each
primer pair listed on Table 2 was fluorescently labeled prior to
being used in the multiplex amplification reaction. In some cases,
a different label was used to label primers to different loci, such
that the alleles produced using the different primers could be
distinguished from one another when detected with a laser-activated
fluorescence detection device.
[0122] Three different fluorescent labels were used in the Examples
below, described as "FL" to indicate fluorescein-labeled, "TMR" to
indicate carboxy-tetramethylrhodamine-labeled, and "R6G" to
indicate 5,6-carboxyrhodamine 6G in Table 2, below. Table 2 also
indicates which primer of each pair of primers used in the
multiplex amplification reaction was so labeled in each Example
(e.g., "FL-69" means the primer with SEQ ID NO:69 was labeled at
its 5' end with fluorescein prior to being used in the multiplex
amplification reaction). In the text of each of the Examples,
however, the label abbreviation is placed immediately before the
SEQ ID NO of the labeled primer used in the amplification reaction
described therein (e.g., "FL-SEQ ID NO:2" instead of "FL-2").
2TABLE 2 Primer Pair: SEQ ID Fluorescent Example Loci Amplified
NO's Used Label(s) Used 1 D3S1358 68, 69 FL-69 HUMTHO1 66, 67 FL-66
D21S11 64, 65 FL-65 D18S51 62, 63 FL-62 HUMvWFA31 76, 40 TMR-40
D8S1179 74, 75 TMR-75 HUMTPOX 72, 73 TMR-73 HUMFIBRA 70, 71 TMR-70
D5S818 84, 85 R6G-85 D13S317 82, 83 R6G-83 D7S820 80, 81 R6G-80
D16S539 29, 79 R6G-79 HUMCSF1PO 77, 78 R6G-78 2, 3 D3S1358 68, 69
FL-69 HUMTHO1 66, 67 FL-66 D21S11 64, 65 FL-65 D18S51 62, 63 FL-62
G475 88, 89 FL-88 Amelogenin 86, 87 TMR-86 HUMvWFA31 76, 40 TMR-40
D8S1179 74, 75 TMR-75 HUMTPOX 72, 73 TMR-73 HUMFIBRA 70, 71 TMR-70
D5S818 84, 85 R6G-85 D13S317 82, 83 R6G-83 D7S820 80, 81 R6G-80
D16S539 29, 79 R6G-79 HUMCSF1PO 77, 78 R6G-78 S159 90, 91 R6G-91 4
D3S1358 68, 69 FL-69 HUMTHO1 66, 67 FL-66 D21S11 64, 65 FL-65
D18S51 62, 63 FL-62 G475 88, 89 FL-88 Amelogenin 86, 87 TMR-86
HUMvWFA31 76, 40 TMR-40 D8S1179 74, 75 TMR-75 HUMTPOX 72, 73 TMR-73
HUMFIBRA 70, 71 TMR-70 D5S818 84, 85 FL-85 D13S317 82, 83 FL-83
D7S820 80, 81 FL-80 D16S539 29, 79 FL-79 HUMCSF1PO 77, 78 FL-78
S159 90, 91 FL-91 5 D3S1358 68, 69 FL-69 HUMTHO1 66, 67 FL-66
D21S11 64, 65 FL-65 D18S51 62, 63 FL-62 G475 88, 94 FL-94
Amelogenin 86, 87 TMR-86 HUMvWFA31 76, 40 TMR-40 D8S1179 74, 75
TMR-75 HUMTPOX 72, 73 TMR-73 HUMFIBRA 70, 71 TMR-70 D5S818 84, 85
FL-85 D13S317 82, 83 FL-83 D7S820 80, 81 FL-80 D16S539 29, 79 FL-79
HUMCSF1PO 77, 78 FL-78 S159 95, 96 FL-96 6 D3S1358 69, 106 FL-69
HUMTHO1 38, 103 FL-38 D21S11 64, 65 FL-65 D18S51 101, 102 FL-101
S159 92, 93 FL-93 Amelogenin 105, 87 TMR-105 HUMvWFA31 76, 40
TMR-40 D8S1179 104, 75 TMR-104 HUMTPOX 72, 73 TMR-72 HUMFIBRA 70,
107 TMR-70 D5S818 84, 85 FL-85 D13S317 3, 4 FL-4 D7S820 80, 81
FL-80 D16S539 29, 97 FL-29 HUMCSF1PO 77, 98 FL-98 C221 99, 100
FL-99
Example 1
Fluorescent Detection of Multiplex Amplification of Loci D3S1358,
HUMTH01, D21S11, D18S51, HUMvWFA31, D8S1179, HUMTPOX, HUMFIBRA,
D5S818, D7S820, D13S317, D16S539, and HUMCSF1PO as Detected with
the ABI PRISM.RTM. 310 Genetic Analyzer
[0123] In this Example, a DNA template was amplified simultaneously
at the individual loci D3S1358, HUMTH01, D21S11, D18S51, HUMvWFA31,
D8S1179, HUMTPOX, HUMFIBRA, D5S818, D7S820, D13S317, D16S539, and
HUMCSF1PO in a single reaction vessel. The PCR amplification was
performed in 25 .mu.l of 1.times. Gold ST*R Buffer (50 mM KCl, 10
mM Tris-HCl (pH 8.3 at 25.degree. C.), 0.1% Triton X-100, 1.5 mM
MgCl.sub.2, 160 .mu.g/ml BSA and 200 .mu.M each of dATP, dCTP, dGTP
and dTTP) using 1 ng template, and 3.25 U AmpliTaq Gold.TM. DNA
Polymerase. A GeneAmp.RTM. PCR System 9600 (Perkin Elmer, Foster
City, Calif.) was employed with the following amplification
protocol: 96.degree. C. for 12 min., then 10 cycles of 94.degree.
