Long distance polymerase chain reaction-based assay for detecting chromosomal rearrangements

Abstract

Methods are presented for determining the presence of an inversion in the factor VIII gene which cause hemophilia A. The methods encompass long distance, multiplex PCR (including overlapping PCR). The use of deaza-dGTP, high levels of DNA polymerases and high levels of DMSO aid in successfully performing the PCR. The use of a novel technique called subcycling PCR can also be applied as part of the methods. The technique allows for the determination of whether a person is homozygous or hemizygous for the inversion and has hemophilia A or whether a person is heterozygous for the inversion and is a carrier. The technique of long distance, multiplex PCR including use of deaza-dGTP, high levels of DNA polymerases and high levels of DMSO are applicable to the determination of the presence of other gross chromosomal aberrations such as deletions/inversions, translocations and inversions. The use of subcycling PCR can achieve efficient and more even amplification than normal two or three temperature PCR and is applicable to long distance, multiplex PCR.

1 . A method for detecting in an individual a chromosomal inversion of a first locus and a second locus, said method comprising:
 subjecting a nucleic acid sample from the individual to long distance polymerase chain reaction (PCR), utilizing a set of primers specific for regions flanking the first and second locus, and wherein each primer of said set of primers is specific for a different region flanking the first or second locus, wherein:
 said polymerase chain reaction forms a first set of products when the inversion is absent in the individual; 
 said polymerase chain reaction forms a second set of products when the individual is homozygous or hemizygous for the inversion; and 
 said polymerase chain reaction forms a third set of products when the individual is heterozygous for the inversion. 
   
     
     
         2 . The method of  claim 1 , wherein either
 (a) 3 primers are used; or   (b) 4 primers are used.   
     
     
         3 . The method of  claim 1 , wherein said PCR is performed in a single reaction vessel. 
     
     
         4 . The method of  claim 1 , wherein said set of primers comprises a first pair of primers which are specific to sequences flanking the first locus, and a second pair of primers which are specific to sequences flanking the second locus. 
     
     
         5 . The method of  claim 1 , wherein said inversion causes hemophilia. 
     
     
         6 . The method of  claim 5 , wherein said first locus comprises Int22h1 of the factor VIII gene and said second locus comprises at least one of Int22h2 and Int22h3. 
     
     
         7 . The method of  claim 1 , further comprising overlapping PCR. 
     
     
         8 . The method of  claim 1 , wherein said method produces at least one amplified product greater than 4 kilobases in length. 
     
     
         9 . The method of  claim 1 , wherein said method produces at least one amplified product greater than 10 kilobases in length. 
     
     
         10 . The method of  claim 1 , wherein said PCR comprises any combination of one, two, or three of the following:
 a) DMSO at a concentration of at least 6%;   b) DNA polymerase at a concentration of at least 0.05 units per μL; and   c) deaza-dGTP at a concentration of 33% or greater of the concentration of dGTP.   
     
     
         11 . The method of  claim 1 , wherein said PCR utilizes two temperatures, three temperatures, or subcycling PCR. 
     
     
         12 . The method of  claim 1 , wherein the first locus comprises Int22h1 of the factor VIII gene. 
     
     
         13 . The method of  claim 1 , wherein the second locus comprises at least one of Int22h2 and Int22h3. 
     
     
         14 . A method for detecting in an individual a chromosomal inversion of a first locus comprising Int22h1 and a second locus comprising at least one of Int22h2 and Int22h3 of the factor VIII gene, said method comprising:
 subjecting a nucleic acid sample from the individual to long distance polymerase chain reaction (PCR) in a single vessel, utilizing a first pair of primers which are specific to sequences flanking the first locus and a second pair of primers which are specific to sequences flanking the second locus, wherein:
 said polymerase chain reaction forms a first set of products when the inversion is absent in the individual; 
 said polymerase chain reaction forms a second set of products when the individual is homozygous or hemizygous for the inversion; and 
 said polymerase chain reaction forms a third set of products when the individual is heterozygous for the inversion.