WO1999004034A1 - Primers for obtaining highly informative dna markers - Google Patents
Primers for obtaining highly informative dna markers Download PDFInfo
- Publication number
- WO1999004034A1 WO1999004034A1 PCT/CA1998/000691 CA9800691W WO9904034A1 WO 1999004034 A1 WO1999004034 A1 WO 1999004034A1 CA 9800691 W CA9800691 W CA 9800691W WO 9904034 A1 WO9904034 A1 WO 9904034A1
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- primer
- dna
- seq
- acid sequence
- nucleic acid
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/16—Primer sets for multiplex assays
Definitions
- the invention relates to a new primer pair consisting of R14B/264 primer and a primer designed from Sextolet 900 marker and to a highly informative DNA marker obtained from amplification of genomic DNA with the primer pair.
- genetic markers in human have helped map part of the human genome, and identify genes responsible for particular diseases. In addition, genetic markers are used to differentiate between individuals at the molecular level .
- DNA polymorphism derives from differences in DNA sequences inherited from each parental chromosome. DNA polymorphism could also arise from a mutation in one defined single nucleotide, an insertion or a deletion of a sequence, or from variations in the length of a stretch of short tandem repeats (STR) .
- Polymorphic DNA markers provide information regarding the segregation pattern of parental chromosomes during the mating process and hence disclose a person's genetic identity.
- the informativity of a specific marker is measured by its heterozigosity (H) , its polymorphic information content (PIC) and the allelic frequency of each allele.
- H heterozigosity
- PIC polymorphic information content
- the total informativity of a marker system (where several markers may be involved) is a function of the informativity extracted from the individual markers. The higher informativity of individual markers, the more informative a marker system is.
- RFLP Restriction fragment length polymorphism
- Southern Blot technique is widely used to identify DNA polymorphism related to genetic diseases, genome mapping, evolution and forensic studies.
- Microsatellite DNA markers revealed by this method are highly informative.
- this method requires considerable amounts of DNA and large sample sizes.
- relatively long analysis times are needed to obtain results and translate into high cost and increases in turn around time.
- PCR polymerase chain reaction
- DNA present in minute amounts of biological material can be amplified into as many copies as needed. Amplified fragments are easy to be analyzed.
- the application of PCR to DNA marker analysis proves to be fast, reliable and cost effective.
- markers which have thus far been analyzed by PCR are of STR polymorphism. These markers consist of more than two alleles with the exceptions of some highly informative ones where more than 35 alleles (Kimpton C, et al . Forensic Science International 71 (2) : 137-52 , 1995) and a heterozigosity less than 0.93 have been described. No marker with more than 40 alleles and heterozigosity greater than 0.96 has been described so far.
- Marker Q900 described by Tang et al . (Tang J.Q., et al . , Mammalian Genome 6:345-349, 1995) reportedly consisted of 23 alleles and had a heterozigosity of 0.95.
- U.S. Patent 5,576,180 in the name of Melan ⁇ on et al discloses a DNA amplification primer pair for the simultaneous amplification of multiple highly polymorphic genomic loci .
- the primer pair consists of R14B/264 and Q560mak. This primer pair allows for the identification of three (3) markers including the marker Q900. However, only 23 alleles with a heterozigosity of 0.95 have been observed using the marker Q900.
- One aim of the present invention is to provide a primer pair allowing for the amplification of a marker having a high discrimination power, with a heterozigosity greater than 0.96 and comprising more than 64 alleles.
- Another aim of the present invention is to provide for a DNA amplification primer pair consisting of primer R14B/264 and a primer designed from Sextolet 900 marker, which corresponds to a sequence of polymorphic loci .
- a DNA amplification primer pair for the amplification of a marker having a heterozigosity of at least 0.97 and comprising more than 64 alleles, the primer pair consisting of a first R14B/264 primer (SEQ ID NO: 1) and a second primer designed from the nucleic acid sequence of SEQ ID NO : 2.