C. for 30 sec., ramp for 68 sec. to 58.degree. C., hold for 30
sec., ramp 50 sec. to 70.degree. C., hold for 45 sec., followed by
20 cycles of 90.degree. C. for 30 sec., ramp 60 sec. to 58.degree.
C., hold for 30 sec., ramp for 50 sec, to 70.degree. C., hold for
45 sec., followed by 1 cycle of 60.degree. C. for 30 min.
[0124] Twenty-six amplification primers were used in combination,
including 0.12 .mu.M each D3S1358 primers 1 [SEQ ID NO:68] and 2
[FL-SEQ ID NO:69], 0.08 .mu.M each HUMTH01 primers 1 [FL-SEQ ID
NO:66] and 2 [SEQ ID NO:67], 0.3 .mu.M each D21S11 primers 1 [SEQ
ID NO:64] and 2 [FL-SEQ ID NO:65], 0.2 .mu.M each D18S51 primers 1
[FL-SEQ ID NO:62] and 2 [SEQ ID NO:63], 1.1 .mu.M each HUMvWFA31
primers 1 [SEQ ID NO:76] and 2 [TMR-SEQ ID NO:40], 1.8 .mu.M each
D8S1179 primers 1 [SEQ ID NO:74] and 2 [TMR-SEQ ID NO:75], 0.6
.mu.M each HUMTPOX primers 1 [SEQ ID NO:72] and 2 [TMR-SEQ ID
NO:73], 2.4 .mu.M each HUMFIBRA primers 1 [TMR-SEQ ID NO:70] and 2
[SEQ ID NO:71], 0.2 .mu.M each D5S818 primers 1 [SEQ ID NO:84] and
2 [R6G-SEQ ID NO:85], 0.1 .mu.M each D13S317 primers 1 [SEQ ID
NO:82] and 2 [R6G-SEQ ID NO:83], 0.2 .mu.M each D7S820 primers 1
[R6G-SEQ ID NO:80] and 2 [SEQ ID NO:81], 0.15 .mu.M each D16S539
primers 1 [SEQ ID NO:29] and 2 [R6G-SEQ ID NO:79], 0.2 .mu.M each
HUMCSF1PO primers 1 [SEQ ID NO:77] and 2 [R6G-SEQ ID NO:78]
[0125] Amplified products were separated using an ABI PRISM.RTM.
310 Genetic Analyzer. DNA samples were mixed with 24 .mu.l of a
loading solution (deionized formamide) and 1.0 .mu.l of an internal
lane size standard, denatured at 95.degree. C. for 3 min., and
chilled on ice prior to injection. Separation was carried out using
Performance Optimized Polymer 4 (POP-4)(Perkin Elmer Biosystems,
Foster City, Calif.) in a 47 cm.times.50 .mu.m capillary. The
manufacturer's GeneScan.RTM. run module GS STR POP4 (Id.) (1 ml) A
was used. Conditions for the electrophoresis were a 5 second
injection, injection kV was 15.0, run kV was 15.0, run temperature
was 60.degree. C., run time was 28 minutes and virtual filter A was
used.
[0126] FIG. 1A is a printout of results of scanning the amplified
fragments of each locus separated and detected with the ABI
PRISM.RTM. 310 Genetic Analyzer, as described above. FIG. 1A shows
amplification products of a DNA sample simultaneously co-amplified
for the loci D3S1358, HUMTH01, D21S11, D18S51, HUMvWFA31, D8S1179,
HUMTPOX, HUMFIBRA, D5S818, D7S820, D13S317, D16S539, and HUMCSF1PO.
Peaks shown in Panel A are labeled with fluorescein, peaks shown in
Panel B are labeled with carboxy-tetramethylrhodamine, and peaks
shown in Panel C are labeled with 5,6 carboxyrhodamine 6G.
