Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-05-30
2003-03-11
Jones, W. Gary (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S005000, C435S091100, C435S091200, C435S810000, C536S024330, C536S023100, C536S025300, C536S025320, C548S427000, C436S519000, C430S093000, C430S580000
Reexamination Certificate
active
06531282
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention is generally directed to the detection of short tandem repeat (STR) genetic markers in a genomic system. The present invention is more specifically directed to the simultaneous amplification of the thirteen specific and distinct polymorphic STR genetic loci of the Combined DNA Index System (CODIS) using the polymerase chain reaction (PCR) and the use of locus specific brackets (LSB) in electrophoretic calibration of their fragment lengths to determine in one, two or four PCR reactions and analytical channels the alleles of each locus contained within the multiplex system.
2. Background of the Invention
Due to their highly polymorphic nature, short tandem repeat (STR) loci are extremely useful as genetic markers. For example, the utilization of STRs has been fundamental to the identification and characterization of many disease genes, and to the development of such sophisticated technologies as linkage mapping and DNA genotyping.
STRs are short, tandemly repeated DNA sequences which are interspersed throughout the human genome at up to several hundred thousand loci (Koreth, et al. 1996; Fregeau, et al., 1997). They are also found in animals and plants where they are similarly useful as genetic markers (Orti, et al., 1997; Powell, et al., 1996). STRs are typically 2-7 base pairs in length. These loci are highly polymorphic with respect to the number of repeat units they contain and may vary in internal structure as well. Variation in the number of STR repeat units at a particular locus causes the length of the DNA at that locus to vary from allele to allele and from individual to individual. Thus, many allelic variants exist within the human population, and STRs provide a rich source of genetic markers.
While the alleles at a single STR locus may be the same for two different individuals in a population, especially if the individuals are genetically related, the probability that the alleles of two individuals will be identical at several different loci becomes smaller and smaller as the number of loci which are examined increases. If a sufficient number of loci are examined, the overall allelic pattern will be unique for each individual. As a result, and of particular importance in forensic analysis, by determining the alleles at a sufficiently large number of loci in two different DNA samples it is possible to establish with virtual certainty whether or not the two samples originally came from the same individual.
Characterization of the alleles at specific STR loci for purposes of individual identification usually begins with their PCR amplification from genomic DNA of the individual whose genome contains those loci. Although a particular repeat unit may be common to several different STR loci, identification of a particular STR locus may be effected via PCR amplification by utilizing primer pairs which hybridize to unique DNA sequences which flank the repeat region, i.e. unique sequences located 5′ and 3′ to the repeat units. Use of such unique primers makes it possible to simultaneously amplify many different STR loci in a single DNA sample, a technique referred to as multiplexing. The resulting PCR products (amplicons) from the various loci may then be separated by electrophoresis and identified by determining their lengths in comparison to known DNA standards.
While the process is in theory straightforward, several factors must be considered in order to ensure correct identification of the STR loci. For example, STR alleles are typically categorized by the number of repeat units they contain, which is convenient for entry into databases. In forensic applications, the preferred alleles are for the most part composed of regular repeat units of a size that is optimally resolved with current electrophoretic technology, usually four bases long (Edwards et al., 1991; Perez-Lezaun, et al., 1997). However, some of the tetrameric STR loci useful in forensic analysis contain non-integer alleles which differ in size by only 1 or 2 nucleotides (Puers,
Science
272: 1755-1762, 1993). Therefore, an error of <0.5 nucleotide is necessary for accurate sizing of these alleles. This level of resolution has been reliably obtained only with instruments intended for automated DNA sequencing analysis. Such instruments are designed to analyze the length of DNA fragments produced by the Sanger chain termination chemistry employed in DNA sequencing (Connell, et al., 1987). This sequencing chemistry produces a set of DNA fragments each of which terminates with 1 of the 4 dideoxyribonucleotides. The fragments in the set produced differ in length by only one nucleotide and form a “ladder” of successively longer fragments which must be reliably resolved from one another by electrophoresis.
Electrophoresis instruments used to separate sequencing fragments utilize a slab gel or capillary format, but vary in their method of detection. For example, with the ALF™ and ALFexpress™ slab gel systems (Amersham Pharmacia Biotech, Piscataway, N.J.) all 4 dideoxyribonucleotide-terminating fragment types are labeled with the same fluorophore. The 4 fragments types must therefore be assigned to 4 different lanes for electrophoresis in order to distinguish among them. The ABI Prism 310® capillary and ABI Prism 377® slab gel systems (PE Applied Biosystems, Foster City, Calif.) allow electrophoresis of all fragments in the same channel or lane because different fluorophores are assigned to each of the 4 dideoxyribonucleotides. The newer Visible Genetics Microgene Clipper™ (Visible Genetics, Inc., Toronto, Canada) employs two fluorophores to identify two sets of fragments which are electrophoresed in two gel lanes. The Hitachi FMBIO® II Fluorescent Scanner employs 3 fluorophores. All of these instruments employ computerized measurement of the migration time of each fragment over a fixed distance or time (Hitachi) in order to “call” the nucleotide sequence of the DNA molecule under analysis. Since consecutive fragments differ from each other by only one nucleotide (their sequences being otherwise homologous) their relative mobilities are almost identical. Thus the small differences in length can be measured accurately and allow the alignment of fragments relative to each other when constructing the sequencing “ladder” which represents the oligonucleotide sequence.
Sequencing gel electrophoretic instruments therefore provide the resolution necessary to discriminate between DNA fragments which differ in length by only 1 nucleotide (Carrano et al., 1989). When these instruments were later adapted to STR analysis a problem arose, because there was no longer an entire series of similarly mobile fragments to be aligned in correct series, but rather only one or two alleles from each amplified locus, depending upon whether the subject was homo- or heterozygous. Now the lengths of these fragments had to be measured by means of calibration standards in order to assign them the correct allele number. The standards employed were no longer almost identical in sequence like the series of Sanger gene termination fragments, but were heterologous, usually produced by restriction enzymatic digestion of microbial DNA. Sequence differences and the ensuing electrophoretic mobility differences (Frank, R. et al., 1979) between the calibration standards and their target DNA caused a large and variable calibration error of up to 3 nt (AMPFISTR® User's Manual, 1998). Manufacturers have corrected this error with either a heterologous or chemically compounded internal and external lane standard labeled with its own fluorophore distinct from that of their target alleles combined with an external lane allelic ladder (Schumm, J. W., 1997). Allelic ladders themselves are not co-electrophoresed as internal lane standards because by migrating in the same position as the sample alleles they could interfere with their measurement by obscuring small sample peaks, by spectral interference or by peak broadening.
LSB overcome many of the difficulties in calibration
Dau Peter C.
Liu Debang
Jones W. Gary
Oligotrail, LLC
Taylor Janell E.
Whitham Curtis & Christofferson, P.C.
LandOfFree
Multiplex amplification and analysis of selected STR loci does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Multiplex amplification and analysis of selected STR loci, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Multiplex amplification and analysis of selected STR loci will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3055805