- the second primer may be designed from a nucleic acid sequence comprised between nucleic acids 661 and 960 of the nucleic acid sequence of SEQ ID NO: 2 and is preferably the Sext.900E primer having the nucleic acid sequence of SEQ ID NO: 3.
- the marker amplified with the DNA amplification primer pair of the present invention is preferably Sextolet 900.
- a method for the DNA fingerprinting identification of genetically related or unrelated individuals which comprises the steps of: a) performing DNA amplification of DNA samples collected from individuals using a first primer R14B/264 and a second primer designed from the nucleic acid sequence of SEQ ID NO : 2 ; and b) separating the amplified DNA samples of step a) whereby polymorphic regions of different size are amplified and serve as DNA fingerprinting of said individuals .
- the second primer may be labeled either by radioactive materials or by fluorescent dyes for the purpose of separating the amplified DNA samples.
- step a) mentioned above may be effected by any PCR procedures, such as regular or asymmetric PCR procedures .
- the DNA separating step b) mentioned above may be effected using a DNA sequencer or a gel electrophoresis procedure, such as a high resolution polyacrylamide sequencing gel type electrophoresis procedure .
- a method for the DNA fingerprinting identification of genetically related or unrelated individuals which comprises the steps of: a) performing DNA amplification of DNA samples collected from individuals using a first primer R14B/264 and a second primer designed from the nucleic acid sequence of SEQ ID NO : 2 ; and b) separating the amplified DNA samples of step a) whereby polymorphic regions of different size are amplified and serve as an internal standard in genetic analyses allowing to detect samples mix up.
- a method for the DNA fingerprinting identification of genetically related or unrelated individuals which comprises the steps of: a) performing DNA amplification of DNA samples from blood collected from individuals using a first primer R14B/264 and a second primer designed from the nucleic acid sequence of SEQ ID NO : 2 , wherein the blood is collected as blood spots on paper; and b) separating the amplified DNA samples of step a) whereby polymorphic regions of different size are amplified and serve as DNA fingerprinting for baby individualization or identification.
- kit for amplification of Sextolet 900 marker which comprises: a) a first primer R14B/264 having the nucleic acid sequence of SEQ ID NO : 1 ; and b) a second primer designed from the nucleic acid sequence of SEQ ID NO: 2.
- the kit may further comprises: c) typed Sextolet 900 DNAs .
- primer is intended to mean any oligonucleotide which can be used to direct DNA polymerization, more specifically the DNA amplification primer of the present invention consists in R14B/264 (SEQ ID NO:l) and a primer designed from Sextolet 900 marker (SEQ ID NO: 2) .
- primer designed from Sextolet 900 marker is intended to mean any primer suitable to be used with primer R14B/264 and which allows for the amplification of a marker having a high discrimination power, with a heterozigosity greater than 0.97 and comprising more than 64 alleles, such as primer Sext.900E (SEQ ID NO : 3 ) , without limitation.
- marker is intended to mean any polymorphic genomic locus, which may vary among individuals and serve as a DNA fingerprinting identification mean.
- asymmetric PCR is intended to mean any DNA amplification reaction using unequal concentration of primers.
- Fig. 1 illustrates the nucleic acid sequence of the primer R14B/264 (SEQ ID NO:l);
- Fig. 2 illustrates the complete nucleic acid sequence of Sextolet 900 marker (SEQ ID NO: 2) ;
- Fig. 3 illustrates a nucleic acid sequence (SEQ ID NO: 3) of a primer designed from the sequence of the Sextolet 900 marker (SEQ ID NO: 2) .
- Fig. 1 SEQ ID NO:l
- a primer designed from the nucleic acid sequence Sextolet 900 marker Fig. 2; SEQ ID NO: 2.