[0127] FIG. 1B is a printout of the results of scanning a sample
prepared in the same way as the sample scanned in FIG. 1A, except
that no DNA template was used in the amplification reaction. Peaks
in this figure are background products resulting from dye
conjugation and purification procedures and from undefined
causes.
Example 2
Fluorescent Detection of Multiplex Amplification of Loci D3S1358,
HUMTH01, D21 S11, D18S51, G475, Amelogenin, HUMvWFA31, D8S1179,
HUMTPOX, HUMFIBRA, D5S818, D7S820, D13S317, D16S539, HUMCSF1PO, and
S159 as Detected with the ABI PRISM.RTM. 310 Genetic Analyzer
[0128] In this Example, a DNA template was amplified simultaneously
at the individual loci D3S1358, HUMTH01, D21S11, D18S51, G475,
Amelogenin, HUMvWFA31, D8S1179, HUMTPOX, HUMFIBRA, D5S818, D7S820,
D13S317, D16S539, HUMCSF1PO and S159 in a single reaction vessel.
The PCR amplification was performed in 25 .mu.l of 1.times. Gold
ST*R Buffer (50 mM KCl, 10 mM Tris-HCl (pH 8.3 at 25.degree. C.),
0.1% Triton X-100, 1.5 mM MgCl.sub.2, 160 .mu.g/ml BSA and 200
.mu.M each of dATP, dCTP, dGTP and dTTP) using 1 ng template, and 4
U AmpliTaq Gold.TM. DNA Polymerase. A GeneAmp.RTM. PCR System 9600
(Perkin Elmer, Foster City, Calif.) was employed with the following
amplification protocol: 96.degree. C. for 12 min., then 10 cycles
of 94.degree. C. for 30 sec., ramp for 68 sec. to 58.degree. C.,
hold for 30 sec., ramp 50 sec. to 70.degree. C., hold for 45 sec.,
followed by 20 cycles of 90.degree. C. for 30 sec., ramp 60 sec. to
58.degree. C., hold for 30 sec., ramp for 50 sec, to 70.degree. C.,
hold for 45 sec., followed by 1 cycle of 60.degree. C. for 30
min.
[0129] Thirty-two amplification primers were used in combination,
including 0.12 .mu.M each D3S1358 primers 1 [SEQ ID NO:68] and 2
[FL-SEQ ID NO:69], 0.08 .mu.M each HUMTH01 primers 1 [FL-SEQ ID
NO:66] and 2 [SEQ ID NO:67], 0.3 .mu.M each D21S11 primers 1 [SEQ
ID NO:64] and 2 [FL-SEQ ID NO:65], 0.2 .mu.M each D18S51 primers 1
[FL-SEQ ID NO:62] and 2 [SEQ ID NO:63], 0.24 .mu.M each G475
primers 1 [FL-SEQ ID NO:88] and 2 [SEQ ID NO:89], 0.6 .mu.M each
Amelogenin primers 1 [TMR-SEQ ID NO:86] and 2 [SEQ ID NO:87], 1.1
.mu.M each HUMvWFA31 primers 1 [SEQ ID NO:76] and 2 [TMR-SEQ ID
NO:40], 1.8 .mu.M each D8S1179 primers 1 [SEQ ID NO:74] and 2
[TMR-SEQ ID NO:75], 0.6 .mu.M each HUMTPOX primers 1 [SEQ ID NO:72]
and 2 [TMR-SEQ ID NO:73], 2.4 .mu.M each HUMFIBRA primers 1
[TMR-SEQ ID NO:70] and 2 [SEQ ID NO:71], 0.2 .mu.M each D5S818
primers 1 (SEQ ID NO:84] and 2 [R6G-SEQ ID NO:85], 0.1 .mu.M each
D13S317 primers 1 [SEQ ID NO:82] and 2 [R6G-SEQ ID NO:83], 0.2
.mu.M each D7S820 primers 1 [R6G-SEQ ID NO:80] and 2 [SEQ ID
NO:81], 0.15 .mu.M each D16S539 primers 1 [SEQ ID NO:29] and 2
[R6G-SEQ ID NO:79], 0.2 .mu.M each HUMCSF1PO primers 1 [SEQ ID
NO:77] and 2 [R6G-SEQ ID NO:78] 0.1 .mu.M each S159 primers 1 [SEQ
ID NO:90] and 2 [R6G-SEQ ID NO:91]
[0130] Amplified products were separated using an ABI PRISM.RTM.
