Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-06-07
2001-04-24
Jones, W. Gary (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S091200, C536S023100, C536S024310, C536S024330
Reexamination Certificate
active
06221598
ABSTRACT:
FIELD OF THE INVENTION
The present invention is generally directed to the detection of genetic markers in a genomic system. The present invention is more specifically directed to the simultaneous amplification of multiple distinct polymorphic genetic loci using the polymerase chain reaction or other amplification systems to determine in one reaction the alleles of each locus contained within the multiplex system.
CITED REFERENCES
A full bibliographic citation of the references cited in this application can be found in the section preceding the claims.
DESCRIPTION OF THE PRIOR ART
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.
Many loci, at least in the human genome, contain a polymorphic STR region. STR loci consist of short, repetitive sequence elements of 3 to 7 base pairs in length. It is estimated that there are 2,000,000 expected trimeric and tetrameric STRs present as frequently as once every 15 kilobases (kb) in the human genome (Edwards et al. 1991; Beckmann and Weber 1992). Nearly half of the STR loci studied by Edwards et al. (1991) are polymorphic, which provides a rich source of genetic markers. Variation in the number of repeat units at a particular locus is responsible for the observed polymorphism reminiscent of VNTR loci (Nakamura et al. 1987) and minisatellite loci (Jeffreys et al. 1985), which contain longer repeat units, and microsatellite or dinucleotide repeat loci (Litt and Luty 1989, Tautz 1989, Weber and May 1989, Beckmann and Weber 1992).
Polymorphic STR loci are extremely useful markers for human identification, paternity testing and genetic mapping. STR loci may be amplified via the polymerase chain reaction (PCR) by employing specific primer sequences identified in the regions flanking the tandem repeat.
Alleles of these loci are differentiated by the number of copies of the repeat sequence contained within the amplified region and are distinguished from one another following electrophoretic separation by any suitable detection method including radioactivity, fluorescence, silver stain, and color.
To minimize labor, materials and analysis time, it is desirable to analyze multiple loci and/or more samples simultaneously. One approach for reaching this goal involves amplification of multiple loci simultaneously in a single reaction. Such “multiplex” amplifications have been described extensively in the literature. Multiplex amplification sets have been extensively developed for analysis of genes related to human genetic diseases such as Duchenne Muscular Dystrophy (Chamberlain et al. 1988, Chamberlain et al. 1989, Beggs et al. 1990, Clemens et al. 1991, Schwartz et al. 1992, Covone et al. 1992), Lesch-Nyhan Syndrome (Gibbs et al. 1990), Cystic Fibrosis (Estivill et al. 1991, Fortina et al. 1992, Ferrie et al. 1992, Morral and Estivill, 1992), and Retinoblasma (Lohmann et al. 1992). Multiplex amplification of polymorphic microsatellite markers (Clemens et al. 1991, Schwartz et al. 1992, Huang et al. 1992) and even STR markers (Edwards et al. 1992, Kimpton et al. 1993, Hammond et al. 1994) have been described.
These amplified products are generally separated by one of several methods of electrophoresis known to those skilled in the art. Several well-known methods of detection of the amplified products have also been described. While ethidium bromide staining of amplified fragments is employed in some cases, in others it is preferred to use methods which label only one of the two strands of the amplified material. Examples of this include radioactive or fluorescent labeling of one of the two primers prior to the amplification of a locus. One of the more sophisticated approaches to detection is the use of different fluorescent labels to allow detection of amplified materials representing different loci, but existing in the same space following electrophoresis. The products of the different loci are differentiated with the use of filters, which allow visualization of one fluorescent label at a time.
Reference is made to International Publications WO 93/18177 and WO 93/18178 to Fortina et al., which are directed to methods and kits for diagnosing diseases such as Cystic Fibrosis and &bgr;-thalassemia, respectively, using an allele-specific multiplex polymerase chain reaction system. According to Fortina et al., multiplex PCR has also been used for simultaneous amplification of multiple target sequences, permitting mutant allele scanning using two lanes of an agarose gel.
Ballabio et al. (1991), disclose a single-tube, multiplex allele specific PCR test using two different dye-tagged fluorescent primers for detection of the ▴F508 cystic fibrosis mutation.