- the primer pair consists of R14B/264 and Sext.900E primers having respectively the following sequences:
- Marker Q900 has been amplified by polymerase chain reaction (PCR) using primers R14B/264 and Q560mak
- the complete sequence of the non-polymorphic part of the clone Q900B3.2 revealed a 960 base pairs DNA sequence (Fig. 2; SEQ ID NO : 2 ) , differing from the sequence described by Tang et al . (Tang J.Q., et al . , Mammalian Genome 6:345-349, 1995).
- different primers designed from the sequence of Sextolet 900 marker (Fig. 2; SEQ ID NO: 2) have been tested.
- These primers combined with the 5' primer R14B/264 were able to amplify the same DNA patterns using samples from genetically related and non-related individuals.
- Sext.900E chromosomal location of the Sextolet 900 marker, amplified by one of the designed primers, hereinafter referred to as Sext.900E (Fig. 3; SEQ ID N0:3), has been mapped to chromosome 19, the same chromosome as the one marker Q900 was assigned to.
- the asymmetric PCR and labeled Sext.900E optimize the amplification of Sextolet 900 while the amplification with R14B/264 is inhibited and invisible (Tang J.Q., et al . , Mammalian Genome 6:345- 349, 1995) .
- a protocol using a fluorescent labeled Sext.900E in which the amplification products are determined using an automatic DNA sequencer has also been developed. The amplified products were analyzed directly with the program. This fluorescent-label protocol allows for a direct identification of each individual allele.
- the primer Sext.900E can be either labeled by fluorescent dye(s) or radioactivity allowing for the analysis of the marker using an automatic sequencer or conventional autoradiography .
- Multiplex PCRs have also been tested with other markers developed. Independent amplifications were always achieved from each marker and no background is found so far in a multiplex of four (4) markers.
- this marker can be used in various ways. Due to its high discrimination power, the marker will be very useful where individualization is required, especially in forensic identification procedures and in paternity testing. It can also be used in linkage analysis where any interesting DNA sequences are tightly linked to this marker. The fact that this marker can be multiplexed with other markers greatly increases the overall performance of the identity test.
- the marker of the present invention can also be used as an internal control for PCR where the detection of the contamination of undesired genomic DNA source is very important for the experiments. Such use can be seen in virtually all PCR application from specific amplification of a single fragment in detection of specific mutations to one-cell-PCR or preimplantation diagnostic .
- primers other than Sext.900E may be found. Table 2 lists without limitation some of the primer pairs that may be used in accordance with the present invention.
- the reaction is carried out in a 200 ⁇ l thin wall tube in a RobocyclerTM (Stratagene) .
- the reaction mixture (20 ⁇ ) includes 20ng of genomic DNA, 1 unit of Taq DNA polymerase, and 0.2mM each of four dNTPs, in lOmM Tris-HCl, pH 9.0, 50mM KC1 , 1.5mM MgCl2, 2% formamide, 0.01% gelatin with 0.5 ⁇ M of the primer R14B/264 (5' CAG AGC GAG ACT CT 3 ' , SEQ ID NO:l), 0.5 ⁇ M of the primer Sext.900E (5' ATG CGT TTT GGA TGA AAT GAG ATG 3', SEQ ID NO : 3 ) and 0.5 ⁇ M of 32 P end-labeled Sext.900E.
- the reaction is carried out with 30 cycles at 94°C for 30 sec, 53°C for 45 sec, and 72°C for 45 sec. An additional 72°C incubation for 5 min.
- lO ⁇ l of loading buffer (95% formamide 0.04% Bromophenol blue, 0.04% Xylene cyanol and 5mM EDTA) was added.
- the sample was heated at 94°C for 5 min. and immediately cooled to 4-6°C.
- Five to six (5-6) ⁇ l of the sample were loaded into a sequencing type gel (6% polyacrylamide :bis-acrylamide 19:1, 8M urea and 0.5 X TBE) .
- a sequence ladder (with 1 bp resolution) is always included to identify the size of the alleles.