310 Genetic Analyzer. DNA samples were mixed with 24 .mu.l of a
loading solution (deionized formamide) and 1.0 .mu.l of an internal
lane size standard, denatured at 95.degree. C. for 3 min., and
chilled on ice prior to injection. Separation was carried out using
Performance Optimized Polymer 4 (POP-4) (Perkin Elmer Biosystems,
Foster City, Calif.) in a 47 cm.times.50 .mu.m capillary. The
manufacturer's GeneScan.RTM. run module GS STR POP4 (Id.)(1 ml) A
was used. Conditions for the electrophoresis were a 5 second
injection, injection kV was 15.0, run kV was 15.0, run temperature
was 60.degree. C., run time was 28 minutes and virtual filter A was
used.
[0131] FIG. 2A is a printout of results of scanning the amplified
fragments of each locus separated and detected with the ABI
PRISM.RTM. 310 Genetic Analyzer, as described above. FIG. 2A shows
amplification products of a DNA sample simultaneously co-amplified
for the loci D3S1358, HUMTH01, D21 S11, D18S51, G475, Amelogenin,
HUMvWFA31, D8S1179, HUMTPOX, HUMFIBRA, D5S818, D7S820, D13S317,
D16S539, HUMCSF1PO, and S159. Peaks shown in Panel A are labeled
with fluorescein, peaks shown in Panel B are labeled with
carboxy-tetramethylrhodamine, and peaks shown in Panel C are
labeled with 5,6 carboxyrhodamine 6G.
[0132] FIG. 2B is a printout of the results of scanning a sample
prepared in the same way as the sample scanned in FIG. 2A, except
that no DNA template was used in the amplification reaction. Peaks
in this figure are background products resulting from dye
conjugation and purification procedures and from undefined
causes.
Example 3
Fluorescent Detection of Multiplex Amplification of Loci
D3S1358,
[0133] HUMTH01, D21S11, D18S51, G475, Amelogenin, HUMvWFA31,
D8S1179.,HUMTPOX, HUMFIBRA, D5S818, D7S820, D13S317, D16S539,
HUMCSF1PO, and S159 as Detected with the ABI PRISM.RTM. 377 DNA
Sequencer
[0134] In this Example, a DNA template was amplified as in Example
2. Amplified products were separated using an ABI PRISM.RTM. 377
DNA Sequencer. This was carried out using a 0.2 mm thick, 5% Long
Ranger.TM. Acrylamide (FMC BioProducts, Rockland, Me.), 7M urea
gel. DNA samples were mixed with 1.5 .mu.l of a loading solution
(88.25% formamide, 4.1 mM EDTA, 15 mg/ml Blue Dextran) and 0.5
.mu.l of an internal lane size standard, denatured at 95.degree. C.
for 2 min., and chilled on ice prior to loading. Electrophoresis
was carried out using the manufacturer's GeneScan.RTM. modules for
Prerun (PR GS 36A-2400) and Run (GS 36A-2400). Run time was 3 hours
and virtual filter A was used.
[0135] FIG. 3A is a printout of results of scanning the amplified
fragments of each locus separated and detected with the ABI
PRISM.RTM. 377 DNA Sequencer, as described above. FIG. 3A shows
amplification products of a DNA sample simultaneously co-amplified
for the loci D3S1358, HUMTH01, D21S11, D18S51, G475, Amelogenin,
HUMvWFA31, D8S1179, HUMTPOX, HUMFIBRA, D5S818, D7S820, D13S317,
D16S539, HUMCSF1PO, and S159. Peaks shown in Panel A are labeled
with fluorescein, peaks shown in Panel B are labeled with
carboxy-tetramethylrhodamine, and peaks shown in Panel C are
labeled with 5,6 carboxyrhodamine 6G.
[0136] FIG. 3B is a printout of the results of scanning a sample
prepared in the same way as the sample scanned in FIG. 3A, except
that no DNA template was used in the amplification reaction. Peaks
in this figure are background products resulting from dye
conjugation and purification procedures and from undefined
causes.
Example 4
Fluorescent Detection of Multiplex Amplification of Loci D3S1358,
HUMTH01, D21S11, D18S51, G475, Amelogenin, HUMvWFA31, D8S1179,
HUMTPOX, HUMFIBRA, D5S818. D7S820, D13S317, D16S539, HUMCSF1PO, and
S159 as detected with the Hitachi FMBIO.RTM. II Fluorescent
Scanner
[0137] In this example, two DNA templates were each amplified
simultaneously at each of three different locus combinations
selected from the loci D3S1358, HUMTH01, D21S11, D18S51, G475,
Amelogenin, HUMvWFA31, D8S1179, HUMTPOX, HUMFIBRA, D5S818, D7S820,
D13S317, D16S539, HUMCSF1PO and S159. Amplification of each locus
combination included 5 ng template in a single reaction vessel
containing 25 .mu.l of 1.times. Gold ST*R Buffer (50 mM KCl, 10 mM
Tris-HCl (pH 8.3 at 25.degree. C.), 0.1% Triton X-100, 1.5 mM
MgCl.sub.2, 160 .mu.g/ml BSA and 200 .mu.M each of dATP, dCTP, dGTP
and dTTP).
