(19) United States
`(12) Patent Application Publication (10) Pub. No.: US 2004/0137470 A1
`Dhallan
`(43) Pub. Date:
`Jul. 15, 2004
`
`US 2004O13747OA1
`
`(54) METHODS FOR DETECTION OF GENETIC
`DSORDERS
`
`(76) Inventor: Ravinder S. Dhallan, Bethesda, MD
`(US)
`Correspondence Address:
`MORRISON & FOERSTER LLP
`755 PAGE MILL RD
`PALO ALTO, CA 94304-1018 (US)
`(21) Appl. No.:
`10/661,165
`(22) Filed:
`Sep. 11, 2003
`
`(60) Provisional application No. 60/378,354, filed on May
`8, 2002. Provisional application No. 60/360,232, filed
`on Mar. 1, 2002. Provisional application No. 60/378,
`354, filed on May 8, 2002. Provisional application
`No. 60/360,232, filed on Mar. 1, 2002.
`Publication Classification
`
`(51) Int. Cl." ....................................................... C12O 1/68
`(52) U.S. Cl. .................................................................. 435/6
`
`(57)
`
`ABSTRACT
`
`Related U.S. Application Data
`(63) Continuation-in-part of application No. PCT/US03/
`06198, filed on Feb. 28, 2003, and which is a con
`tinuation of application No. 10/093,618, filed on Mar.
`11, 2002.
`Continuation of application No. PCT/US03/27308,
`filed on Aug. 29, 2003.
`Continuation-in-part of application No. 10/376,770,
`filed on Feb. 28, 2003, and which is a continuation
`in-part of application No. 10/093,618, filed on Mar.
`11, 2002.
`
`The invention provides a method useful for detection of
`genetic disorders. The method comprises determining the
`Sequence of alleles of a locus of interest, and quantitating a
`ratio for the alleles at the locus of interest, wherein the ratio
`indicates the presence or absence of a chromosomal abnor
`mality. The present invention also provides a non-invasive
`method for the detection of chromosomal abnormalities in a
`fetus. The invention is especially useful as a non-invasive
`method for determining the sequence of fetal DNA. The
`invention further provides methods of isolation of free DNA
`from a Sample.
`
`PROGENITY - EXHIBIT 1098
`Progenity, Inc. v. Natera, Inc.
`IPR2021-00282
`
`

`

`Patent Application Publication
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`Jul. 15, 2004 Sheet 1 of 33
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`US 2004/O137470 A1
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`METHODS FOR DETECTION OF GENETIC
`DISORDERS
`
`CROSS-REFERENCE TO RELATED
`APPLICATIONS
`0001. This application is a continuation-in-part of PCT/
`US03/06198, filed Feb. 28, 2003, which claims benefit under
`35 U.S.C. S119(e) of U.S. Provisional Patent Application
`No. 60/378,354, filed May 8, 2002, and which is a continu
`ation-in-part of U.S. patent application Ser. No. 10/093,618,
`filed Mar. 11, 2002, which claims benefit under 35 U.S.C. S
`119(e) of U.S. Provisional Patent Application No. 60/360,
`232, filed Mar. 1, 2002; this application is also a continua
`tion of PCT/US
`, Method for Detection of Genetic
`Disorders, filed Aug. 29, 2003 (Attorney Docket No. 54331
`20004.41); this application further is a continuation-in-part
`of U.S. patent application Ser. No. 10/376,770, filed Feb. 28,
`2003, which claims benefit under 35 U.S.C. S 119(e) of U.S.
`Provisional Patent Application No. 60/378,354, filed May 8,
`2002, and which is a continuation-in-part of U.S. patent
`application Ser. No. 10/093,618, filed Mar. 11, 2002, which
`claims benefit under 35 U.S.C. S.119(e) of U.S. Provisional
`Patent Application No. 60/360,232, filed Mar. 1, 2002. The
`contents of these applications are incorporated by reference
`in their entirety.
`
`BACKGROUND OF THE INVENTION
`0002) 1. Field of the Invention
`0003. The present invention is directed to a method for
`the detection of genetic disorders including chromosomal
`abnormalities and mutations. The present invention provides
`a rapid, non-invasive method for determining the Sequence
`of DNA from a fetus. The method is especially useful for
`detection of chromosomal abnormalities in a fetus including
`translocations, transversions, monoSomies, trisomies, and
`other aneuplodies, deletions, additions, amplifications,
`translocations and rearrangements.
`0004 2. Background Art
`0005 Chromosomal abnormalities are responsible for a
`Significant portion of genetic defects in liveborn humans.
`The nucleus of a human cell contains forty-six (46) chro
`mosomes, which contain the genetic instructions, and deter
`mine the operations of the cell. Half of the forty-six chro
`mosomes originate from each parent. Except for the SeX
`chromosomes, which are quite different from each other in
`normal males, the chromosomes from the mother and the
`chromosomes from the father make a matched Set. The pairs
`were combined when the egg was fertilized by the Sperm.
