Cloning multiple control sequences into chromosomes or into artificial centromeres

Abstract

Artificially synthesizing 29 homozygous cystic fibrosis core panel controls demonstrates placing multiple homozygous mutant sequences on the same single control DNA sequence to streamline quality control by minimizing extra control assays, time, and costly formatted test materials and testing all controls during every test. Any rare or unavailable reported DNA sequence can be PCR amplified using primer pairs synthesized with the designated mutation or variant sequence with paired adjacent upstream and downstream primers to amplify target sequences in total genomic DNA.

1 . A method of optimizing quality control in a genetic test assay, the method comprising the steps of:
 testing for the presence of a normal gene nucleotide sequence portion at a pre-selected gene locus;   testing for the presence of at least a first mutant gene nucleotide sequence portion at the pre-selected gene locus; and   testing for interference by at least a first homologous nucleotide sequence portion.   
     
     
         2 . The method of  claim 1 , wherein the genetic test assay is a hybridization based assay. 
     
     
         3 . The method of  claim 1 , wherein the step of testing for interference by homologous nucleotide sequence portion, involves the step of providing a homologous nucleotide sequence control. 
     
     
         4 . The method of  claim 3 , wherein the homologous nucleotide sequence control includes at least a first homologous nucleotide sequence portion which is homologous to the normal gene nucleotide sequence of the pre-selected gene locus, and is devoid of a hybridizing nucleotide sequence portion of the pre-selected gene locus, wherein the hybridizing nucleotide sequence portion is sufficiently large to prevent detection of the pre-selected gene locus by the assay. 
     
     
         5 . The method of  claim 4 , wherein the hybridizing nucleotide sequence portion is the normal gene nucleotide sequence portion. 
     
     
         6 . The method of  claim 4 , wherein the hybridizing nucleotide sequence portion is substantially the entire gene nucleotide sequence of the pre-selected gene locus. 
     
     
         7 . A control comprising:
 At least a first nucleotide sequence, wherein the at least a first nucleotide sequence includes at least a first homologous nucleotide sequence portion and wherein the at least a first nucleotide sequence lacks a sufficiently large segment of the gene nucleotide sequence at an at least a first pre-selected gene locus to preclude detection of the gene nucleotide sequence at the at least a first pre-selected gene locus by an assay, and   Adapted for use in the assay, wherein the assay is for the detection of mutations in the gene nucleotide sequence at the at least a first pre-selected gene locus.   
     
     
         8 . The control of  claim 7 , wherein the at least a first nucleotide sequence is total genomic DNA having the sufficiently large segment of the gene nucleotide sequence at the at least a first pre-selected gene locus removed. 
     
     
         9 . The control of  claim 8  further comprising:
 at least a second nucleotide sequence containing a sufficiently large segment of an at least a first pre-selected mutant gene nucleotide sequence so as to be detectable by the assay.   
     
     
         10 . The control of  claim 9  wherein the at least a second nucleotide sequence contains sufficiently large segments of at least second and third pre-selected mutant gene nucleotide sequences so as to be detectable by the assay. 
     
     
         11 . The control of  claim 10  wherein the number of copies of the at least a first nucleotide sequence is approximately equal to the number of copies of the at least a second nucleotide sequence. 
     
     
         12 . A control comprising:
 a first nucleotide sequence portion, wherein the first nucleotide sequence portion includes at least a first homologous nucleotide sequence portion having a sufficient length to adequately imitate corresponding normal or mutant nucleotide sequence portions found at a pre-selected gene locus being tested in an assay and containing at least a first distinct nucleotide species, wherein the at least a first distinct nucleotide species is not found in either the normal or mutant nucleotide sequence portions and wherein the at least a first distinct nucleotide species is suitably distinct as to provide a means for confirmation that the first homologous nucleotide sequence portion is not being detected in the assay by a normal or a mutant sequence primer used in the assay; and   a second nucleotide sequence portion, wherein the second nucleotide sequence portion includes a sufficiently large segment of a first mutant gene nucleotide sequence portion found at the pre-selected gene locus so as to be detectable by the assay.   
     
     
         13 . The control of  claim 12 , further comprising:
 at least a second homologous nucleotide sequence portion substantially adjacent the first nucleotide sequence portion, wherein the at least a second homologous nucleotide sequence portion has a sufficient length to adequately imitate corresponding normal or mutant nucleotide sequence portions of the pre-selected gene locus being tested in the assay and containing at least a first distinct nucleotide species, wherein the at least a first distinct nucleotide species is not found in either the normal or mutant nucleotide sequence portions and wherein the at least a first distinct nucleotide species is suitably distinct as to provide a means for confirmation that the at least a second homologous nucleotide sequence portion is not being detected in the assay by a normal or a mutant sequence primer used in the assay   
     
     
         14 . The control of  claim 13 , wherein the second nucleotide sequence portion further comprises:
 A sufficiently large segment of an at least a second mutant gene nucleotide sequence portion found at the pre-selected gene locus so as to be detectable by the assay