[0138] A GeneAmp.RTM. PCR System 9600 (Perkin Elmer, Foster City,
Calif.) was employed with the following amplification protocol:
96.degree. C. for 12 min., then 10 cycles of 94.degree. C. for 30
sec., ramp for 68 sec. to 58.degree. C., hold for 30 sec., ramp 50
sec. to 70.degree. C., hold for 45 sec., followed by 22 cycles of
90.degree. C. for 30 sec., ramp 60 sec. to 58.degree. C., hold for
30 sec., ramp for 50 sec, to 70.degree. C., hold for 45 sec.,
followed by 1 cycle of 60.degree. C. for 30 min.
[0139] Thirty-two amplification primers were used in the following
concentrations, including 0.225 .mu.M each D3S1358 primers 1 [SEQ
ID NO:68] and 2 [FL-SEQ ID NO:69], 0.2 .mu.M each HUMTH01 primers 1
[FL-SEQ ID NO:66] and 2 [SEQ ID NO:67], 1.0 .mu.M each D21S11
primers 1 [SEQ ID NO:64] and 2 [FL-SEQ ID NO:65], 1.0 .mu.M each
D18S51 primers 1 [FL-SEQ ID NO:62] and 2 [SEQ ID NO:63], 2.8 .mu.M
each G475 primers 1 [FL-SEQ ID NO:88] and 2 [SEQ ID NO:89], 0.2
.mu.M each Amelogenin primers 1 [TMR-SEQ ID NO:86] and 2 [SEQ ID
NO:87], 0.3 .mu.M each HUMvWFA31 primers 1 [SEQ ID NO:76] and 2
[TMR-SEQ ID NO:40], 1.5 .mu.M each D8S1179 primers 1 [SEQ ID NO:74]
and 2 [TMR-SEQ ID NO:75], 0.2 .mu.M each HUMTPOX primers 1 [SEQ ID
NO:72] and 2 [TMR-SEQ ID NO:73], 2.0 .mu.M each HUMFIBRA primers 1
[TMR-SEQ ID NO:70] and 2 [SEQ ID NO:71], 0.55 .mu.M each D5S818
primers 1 [SEQ ID NO:84] and 2 [FL-SEQ ID NO:85], 1.1 .mu.M each Di
3S317 primers 1 [SEQ ID NO:82] and 2 [FL-SEQ ID NO:83], 1.7 .mu.M
each D7S820 primers 1 [FL-SEQ ID NO:80] and 2 [SEQ ID NO:81], 3.3
.mu.M each D16S539 primers 1 [SEQ ID NO:291 and 2 [FL-SEQ ID
NO:79], 0.5 .mu.M each HUMCSF1PO primers 1 [SEQ ID NO:77] and 2
[FL-SEQ ID NO:78], 2.0 .mu.M each S159 primers 1 SEQ ID NO:90] and
2 [FL-SEQ ID NO:91].
[0140] In the first locus combination, each template was amplified
using 2.5 U of AmpliTaq Gold.TM. DNA Polymerase and primers for
each locus used in the concentrations described above for the loci
D3S1358, HUMTH01, D21S11, D18S51, G475, Amelogenin, HUMvWFA31,
D8S1179, HUMTPOX, and HUMFIBRA. In the second locus combination,
all thirty-two primers, above, at the described concentrations, and
4 U of AmpliTaq Gold.TM. DNA Polymerase were used to amplify DNA
templates at all sixteen loci, D3S1358, HUMTH01, D21S11, D18S51,
G475, Amelogenin, HUMvWFA31, D8S1179, HUMTPOX, HUMFIBRA, D5S818,
D7S820, D13S317, D16S539, HUMCSF1PO and S159 in a single reaction
vessel. In the third combination, each template was amplified using
1.5 U of AmpliTaq Gold.TM. DNA Polymerase and primers for each
locus used in the concentrations described above for the loci
D5S818, D7S820, D13S317, D16S539, HUMCSF1PO and S159.
[0141] 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 which was chemically cross-linked to 2 glass
plates (Kobayashi, Y. (1988), BRL Focus 10: 73-74). DNA samples
were mixed with 3.5 .mu.l of a loading solution (10 mM NaOH, 95%
formamide, 0.05% bromophenol blue) and 0.5 .mu.l of an internal
lane size standard, denatured at 95.degree. C. for 2 min., and
chilled on ice prior to loading. The separated products were
visualized by detection of the fluorescent signals using the
Hitachi FMIBO.RTM. II fluorescent scanner (Hitachi Software
Engineering America, Ltd. South San Francisco, Calif.). Band pass
filters at 505 nm and 585 nm, respectively, were used for the
detection of fluorescein-labeled loci and
carboxy-tetramethylrhodamine-labeled loci, respectively. A band
pass filter of 650 nm was used for detection of the internal lane
standard (size standard data, not shown).