While there are multiplex amplification procedures for specific loci, the use of multiplex amplification procedures is greatly desired for the detection of alleles in other types of loci such as specific STR loci.
SUMMARY OF THE INVENTION
It is therefore an object of the present invention to provide a method for the simultaneous amplification of multiple distinct polymorphic STR loci using PCR or other amplification systems to determine, in one reaction, the alleles of each locus contained within the multiplex. These combinations of specific loci into multiplexes have not been heretofore shown.
It is also an object of the present invention to provide a method and a kit specific for multiplex amplifications comprising specified loci.
These and other objects are addressed by the present invention which is directed to a method of simultaneously analyzing or determining the alleles present at each individual locus of each multiplex. This method comprises the steps of (1) obtaining at least one DNA sample to be analyzed, wherein the DNA sample has at least two loci which can be amplified together; (2) amplifying the STR sequences in the DNA sample; and (3) detecting the amplified materials in a fashion which reveals the polymorphic nature of the systems employed.
The present invention is also directed to a method of simultaneously analyzing multiple STR sequences wherein at least one of the loci is selected from the group consisting of: HUMCSF1PO, HUMTPOX, HUMVWFA31, HUMFESFPS, HUMBFXIII (F13B), HUMLIPOL, HSAC04 (ACTBP2), HUMCYP19, HUMPLA2A1, HUMAPOA2, HUMCD4, HUMF13A01 and HUMMYOPK (Myotonic).
Specifically, the present invention is directed to a method of simultaneously analyzing multiple STR sequences in the following groups of loci: HUMTH01 and HUMCSF1PO; HUMTH01 and HUMCD4; HUMTH01 and HUMTPOX; HUMF13A01 and HUMFABP; HUMF13A01 and HUMMYOPK (Myotonic); HUMF13A01 and HUMBFXIII (F13B); HUMBFXIII (F13B) and HUMFESFPS; HUMBFXIII (F13B) and HUMLIPOL; HUMHPRTB and HUMFESFPS; HSAC04 (ACTBP2) and HUMCYP19; HUMCSF1PO, HUMTPOX and HUMTH01; HUMHPRTB, HUMFESFPS and HUMVWFA31; HSAC04 (ACTBP2), HUMCYP19 and HUMPLA2A1; HSAC04 (ACTBP2) and HUMFABP; HUMAPOA2, HUMCYP19 and HUMPLA2A1; HUMCD4, HUMCSF1PO and HUMTH01; HUMCYP19, HUMFABP and HUMPLA2A1; HUMCYP19, HUMHPRTB and HUMPLA2A1; HUMF13A01, HUMFABP and HUMCD4; HUMHPRTB, HUMFESFPS and HUMLIPOL; HUMF13A01, HUMFABP and HUMCD4; HUMHPRTB, HUMBFXIII (F13B) and HUMPLA2A1; HUMHPRTB, HUMBFXIII (F13B) and HUMTPOX; HUMHPRTB, HUMBFXIII (F13B) and HUMFESFPS; HUMCSF1PO, HUMTPOX and HUMCD4; HUMHPRTB, HUMFESFPS and HUMMYOPK (Myotonic); HUMCSF1PO, HUMTH01 and HUMCD4; HUMCSF1PO, HUMTH01 and HUMVWFA31; HUMHPRTB, HUMBFXIII (F13B) and HUMLIPOL; HUMCSF1PO, HUMTPOX, HUMTH01 and HUMVWFA31; HUMHPRTB, HUMFESFPS, HUMBFXIII (F13B) and HUMLIPOL; HUMCSF1PO, HUMTPOX, HUMTH01 and HUMCD4; and HUMCSF1PO, HUMTH01, HUMTPOX and HUMCD4.
The present invention provides a high throughput method for the detection and analysis of polymorphic genetic markers using specific combinations of loci and specified conditions. By selection of the appropriate detection method, the process can be used in laboratories which have only a power supply and a standard apparatus for polyacrylamide gel
Lins Ann M.
Schumm James W.
Sprecher Cynthia J.
Enewold Jeanine
Frenchick Grady J.
Jones W. Gary
King Karen B.
Michael & Best & Friedrich LLP
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