Comparative genomic hybridization array method for preimplantation genetic screening

Abstract

A method for determining the presence of a copy number imbalance in genomic DNA of a test sample is provided. The method can separately measure hybridization of a single test sample to a first hybridization array and hybridization of a plurality of reference samples to a plurality of other, respective test arrays. A determination of copy number can be based on the best fit reference array, relative to the test array. The best fit can be determined based on the closest or most similar signal-to-noise ratio of the measured signals.

1 . A method for determining the presence of a copy number imbalance in genomic DNA of a test sample, comprising:
 a) labeling sample genomic DNA from a test sample, or an amplification product thereof, to form labeled test DNA;   b) hybridizing the labeled test DNA to a first hybridization array;   c) labeling first reference genomic DNA from a reference sample, or an amplification product thereof, to form labeled first reference DNA;   d) hybridizing the labeled first reference DNA to a second hybridization array;   e) labeling second reference genomic DNA from a second reference sample, or an amplification product thereof, to form labeled second reference DNA;   f) hybridizing the labeled second reference DNA to a third hybridization array;   g) analyzing the first hybridization array after the hybridizing of the labeled test DNA to determine signal intensities produced by hybridization of the labeled test DNA;   h) analyzing the second hybridization array after the hybridizing of the labeled first reference DNA to determine signal intensities produced by hybridization of the labeled first reference DNA;   i) analyzing the third hybridization array after the hybridizing of the labeled second reference DNA to determine signal intensities produced by hybridization of the labeled second reference DNA; and   j) estimating the copy number of at least one region of the sample genomic DNA by comparing the signal intensities of the first hybridization array with the signal intensities of at least one of the second hybridization array and the third hybridization array.   
     
     
         2 . The method of  claim 1 , wherein the labeled first reference DNA includes at least one copy number change in one or more pre-defined regions of the genome, relative to the labeled test DNA. 
     
     
         3 . The method of  claim 2 , wherein the labeled second reference DNA includes at least one pre-defined region which does not have the same copy number change relative to the labeled test DNA, as the labeled first reference DNA does. 
     
     
         4 . The method of  claim 2 , wherein the signal intensity produced by hybridization of the labeled test DNA is compared to the signal intensity produced by hybridization of the labeled first reference DNA in the one or more pre-defined regions, the signal intensity produced by hybridization of the labeled test DNA is compared to the signal intensity produced by hybridization of the labeled second reference DNA in the one or more pre-defined regions, and the method further comprises determining a dynamic range of the method based on an expected copy number. 
     
     
         5 . The method of  claim 1 , wherein the labeled first reference DNA is from a male animal of a first species and the labeled second reference DNA is from a female animal of the first species. 
     
     
         6 . The method of  claim 1 , wherein the labeled first reference DNA and the labeled second reference DNA comprises a mixture of DNA from a male and from a female of a same species of animal. 
     
     
         7 . The method of  claim 1 , wherein the signal intensity produced by hybridization of the labeled test DNA is compared to the signal intensity produced by hybridization of the labeled first reference DNA in the one or more pre-defined regions, to determine a first estimate of copy number, the signal intensity produced by hybridization of the labeled test DNA is compared to the signal intensity produced by hybridization of the labeled second reference DNA in the one or more pre-defined regions, to determine a second estimate of copy number, and the first and second estimates of copy number are combined to obtain an overall estimate of copy number. 
     
     
         8 . The method of  claim 1 , wherein signal intensities are normalized before the copy number is estimated. 
     
     
         9 . The method of  claim 1 , wherein the first reference genomic DNA from a reference sample, or an amplification product thereof, comprises an amplification product produced by a first amplification technique, and the second reference genomic DNA from a reference sample, or an amplification product thereof, comprises an amplification product produced by the same first amplification technique. 
     
     
         10 . The method of  claim 1 , wherein the first reference genomic DNA from a reference sample, or an amplification product thereof, comprises a plurality of different references at different respective concentrations. 
     
     
         11 . The method of  claim 1 , further comprising:
 hybridizing a labeled third reference DNA to a fourth hybridization array;   hybridizing a labeled fourth reference DNA to a fifth hybridization array;   hybridizing a labeled fifth reference DNA to a sixth hybridization array;   wherein the estimating the copy number comprises comparing the signal intensities of the first hybridization array with signal intensities generated by each of the second, third, fourth, fifth, and sixth hybridization arrays.   
     
     
         12 . The method of  claim 1 , further comprising determining an aneuploidy status of a human polar body or embryo based on the copy number estimate. 
     
     
         13 . The method of  claim 12 , further comprising implanting an embryo based on the aneuploidy status determined in an IVF procedure. 
     
     
         14 . The method of  claim 1 , further comprising isolating genomic DNA from the test sample to form the sample genomic DNA or amplification product thereof. 
     
     
         15 . The method of  claim 1 , wherein the test sample comprises at least one cell from an embryo or associated biopsy. 
     
     
         16 . The method of  claim 1 , wherein the first genomic reference DNA comprises DNA obtained from tissue or cells of an animal having a chromosomal anomaly. 
     
     
         17 . The method of  claim 1 , wherein the first genomic reference DNA comprises DNA obtained from mosaic tissue or cells. 
     
     
         18 . The method of  claim 1 , wherein the labeled first reference DNA has a first concentration of DNA and comprises an amplification product of a first concentration of a first DNA, and the labeled second reference DNA comprises an amplification product of the first DNA produced after diluting the first concentration of first DNA. 
     
     
         19 - 21 . (canceled) 
     
     
         22 . A library of reference array data sets stored in a processor, each reference array data set comprising data gathered from a respective reference array during a copy number hybridization assay carried out on the respective reference array, wherein
 (a) each reference array from which a respective data set is gathered, is substantially identical or identical to each other reference array from which a data set is gathered;   (b) each copy number hybridization assay, from which a respective data set is gathered, is carried out under the same or one or more different conditions than each other copy number hybridization assay from which a data set is gathered; and   (c) at least two reference array data sets of the library differ from each other.   
     
     
         23 - 26 . (canceled) 
     
     
         27 . A kit comprising:
 a first copy number hybridization array;   a second copy number hybridization array identical to the first copy number hybridization array;   a third copy number hybridization array identical to the first copy number hybridization array;   a first reference genomic DNA;   a second reference genomic DNA; and   instructions for (a) comparing test results generated from a hybridization assay carried out on the first copy number hybridization array, to test results generated from a hybridization assay carried out on the second copy number hybridization array using the first reference genomic DNA, and for (b) comparing test results generated from a hybridization assay carried out on the first copy number hybridization array, to test results generated from a hybridization assay carried out on the third copy number hybridization array using the second reference genomic DNA.   
     
     
         28 - 30 . (canceled)