`Occasionally, an error occurs in either the formation or
`combination of chromosomes, and the fertilized egg is
`formed with too many or too few chromosomes, or with
`chromosomes that are mixed in Some way. Because each
`chromosome contains many genes, chromosomal abnor
`malities are likely to cause Serious birth defects, affecting
`many body Systems and often including developmental
`disability (e.g., mental retardation).
`0006 Cells mistakenly can rejoin broken ends of chro
`mosomes, both Spontaneously and after exposure to chemi
`cal compounds, carcinogens, and irradiation. When rejoin
`ing occurs within a chromosome, a chromosome Segment
`between the two breakpoints becomes inverted and is clas
`
`sified as an inversion. With inversions, there is no loss of
`genetic material; however, inversions can cause disruption
`of a critical gene, or create a fusion gene that induces a
`disease related condition.
`0007. In a reciprocal translocation, two non-homologous
`chromosomes break and exchange fragments. In this Sce
`nario, two abnormal chromosomes result: each consists of a
`part derived from the other chromosome and lacks a part of
`itself. If the translocation is of a balanced type, the indi
`vidual will display no abnormal phenotypes. However, dur
`ing germ-cell formation in the translocation-bearing indi
`viduals, the proper distribution of chromosomes in the egg
`or Sperm occasionally fails, resulting in miscarriage, mal
`formation, or mental retardation of the offspring.
`0008. In a Robertsonian translocation, the centromeres of
`two acrocentric (a chromosome with a non-centrally located
`centromere) chromosomes fuse to generate one large meta
`centric chromosome. The karyotype of an individual with a
`centric fusion has one less than the normal diploid number
`of chromosomes.
`0009 Errors that generate too many or too few chromo
`Somes can also lead to disease phenotypes. For example, a
`missing copy of chromosome X (monosomy X) results in
`Tumer's Syndrome, while an additional copy of chromo
`Some 21 results in Down's Syndrome. Other diseases such
`as Edwards Syndrome, and Patau Syndrome are caused by
`an additional copy of chromosome 18, and chromosome 13,
`respectively.
`0010. One of the most common chromosome abnormali
`ties is known as Down Syndrome. The estimated incidence
`of Down's syndrome is between 1 in 1,000 to 1 in 1,100 live
`births. Each year approximately 3,000 to 5,000 children are
`born in the U.S. with this chromosomal disorder. The vast
`majority of children with Down syndrome (approximately
`95 percent) have an extra chromosome 21. Most often, the
`extra chromosome originates from the mother. However, in
`about 34 percent of people with Down Syndrome, a trans
`location between chromosome 21 and either 14 or 22 is
`responsible for the genetic abnormality. Finally, another
`chromosome problem, called mosaicism, is noted in about 1
`percent of individuals with Down's syndrome. In this case,
`Some cells have 47 chromosomes and others have 46 chro
`mosomes. Mosiacism is thought to be the result of an error
`in cell division Soon after conception.
`0011 Chromosomal abnormalities are congential, and
`therefore, prenatal diagnosis can be used to determine the
`health and condition of an unborn fetus. Without knowledge
`gained by prenatal diagnosis, there could be an untoward
`outcome for the fetus or the mother or both. Congenital
`anomalies account for 20 to 25% of perinatal deaths. Spe
`cifically, prenatal diagnosis is helpful for managing the
`remaining term of the pregnancy, planning for possible
`complications with the birth process, preparing for problems
`that can occur in the newborn infant, and finding conditions
`that may affect future pregnancies.
`0012. There are a variety of non-invasive and invasive
`techniques available for prenatal diagnosis including ultra
`Sonography, amniocentesis, chorionic Villus Sampling
`(CVS), fetal blood cells in maternal blood, maternal serum
`alpha-fetoprotein, maternal Serum beta-HCG, and maternal
`Serum estriol. However, the techniques that are non-invasive
`
`

`

`US 2004/O137470 A1
`
`Jul. 15, 2004
`
`are leSS Specific, and the techniques with high Specificity and
`high Sensitivity are highly invasive. Furthermore, most
`techniques can be applied only during Specific time periods
`during pregnancy for greatest utility.
`0013 Ultrasonography
`0.014. This is a harmless, non-invasive procedure. High
`frequency Sound waves are used to generate visible images
`from the pattern of the echoes made by different tissues and
`organs, including the fetus in the amniotic cavity. The
`developing embryo can be visualized at about 6 weeks of
`gestation. The major internal organs and extremities can be
`assessed to determine if any are abnormal at about 16 to 20
`weeks gestation.
`0.015. An ultrasound examination can be useful to deter
`mine the size and position of the fetus, the amount of
`amniotic fluid, and the appearance of fetal anatomy; how
`ever, there are limitations to this procedure. Subtle abnor
`malities, Such as Down Syndrome, where the morphologic
`abnormalities are often not marked, but only Subtle, may not
`be detected at all.
`0016 Amniocentesis
`0.017. This is a highly invasive procedure in which a
`needle is passed through the mother's lower abdomen into
`the amniotic cavity inside the uterus. This procedure can be
`performed at about 14 weeks gestation. For prenatal diag
`nosis, most amniocenteses are performed between 14 and 20
`weeks gestation. However, an ultrasound examination is
`performed, prior to amniocentesis, to determine gestational
`age, position of the fetus and placenta, and determine if
`enough amniotic fluid is present. Within the amniotic fluid
`are fetal cells (mostly derived from fetal skin) which can be
`grown in culture for chromosomal, biochemical, and
`molecular biologic analyses.