[0142] Reference is made to FIGS. 4A and 4B, which display the
fragments resulting from each amplification reaction. FIG. 4A shows
the results from the 505 nm scan (Fluorescein channel) and FIG. 4B
shows the results from the 585 nm scan
(carboxy-tetramethylrhodamine channel) of the same lanes of the
polyacrylamide gel. For each DNA template, lane 1 shows the results
of the DNA sample which has been simultaneously co-amplified for
the loci D3S1358, HUMTH01, D21S11, D18S51, G475, Amelogenin,
HUMvWFA31, D8S1179, HUMTPOX, and HUMFIBRA. Lane 2 shows the results
of the DNA sample simultaneously co-amplified for the loci D3S1358,
HUMTH01, D21 S11, D18S51, G475, Amelogenin, HUMvWFA31, D8S1179,
HUMTPOX, HUMFIBRA, D5S818, D13S317, D7S820, D16S539, HUMCSF1PO, and
S159. Lane 3 shows the results of the DNA sample simultaneously
co-amplified for the loci D5S818, D13S317, D7S820, D16S539,
HUMCSF1PO, and S159.
Example 5
Fluorescent Detection of Multiplex Amplification of Loci
D3S1358,
[0143] HUMTH01, D21 S11, D18S51, G475, Amelogenin, HUMvWFA31,
D8S1179, HUMTPOX, HUMFIBRA, D5S818, D7S820, D13S317, D16S539,
HUMCSF1PO, and S159 as Detected with the Hitachi FMBIO.RTM. II
Fluorescent Scanner
[0144] In this example, two DNA templates were each amplified
simultaneously at each of two different locus combinations selected
from the loci D3S1358, HUMTH01, D21S11, D18S51, G475, Amelogenin,
HUMvWFA31, D8S1179, HUMTPOX, HUMFIBRA, D5S818, D7S820, D13S317,
D16S539, HUMCSF1PO and S159. Amplification of each locus
combination included 5 ng template in a single reaction vessel
containing 25 .mu.l of 1.times. Gold ST*R Buffer (50 mM KCl, 10 mM
Tris-HCl (pH 8.3 at 25.degree. C.), 0.1% Triton X-100, 1.5 mM
MgCl.sub.2, 160 .mu.g/ml BSA and 200 .mu.M each of dATP, dCTP, dGTP
and dTTP).
[0145] A GeneAmp.RTM. PCR System 9600 (Perkin Elmer, Foster City,
Calif.) was employed with the following amplification protocol:
96.degree. C. for 12 min., then 10 cycles of 94.degree. C. for 30
sec., ramp for 68 sec. to 58.degree. C., hold for 30 sec., ramp 50
sec. to 70.degree. C., hold for 45 sec., followed by 22 cycles of
90.degree. C. for 30 sec., ramp 60 sec. to 58.degree. C., hold for
30 sec., ramp for 50 sec, to 70.degree. C., hold for 45 sec.,
followed by 1 cycle of 60.degree. C. for 30 min.
[0146] Thirty-two amplification primers were used in the following
concentrations, including 0.225 .mu.M each D3S1358 primers 1 [SEQ
ID NO:68] and 2 [FL-SEQ ID NO:69], 0.2 .mu.M each HUMTH01 primers 1
[FL-SEQ ID NO:66] and 2 [SEQ ID NO:67], 1.0 .mu.M each D21 S11
primers 1 [SEQ ID NO:64] and 2 [FL-SEQ ID NO:65], 1.0 .mu.M each D1
8S51 primers 1 [FL-SEQ ID NO:62] and 2 [SEQ ID NO:63], 2.8 .mu.M
each G475 primers 1 [SEQ ID NO:88] and 2 [FL-SEQ ID NO:94], 0.2
.mu.M each Amelogenin primers 1 [TMR-SEQ ID NO:86] and 2 [SEQ ID
NO:87], 0.3 .mu.M each HUMvWFA31 primers 1 [SEQ ID NO:76] and 2
[TMR-SEQ ID NO:40], 1.5 .mu.M each D8S1179 primers 1 [SEQ ID NO:74]
and 2 [TMR-SEQ ID NO:75], 0.2 .mu.M each HUMTPOX primers 1 (SEQ ID
NO:72] and 2 [TMR-SEQ ID NO:73], 2.0 .mu.M each HUMFIBRA primers 1
[TMR-SEQ ID NO:70] and 2 [SEQ ID NO:71], 0.55 .mu.M each D5S818
primers 1 [SEQ ID NO:84] and 2 [FL-SEQ ID NO:85], 1.1 .mu.M each
D13S317 primers 1 [SEQ ID NO:82] and 2 [FL-SEQ ID NO:83], 1.7 .mu.M
each D7S820 primers 1 [FL-SEQ ID NO:80] and 2 [SEQ ID NO:81], 3.3
.mu.M each D16S539 primers 1 [SEQ ID NO:29] and 2 [FL-SEQ ID
NO:79], 0.5 .mu.M each HUMCSF1PO primers 1 [SEQ ID NO:77] and 2
[FL-SEQ ID NO:78], 2.0 .mu.M each S159 primers 1 [SEQ ID NO:95] and
2 [FL-SEQ ID NO:96].
