| US 7,468,249 B2 | ||
| Detection of chromosomal disorders | ||
| Zhiyi Xie, Carlsbad, Calif. (US); Soonkap Hahn, San Clemente, Calif. (US); and Tim Watanaskul, Oceanside, Calif. (US) | ||
| Assigned to Biocept, Inc., San Diego, Calif. (US) | ||
| Filed on May 05, 2004, as Appl. No. 10/840,208. | ||
| Prior Publication US 2005/0250111 A1, Nov. 10, 2005 | ||
| Int. Cl. C12Q 1/68 (2006.01) | ||
| U.S. Cl. 435—6 | 11 Claims |
| 1. A method for detecting any one of multiple chromosomal disorders in a single assay, which method comprises the steps of:
a. making a polymerase chain reaction (PCR) mixture by mixing in a vessel components comprising:
(i) eukaryotic genomic DNA;
(ii) a plurality of pairs of forward and reverse DNA primer oligonucleotides wherein one primer of each said pair is complementary
to a 3′ sequence of a targeted segment of a first DNA strand of the eukaryotic DNA and the other primer is complementary to
the 3′ sequence of the second strand of the targeted segment, the length of the segment of eukaryotic DNA being between about
50 and about 300 base pairs, wherein one of the primers of each pair has a detectable label attached to its 5′ end, wherein
of said plurality of different pairs of primers there are pairs targeted to a segments of a selected different chromosomes
of interest, each of which is indicative of a potential chromosomal disorder, and wherein one pair is targeted for a segment
of a single control gene which is present on a chromosome other than one on which there is a targeted segment and does not
target any chromosome segment that might be indicative of a potential aneuploidy; and
(iii) PCR buffers and enzymes necessary to carry out PCR amplification;
b. conducting a PCR for between about 5 and about 60 temperature cycles to create amplified PCR products;
c. purifying said products of step (b) and obtaining single-stranded DNA having the detectable labels,
d. contacting a microarray with products of step (c), which microarray has a plurality of spots that each contain DNA oligonucleotide
probes having nucleotide sequences complementary to a nucleotide sequence of one of said strands of each of said targeted
segments;
e. hybridizing said DNA oligonucleotide probes and said PCR-amplified label-containing single-stranded products;
f. detecting the presence and relative quantity of the PCR-amplified products hybridized to the microarray by imaging the
microarray; and
g. diagnosing whether or not a chromosomal disorder exists with respect to one or more of said selected different chromosomes
by comparing said imaging of the relevant spots on said microarray for each said targeted segment of a selected chromosome
to the imaging of spots relevant to said control gene to determine a ratio of such intensities for each of a plurality of
said targeted segments, termed an I-ratio, then dividing each said I-ratio by an N-ratio obtained from similar testing of
multiple samples of genomic DNA of the same gender known to be normal to obtain a C-factor for each, averaging all said C-factors
to obtain an average C-factor, then adjusting each said I-ratio by said average C-factor, and then comparing each said adjusted
I-ratio to the respective N-ratio to determine if a disorder exists.
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