- the gel was preheated and run under 1 X TBE (90mM Tris-borate pH 8.3, 2 mM EDTA) at constant power of 70W for 2-3 hrs .
- the gel was dried and autoradiographed at room temperature overnight .
- the DNA from this person is amplified by the method of the present invention and the DNA profile of the father, using the Sextolet 900 marker, is compared with that of the child. If the mother's DNA is available, one could achieve a paternity index (PI) from 3.71 to 178, depending on the frequency of the paternal allele, leading to a probability of paternity from 79% to 99.4% with only the Sextolet 900 marker.
- PI paternity index
- Sextolet 900 marker has a high discrimination power (>0.97) and theoretically more than 2,000 genotypes in the general population, the exclusion of a sample from different source is more likely to be achieved by using Sextolet 900 marker than any other PCR generated STR markers identified so far.
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Abstract
Description
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Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP98933416A EP1002130A1 (en) | 1997-07-17 | 1998-07-16 | Primers for obtaining highly informative dna markers |
AU83297/98A AU8329798A (en) | 1997-07-17 | 1998-07-16 | Primers for obtaining highly informative dna markers |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US08/896,095 US6060243A (en) | 1997-07-17 | 1997-07-17 | Primers for obtaining highly informative DNA markers |
US08/896,095 | 1997-07-17 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO1999004034A1 true WO1999004034A1 (en) | 1999-01-28 |
Family
ID=25405619
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/CA1998/000691 WO1999004034A1 (en) | 1997-07-17 | 1998-07-16 | Primers for obtaining highly informative dna markers |
Country Status (5)
Country | Link |
---|---|
US (1) | US6060243A (en) |
EP (1) | EP1002130A1 (en) |
AU (1) | AU8329798A (en) |
OA (1) | OA11358A (en) |
WO (1) | WO1999004034A1 (en) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1999061659A1 (en) * | 1998-05-26 | 1999-12-02 | Procrea Biosciences Inc. | A novel str marker system for dna fingerprinting |
WO2003031646A1 (en) * | 2001-10-12 | 2003-04-17 | The University Of Queensland | Multiple genetic marker selection and amplification |
KR100459106B1 (en) * | 2002-03-21 | 2004-12-03 | 한국해양연구원 | Identification of an organism by use of the intron dna sequence of the clock gene as dna fingerprints |
Families Citing this family (14)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1997040385A1 (en) | 1996-04-25 | 1997-10-30 | Bioarray Solutions, Llc | Light-controlled electrokinetic assembly of particles near surfaces |
EP1311839B1 (en) | 2000-06-21 | 2006-03-01 | Bioarray Solutions Ltd | Multianalyte molecular analysis using application-specific random particle arrays |
US9709559B2 (en) | 2000-06-21 | 2017-07-18 | Bioarray Solutions, Ltd. | Multianalyte molecular analysis using application-specific random particle arrays |
US7262063B2 (en) | 2001-06-21 | 2007-08-28 | Bio Array Solutions, Ltd. | Directed assembly of functional heterostructures |
JP4377689B2 (en) | 2001-10-15 | 2009-12-02 | バイオアレイ ソリューションズ リミテッド | Combined analysis of polymorphic loci with simultaneous interrogation and enzyme-mediated detection |
US7157228B2 (en) * | 2002-09-09 | 2007-01-02 | Bioarray Solutions Ltd. | Genetic analysis and authentication |
WO2004047007A1 (en) | 2002-11-15 | 2004-06-03 | Bioarray Solutions, Ltd. | Analysis, secure access to, and transmission of array images |