[0147] In the first locus combination, each template was amplified
using 2.5 U of AmpliTaq Gold.TM. DNA Polymerase and primers for
each locus used in the concentrations described above for the loci
D3S1358, HUMTH01, D21S11, D18S51, G475, Amelogenin, HUMvWFA31,
D8S1179, HUMTPOX, and HUMFIBRA. In the second locus combination,
all thirty-two primers, above, at the described concentrations, and
4 U of AmpliTaq Gold.TM. DNA Polymerase were used to amplify DNA
templates at all sixteen loci, D3S1358, HUMTH01, D21S11, D18S51,
G475, Amelogenin, HUMvWFA31, D8S1179, HUMTPOX, HUMFIBRA, D5S818,
D7S820, D13S317, D16S539, HUMCSF1PO and S159 in a single reaction
vessel.
[0148] The separation and visualization of amplified products were
as described in Example 4.
[0149] Reference is made to FIG. 5A and 5B, which display the
fragments resulting from each amplification reaction. FIG. 5A shows
the results from the 505 nm scan (Fluorescein channel) and FIG. 5B
shows the results from the 585 nm scan
(carboxy-tetramethylrhodamine channel) of the same lanes of the
polyacrylamide gel. For each template, lane 1 shows the results of
the DNA sample simultaneously co-amplified for the loci D3S1358,
HUMTH01, D21S11, D18S51, G475, Amelogenin, HUMvWFA31, D8S1179,
HUMTPOX, and HUMFIBRA and lane 2 shows the results of the DNA
sample simultaneously co-amplified for the loci D3S1358, HUMTH01,
D21S11, D18S51, G475, Amelogenin, HUMvWFA31, D8S1179, HUMTPOX,
HUMFIBRA, D5S818, D13S317, D7S820, D16S539, HUMCSF1PO, and
S159.
Example 6
Fluorescent Detection of Multiplex Amplification of Loci D3S1358,
HUMTH01, D21 S11, D18S51, S159. Amelogenin, HUMvWFA31, D8S1179,
HUMTPOX, HUMFIBRA, D5S818, D7S820, D13S317, D16S539, HUMCSF1PO, and
C221 as Detected with the Hitachi FMBIO.RTM. II Fluorescent
Scanner
[0150] In this example, two DNA templates were each amplified
simultaneously at each of three different locus combinations
selected from the loci D3S1358, HUMTH01, D21S11, D18S51, S159,
Amelogenin, HUMvWFA31, D8S1179, HUMTPOX, HUMFIBRA, D5S818, D7S820,
D13S317, D16S539, HUMCSF1PO and C221. Amplification of each locus
combination included 10 ng template in a single reaction vessel
containing 25 .mu.l of 1.times. Gold ST*R Buffer (50 mM KCl, 10 mM
Tris-HCl (pH 8.3 at 25.degree. C.), 0.1% Triton X-100, 1.5 mM
MgCl.sub.2, 160 .mu.g/ml BSA and 200 .mu.M each of dATP, dCTP, dGTP
and dTTP).
[0151] A GeneAmp.RTM. PCR System 9600 (Perkin Elmer, Foster City,
Calif.) was employed with the following amplification protocol:
96.degree. C. for 12 min., then 10 cycles of 94.degree. C. for 30
sec., ramp for 68 sec. to 60.degree. C., hold for 30 sec., ramp 50
sec. to 70.degree. C., hold for 45 sec., followed by 20 cycles of
90.degree. C. for 30 sec., ramp 60 sec. to 60.degree. C., hold for
30 sec., ramp for 50 sec, to 70.degree. C., hold for 45 sec.,
followed by 1 cycle of 60.degree. C. for 30 min.
