Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-05-26
2001-06-26
Arthur, Lisa B. (Department: 1658)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C536S023100, C536S024300
Reexamination Certificate
active
06251592
ABSTRACT:
BACKGROUND OF THE INVENTION
(a) Field of the Invention
The invention relates to seven novel STR markers for DNA fingerprinting.
(b) Description of Prior Art
Creating a reliable, informative system for human identification has been long envisaged in forensic science. Polymorphism at the DNA level provides information regarding the segregation pattern of parental chromosomes during the mating process and hence discloses a person's genetic identity and thus, becomes a powerful tool for DNA fingerprinting. The information extracted from a specific DNA marker can be measured by the frequencies of each allele, and its heterozigosity and matching probability in a population. When several markers are being used together in a fingerprinting procedure, the global informativeness of the system depends on the informativeness extracted from each individual marker. Higher informativeness can only be obtained if more markers or more informative markers are used.
The Southern blot technique has been used to analyze restriction fragment length polymorphism (RFLP), a highly informative system which, however, requires considerable amounts of DNA and long periods of time to obtain, analyze and interpret the results.
The Polymerase chain reaction (PCR) has been widely used since the late 80s and proved to be a highly efficient and sensitive method to disclose and analyze DNA polymorphism. Most markers having been so far analyzed by PCR are of STR (Short Tandem Repeats) polymorphism. In a STR marker, the polymorphism arises from the number of repeats of short stretches of DNA. The number of repeats varies between individuals in the general population and thus provides a source for human identification at the DNA level. Ordinary PCR analyzes one marker at a time. More recently, primers were pooled and multiple markers were amplified in one PCR reaction. Since optimized multiplex PCR consumes considerably less reagent and time for same quality results, it greatly improves DNA fingerprinting procedures. The STR markers analyzed using multiplex PCR techniques proved to be fast, reliable and cost effective.
Markers and multiplex systems have been formulated by different suppliers for DNA forensic studies or paternity testing. The matching probabilities of individual systems reach a level of 1 in 1×10
8
−10
9
. These multiplex systems combine the same 8 to 9 markers with one or two markers differing from one system to another. The need for a unique, independent and highly informative marker system was overdue. Its availability will not only strengthen the existing DNA forensic/paternity testing services, but also provide an independent counter-expertise often required in various situations.
SUMMARY OF THE INVENTION
In accordance with the present invention, we have recently invented a PCR system that fulfills these requirements. This multiplex PCR system analyzes seven markers located on 6 different human chromosomes. Two markers, Sextolet 900 and Sextolet 100 have been reported previously and greatly improved in their present forms. Five additional markers, Sextolet 110, Sextolet 150, Sextolet 180, Sextolet 700 and Sextolet 800 have been discovered and analyzed. Optional multiplex PCRs amplify diverse combinations of these seven markers. The system has been made suitable for different levels of laboratory equipment. The conventional radio-labeled multiplex PCR procedure can be used in routine genetic diagnostic laboratories where a DNA sequencer is not available. The fluorescent-dye labeled multiplex PCR procedure improves the convenience of manipulation, allows for automation and considerably accelerates the output. Data from a population study have yielded a matching probability in the 10
10
scale with the seven markers, and a typical paternity index of over 4250.
In accordance with the present invention there is provided a STR marker system for DNA fingerprinting of a sample DNA, which comprises a sequence selected from the group consisting of SEQ ID NOS:1 to 7 as set forth in
FIGS. 1A-1B
and complementary sequences thereto.
The DNA is from a human patient, such as a foetus, embryo, newborn, children, adult, live or deceased for identification thereof.
In accordance with the present invention there is provided the use of the STR marker system of the present invention to design primers to amplify the marker.
In accordance with the present invention there is provided a DNA amplification primer pair for the simultaneous amplification of at least one STR marker, which comprises a pair of 5′ and 3′ primers selected from the group consisting of:
5′ Primer (5′-3′)
3′ primer (5′-3′)
CAGAGCGAGACTCT (SEQ ID NO:5)
GCCGGGCGCCCAAGAGAAGCCGCCAGGAAC
(SEQ ID NO:8)
GGCCGCCGGAAAAGCCTCTTGGGAAAG
CCGCCGCGCATTTAGGATGGTGGATTTAC
TA (SEQ ID NO:6)
(SEQ ID NO:9)
CAGAGCGAGACTCT (SEQ ID NO:5)
GTACTTATCAGAAACATTTGTTTC
(SEQ ID NO:10)
CAGAGCGAGACTCT (SEQ ID NO:5)
TGCTTCTTAATTTATGGACTATCT
(SEQ ID NO:11)
CAGAGCGAGACTCT (SEQ ID NO:5)
CCGGCGGCCGGGGCCCTGCTTCTTAATTTATG
GACTATCT (SEQ ID NO:12)
CAGAGCGAGACTCTGTCA
ATCTGTCCAGAGACAGACGTCAAT
(SEQ ID NO:7)
(SEQ ID NO:13)
CAGAGCGAGACTCTGTCA
GCCTCAAGAGTGTCCAAACTGAGAC
(SEQ ID NO:7)
(SEQ ID NO:14)
CAGAGCGAGACTCTGTCA
CGGCCGGGGCCCATGCCTGCATTCACACCTCT
(SEQ ID NO:7)
TCCAGT (SEQ ID NO:15)
CAGAGCGAGACTCT (SEQ ID NO:5)
ATGCGTTTTGGATGAAATGAGATG
(SEQ ID NO:16)
In accordance with the present invention there is provided a method for the DNA fingerprinting identification of genetically related or unrelated individuals, which comprises the steps of:
a) collecting genomic DNA sample of the individuals;
b) performing DNA amplification of the DNA samples of step a) using at least two primers for the amplification of at least one marker or a primer pair of the present invention; and
c) separating the amplified DNA segments of step b);
whereby seven markers of the genomic DNA of different size are amplified and serve as DNA finger-printing of the individuals.
The DNA amplification of step b) may be effected by PCR or by asymmetric PCR procedure.
The DNA separation of step c) may be effected using an automated genetic analyzer or a gel electrophoresis procedure.
The gel electrophoresis procedure may be a urea-PAG separation method.
In accordance with the present invention there is provided a method for the DNA fingerprinting identification of human DNA samples, which comprises the steps of:
a) performing DNA amplification of the DNA samples using at least two primers for the amplification of at least one marker or a primer pair of the present invention; and
b) separating the amplified DNA segments of step a);
whereby seven markers of the genomic DNA of different size are amplified and serve as DNA finger-printing of the DNA samples.
The DNA finger-printing of the DNA samples may be for verifying transplanted tissues in research or therapeutic procedures; for single cell genetic profiling in research or therapeutic procedure; for verifying sample mix-up or contamination; or for testing, establishing or verifying paternity, maternity or consanguinity of individuals.
In accordance with the present invention there is provided a kit for simultaneous amplification of STR markers, which comprises:
a) the primer pairs listed above; and
b) typed DNA sequences of the present invention.
The kit may further comprise
c) the typed DNA sequences of all alleles of the seven markers.
REFERENCES:
patent: 5576180 (1996-11-01), Melançon et al.
Busque et al., J. of Forensic Sciences, vol. 42, pp 1147-1153, 1997.*
Tang J.Q., et al.,Mammalian Genome6:345-349, 1995.
Melancon Serge B.
Tang JianQing
Arthur Lisa B.
Cawthorn Christian
Procrea BioSciences Inc.
Renault Swabey Ogilvy
Souaya Jehanne
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