US20040157220A1 (en) | 2003-02-10 | 2004-08-12 | Purnima Kurnool | Methods and apparatus for sample tracking |
WO2005029705A2 (en) | 2003-09-18 | 2005-03-31 | Bioarray Solutions, Ltd. | Number coding for identification of subtypes of coded types of solid phase carriers |
JP4564959B2 (en) | 2003-09-22 | 2010-10-20 | バイオアレイ ソリューションズ リミテッド | Surface-immobilized polyelectrolyte with multiple functional groups that can be covalently bonded to biomolecules |
CA2899287A1 (en) | 2003-10-28 | 2005-05-12 | Bioarray Solutions Ltd. | Optimization of gene expression analysis using immobilized capture probes |
AU2004287069B2 (en) | 2003-10-29 | 2009-07-16 | Bioarray Solutions, Ltd. | Multiplexed nucleic acid analysis by fragmentation of double-stranded DNA |
US7848889B2 (en) | 2004-08-02 | 2010-12-07 | Bioarray Solutions, Ltd. | Automated analysis of multiplexed probe-target interaction patterns: pattern matching and allele identification |
US8486629B2 (en) | 2005-06-01 | 2013-07-16 | Bioarray Solutions, Ltd. | Creation of functionalized microparticle libraries by oligonucleotide ligation or elongation |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1995017522A2 (en) * | 1993-12-21 | 1995-06-29 | University Of Leicester | Identification of simple tandem repeats |
EP0714987A2 (en) * | 1994-09-26 | 1996-06-05 | IMMUNO Aktiengesellschaft | Method for quantifying genomic DNA |
WO1996034979A2 (en) * | 1995-05-01 | 1996-11-07 | Centre De Recherche De L'hopital Sainte-Justine | Primers and methods for simultaneous amplification of multiple markers for dna fingerprinting |
-
1997
- 1997-07-17 US US08/896,095 patent/US6060243A/en not_active Expired - Fee Related
-
1998
- 1998-07-16 AU AU83297/98A patent/AU8329798A/en not_active Abandoned
- 1998-07-16 EP EP98933416A patent/EP1002130A1/en not_active Ceased
- 1998-07-16 WO PCT/CA1998/000691 patent/WO1999004034A1/en not_active Application Discontinuation
-
2000
- 2000-01-17 OA OA1200000010A patent/OA11358A/en unknown
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1995017522A2 (en) * | 1993-12-21 | 1995-06-29 | University Of Leicester | Identification of simple tandem repeats |
EP0714987A2 (en) * | 1994-09-26 | 1996-06-05 | IMMUNO Aktiengesellschaft | Method for quantifying genomic DNA |
WO1996034979A2 (en) * | 1995-05-01 | 1996-11-07 | Centre De Recherche De L'hopital Sainte-Justine | Primers and methods for simultaneous amplification of multiple markers for dna fingerprinting |
Non-Patent Citations (2)
Title |
---|
TANG ET AL.: "A PCR BASED HIGHLY INFORMATIVE DNA MARKER WITH MORE THAN 65 ALLELES", JOURNAL OF HUMAN GENETICS, vol. 61, no. 4 suppl.., 28 October 1997 (1997-10-28), pages A402, XP002080165 * |
TANG J Q ET AL: "ALU-PCR COMBINED WITH NON-ALU PRIMERS REVEALS MULTIPLE POLYMORPHIC LOCI", MAMMALIAN GENOME, vol. 6, no. 5, 1 January 1995 (1995-01-01), pages 345 - 349, XP000607150 * |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1999061659A1 (en) * | 1998-05-26 | 1999-12-02 | Procrea Biosciences Inc. | A novel str marker system for dna fingerprinting |
WO2003031646A1 (en) * | 2001-10-12 | 2003-04-17 | The University Of Queensland | Multiple genetic marker selection and amplification |
KR100459106B1 (en) * | 2002-03-21 | 2004-12-03 | 한국해양연구원 | Identification of an organism by use of the intron dna sequence of the clock gene as dna fingerprints |
Also Published As
Publication number | Publication date |
---|---|
AU8329798A (en) | 1999-02-10 |
US6060243A (en) | 2000-05-09 |
EP1002130A1 (en) | 2000-05-24 |
OA11358A (en) | 2003-12-17 |
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