[0152] Thirty-two amplification primers were used in the following
concentrations, including 0.75 .mu.M each D3S1358 primers 1 [SEQ ID
NO:106] and 2 [FL-SEQ ID NO:69], 0.3 .mu.M each HUMTH01 primers 1
[FL-SEQ ID NO:38] and 2 [SEQ ID NO:103], 2.0 .mu.M each D21S11
primers 1 [SEQ ID NO:64] and 2 [FL-SEQ ID NO:65], 0.3 .mu.M each
D18S51 primers 1 [FL-SEQ ID NO:101] and 2 [SEQ ID NO:102], 2.0
.mu.M each S159 primers 1 [SEQ ID NO:92] and 2 [FL-SEQ ID NO:93],
0.15 .mu.M each Amelogenin primers 1 [TMR-SEQ ID NO:105] and 2 [SEQ
ID NO:87], 1.0 .mu.M each HUMvWFA31 primers 1 [SEQ ID NO:76] and 2
[TMR-SEQ ID NO:40], 1.25 .mu.M each D8S1179 primers 1 [TMR-SEQ ID
NO:104] and 2 [SEQ ID NO:75], 0.75 .mu.M each HUMTPOX primers 1
[TMR-SEQ ID NO:72] and 2 [SEQ ID NO:73], 1.5 .mu.M each HUMFIBRA
primers 1 [TMR-SEQ ID NO:70] and 2 [SEQ ID NO:107], 0.55 .mu.M each
D5S818 primers 1 [SEQ ID NO:84] and 2 [FL-SEQ ID NO:85], 1.1 .mu.M
each D13S317 primers 1 [SEQ ID NO:3] and 2 [FL-SEQ ID NO:4], 1.7
.mu.M each D7S820 primers 1 [FL-SEQ ID NO:80] and 2 [SEQ ID NO:81],
3.3 .mu.M each D16S539 primers 1 [FL-SEQ ID NO:29] and 2 [SEQ ID
NO:97], 0.25 .mu.M each HUMCSF1PO primers 1 [SEQ ID NO:77] and 2
[FL-SEQ ID NO:98], 1.0 .mu.M each C221 primers 1 [FL-SEQ ID NO:99]
and 2 [SEQ ID NO:100].
[0153] In the first locus combination, each template was amplified
using 2.5 U of AmpliTaq Gold.TM. DNA Polymerase and primers for
each locus used in the concentrations described above for the loci
D3S1358, HUMTH01, D21S11, D18S51, S159, Amelogenin, HUMvWFA31,
D8S1179, HUMTPOX, and HUMFIBRA. In the second locus combination,
all thirty-two primers, above, at the described concentrations and
4 U of AmpliTaq Gold.RTM. DNA Polymerase were used to amplify DNA
templates at all sixteen loci, D3S1358, HUMTH01, D21 S11, D18S51,
S159, Amelogenin, HUMvWFA31, D8S1179, HUMTPOX, HUMFIBRA, D5S818,
D7S820, D13S317, D16S539, HUMCSF1PO and C221 in a single reaction
vessel. In the third combination, each template was amplified using
1.5 U of AmpliTaq Gold.TM. DNA Polymerase and primers for each
locus used in the concentrations described above for the loci
D5S818, D7S820, D13S317, D16S539, HUMCSF1PO and C221.
[0154] The amplification products were separated and detected as
described in Example 4, except that each sample of amplification
products was diluted 1:4 in 1.times. STR Buffer (50 mM KCl, 10 mM
Tris-HCl (pH 8.3 at 25.degree. C.), 0.1% Triton X-1 00, 1.5 mM
MgCl.sub.2, and 200 .mu.M each of dATP, dCTP, dGTP and dTTP). The
diluted amplification products (2.5 .mu.l) were mixed with 2.5
.mu.l of a loading solution (10 mM NaOH, 95% formamide, 0.05%
bromophenol blue), without an internal lane standard, denatured at
95.degree. C. for 2 min., and chilled on ice prior to loading.
[0155] Reference is made to FIGS. 6A and 6B, which display the
fragments resulting each amplification reaction. FIG. 6A shows the
results from the 505 nm scan (Fluorescein channel) and FIG. 6B
shows the results from the 585 nm scan
(carboxy-tetramethylrhodamine channel) of the same lanes of the
polyacrylamide gel. For each DNA template, lane 1 shows the results
of the DNA sample simultaneously co-amplified for the loci D3S1358,
HUMTH01, D21 S11, D18S51, S159, Amelogenin, HUMvWFA31, D8S1179,
HUMTPOX, and HUMFIBRA. Lane 2 shows the results of the DNA sample
simultaneously co-amplified for the loci D3S1358, HUMTH01, D21S11,
D18S51, G475, Amelogenin, HUMvWFA31, D8S1179, HUMTPOX, HUMFIBRA,
D5S818, D13S317, D7S820, D16S539, HUMCSF1PO, and C221. Lane 3 shows
the results of the DNA sample simultaneously co-amplified for the
loci D5S818, D13S317, D7S820, D16S539, HUMCSF1PO, and C221.
Sequence CWU 0
0
* * * * *