Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-11-30
2002-03-12
Jones, W. Gary (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S091200, C435S007100, C435S007200, C536S023100, C536S024300, C536S024310, C536S024320
Reexamination Certificate
active
06355422
ABSTRACT:
BACKGROUND OF THE INVENTION
Hemophilia A is one of the most common coagulation disorders with an incidence of about one in 5,000 males. The disease is caused by mutations in the factor VIII gene located on the X chromosome. About half the families with severe disease have a large genomic inversion of the factor VIII gene which separates the first 22 exons from the final four exons. This inversion results from a hotspot of recombination between a 9.5 kb region in intron 22 (Int22h1) and either of two extragenic, distal homologs, Int22h2 and Int22h3 near the Xq telomere which are repeats of Int22h1. These repeated sequences are more than 99% identical with one another (Naylor et al., 1995). Int22h2 and Int22h3 are in the opposite orientation of Int22h1 and therefore recombination produces an inversion. Intrachromosomal homologous recombination occurs between Int22h1 and the distal extragenic homolog (Int22h3), or between Int22h1 and the proximal Int22h2 homolog (types 1 and 2 inversions, respectively) (Antonarakis et al., 1995; Naylor et al., 1993; Lakich et al., 1993). Some patients have more than two copies of the extragenic homologs causing inversion types 3A and 3B.
The inversions disrupt the factor VIII gene and cause almost half of all cases of severe hemophilia A. They are detected routinely by time-consuming and expensive Southern blots using a probe from Int22h1. A rapid and inexpensive test is of particular clinical utility because carrier testing is often paid out-of-pocket due to insurance issues and confidentiality. A low cost test may facilitate more optimal use of genetic services. Successful polymerase chain reaction (PCR) amplification spanning these regions has not been reported, presumably because the homologs contain a 3.5 kb GC island of 65% G+C content and there is a 1 kb region of 79% GC within the GC island (see FIG.
4
).
A single-tube PCR assay is disclosed that combines multiplex PCR with long distance PCR (Cheng et al., 1994; Barnes, 1994) to differentiate wild-type males and females from affected males and from carrier females (FIG.
3
A).
Multiplex PCR is a rapid and convenient method, but uneven amplification is common (Chamberlain et al., 1998). Efforts have been made to achieve uniform amplification. Since primer concentration is often difficult to optimize, Shuber et al. (1995) developed a simplified optimization procedure based on the use of chimeric primers. Each primer contains a 3′ region complementary to sequence-specific recognition sites and a 5′ region made up of a universal 20-nucleotide sequence. Each individual PCR was first optimized by adjusting primer concentrations, cycling times, and annealing temperatures (Shuber et al., 1995). In another approach, two detergents, DMSO and betaine, were combined to achieve uniform amplification for three templates differing in GC contents (Baskaran et al., 1996). Additional approaches include adjusting the annealing temperature, KCl (salt) concentration, and primer concentration for each locus encountered in developing multiplex PCR of small sizes (Henegariu et al., 1997). The instant disclosure sets out a detailed, novel method, termed S-PCR, to more evenly and efficiently amplify the multiplex segments. Although S-PCR results in more even amplification, it is not a necessary step in any of the assays described herein.
The publications and other materials used herein to illuminate the background of the invention or provide additional details respecting the practice, are incorporated by reference, and for convenience are respectively grouped in the appended List of References.
SUMMARY OF THE INVENTION
The present invention provides methods for performing long distance, multiplex PCR to detect the presence of chromosomal abnormalities such as inversions, deletions/inversions and translocations.
In one aspect of the invention, chromosomal deletions/inversions are detected by performing long distance, multiplex PCR using primers which flank the site of the deletion/inversion, wherein the PCR products are used to detect the presence of the deletion/inversion.
In accordance with another aspect of the invention, inversions within a chromosome are detected by performing long distance, multiplex PCR using primers which flank the site of the inversion, wherein the pattern of PCR products which result are used to detect the presence or absence of said inversion.
In accordance with yet another aspect of the invention, translocations between two chromosomes are detected by performing long distance, multiplex polymerase chain reaction using primers which flank the site of the chromosomal breakpoints of the translocation, wherein the pattern of PCR products enables one to determine whether a translocation is present.
Other aspects of the invention are specifically directed to determining the presence of an inversion in the factor VIII gene which causes hemophilia A. These methods comprise performing long distance PCR with 2 primers, 3 primers, 4 primers, or more than 4 primers. These methods allow one to detect the presence of males who have the inversion and therefore have hemophilia A and these methods allow one to determine whether females are carriers of the inversion.
A further aspect of the invention is the use of relatively high levels of DMSO, relatively high levels of DNA polymerases, and/or the use of deaza-dGTP in long distance PCR.
The invention also provides a method of PCR, called subcycling PCR, wherein the temperature of the elongation step (or of a combined annealing/elongation step) is subcycled between at least two temperatures wherein these temperatures are below the denaturation temperature of the PCR product for each full cycle of PCR.
Yet a further aspect of the invention is the determination of DNA sequences flanking intron 22 of the factor VIII gene (Int22h1) and of sequence flanking homologs (Int22h2 and Int22h3) of this region.
Another aspect of the invention is that the long distance, multiplex PCR can be performed in a single reaction vessel.
Yet another aspect of the invention is the determination of primers which are useful in performing long distance PCR to determine the presence of an inversion in the factor VIII gene which inversion causes hemophilia A in males.
A PCR assay method is presented for detecting the inversion in the factor VIII gene which is a common cause of hemophilia A. This protocol comprises a novel single-tube PCR assay that combines overlapping PCR with long distance PCR to differentiate the wild-type, inversion and carrier. The PCR amplifies overlapping and multiplex segments of PQ (12 kb), AB (10 kb), PB (11 kb) and AQ (11 kb) with four primers P, Q, A and B directly from genomic DNA template. Performing a PCR assay to detect this inversion is challenging due to the size of the amplification (10-12 kb), the varying GC content (30-80%) and the multiplex PCR products involved (four for carrier female) and performance of a successful PCR across this region has not been previously reported. Efficient amplification of the four segments depends on three modifications to standard long distance PCR protocols: i) relatively high concentrations of DMSO; ii) addition of deaza-dGTP; and iii) relatively high concentrations of Taq/Pwo DNA polymerases. One of the segments was amplified much more efficiently than the others under standard three-temperature cycling conditions (12 seconds at 94° C., 30 seconds at 65° C., 14 minutes at 68° C.). To facilitate the uniform amplification of the multiple regions, subcycling-PCR (S-PCR) was developed. In S-PCR, the combined annealing/elongation step (or the elongation step alone if annealing and elongation are performed at separate temperatures) is composed of subcycles of shuttling between a low and a high temperature wherein these subcycling temperatures remain below the denaturation temperature, e.g., shuttling four times between 60° C. and 65° C. S-PCR produces consistent robust amplification of the various segments produced by wild-type, mutant, and carrier individuals. S-PCR generally may be advantageous i
Liu Qiang
Sommer Steve S.
City of Hope
Jones W. Gary
Rothwell Figg Ernst & Manbeck
Taylor Janell E.
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