`0.018. Large chromosomal abnormalities, such as extra or
`missing chromosomes or chromosome fragments, can be
`detected by karyotyping, which involves the identification
`and analysis of all 46 chromosomes from a cell and arranges
`them in their matched pairs, based on Subtle differences in
`Size and structure. In this Systematic display, abnormalities
`in chromosome number and Structure are apparent. This
`procedure typically takes 7-10 days for completion.
`0.019 While amniocentesis can be used to provide direct
`genetic information, risks are associated with the procedure
`including fetal loSS and maternal Rh Sensitization. The
`increased risk for fetal mortality following amniocentesis is
`about 0.5% above what would normally be expected. Rh
`negative mothers can be treated with RhoGam.
`0020 Chorionic Villus Sampling (CVS)
`0021. In this procedure, a catheter is passed via the
`vagina through the cervix and into the uterus to the devel
`oping placenta with ultrasound guidance. The introduction
`of the catheter allows cells from the placental chorionic villi
`to be obtained and analyzed by a variety of techniques,
`including chromosome analysis to determine the karyotype
`of the fetus. The cells can also be cultured for biochemical
`or molecular biologic analysis. Typically, CVS is performed
`between 9.5 and 12.5 weeks gestation.
`0022 CVS has the disadvantage of being an invasive
`procedure, and it has a low but Significant rate of morbidity
`
`for the fetus; this loss rate is about 0.5 to 1% higher than for
`Women undergoing amniocentesis. Rarely, CVS can be
`associated with limb defects in the fetus. Also, the possibil
`ity of maternal Rh Sensitization is present. Furthermore,
`there is also the possibility that maternal blood cells in the
`developing placenta will be sampled instead of fetal cells
`and confound chromosome analysis.
`0023 Maternal Serum Alpha-Fetoprotein (MSAFP)
`0024. The developing fetus has two major blood pro
`teins-albumin and alpha-fetoprotein (AFP). The mother
`typically has only albumin in her blood, and thus, the
`MSAFP test can be utilized to determine the levels of AFP
`from the fetus. Ordinarily, only a small amount of AFP gains
`access to the amniotic fluid and crosses the placenta to
`mother's blood. However, if the fetus has a neural tube
`defect, then more AFP escapes into the amniotic fluid.
`Neural tube defects include anencephaly (failure of closure
`at the cranial end of the neural tube) and spina bifida (failure
`of closure at the caudal end of the neural tube). The
`incidence of such defects is about 1 to 2 births per 1000 in
`the United States. Also, if there are defects in the fetal
`abdominal wall, the AFP from the fetus will end up in
`maternal blood in higher amounts.
`0025. The amount of MSAFP increases with gestational
`age, and thus for the MSAFP test to provide accurate results,
`the gestational age must be known with certainty. Also, the
`race of the mother and presence of gestational diabetes can
`influence the level of MSAFP that is to be considered
`normal. The MSAFP is typically reported as multiples of the
`mean (MoM). The greater the MoM, the more likely a defect
`is present. The MSAFP test has the greatest sensitivity
`between 16 and 18 weeks gestation, but can be used between
`15 and 22 weeks gestation. The MSAFP tends to be lower
`when Down's Syndrome or other chromosomal abnormali
`ties is present.
`0026. While the MSAFP test is non-invasive, the MSAFP
`is not 100% specific. MSAFP can be elevated for a variety
`of reasons that are not related to fetal neural tube or
`abdominal wall defects. The most common cause for an
`elevated MSAFP is a wrong estimation of the gestational age
`of the fetus. Therefore, results from an MSAFP test are never
`considered definitive and conclusive.
`0027 Maternal Serum Beta-HCG
`0028 Beginning at about a week following conception
`and implantation of the developing embryo into the uterus,
`the trophoblast will produce detectable beta-HCG (the beta
`Subunit of human chorionic gonadotropin), which can be
`used to diagnose pregnancy. The beta-HCG also can be
`quantified in maternal Serum, and this can be useful early in
`pregnancy when threatened abortion or ectopic pregnancy is
`suspected, because the amount of beta-HCG will be lower
`than normal.
`0029. In the middle to late second trimester, the beta
`HCG can be used in conjunction with the MSAFP to screen
`for chromosomal abnormalities, in particular for Down
`syndrome. An elevated beta-HCG coupled with a decreased
`MSAFP suggests Down syndrome. High levels of HCG
`Suggest trophoblastic disease (molar pregnancy). The
`absence of a fetus on ultrasonography along with an elevated
`HCG Suggests a hydatidiform mole.
`
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`US 2004/O137470 A1
`
`Jul. 15, 2004
`
`0030 Maternal Serum Estriol
`0031. The amount of estriol in maternal serum is depen
`dent upon a viable fetus, a properly functioning placenta,
`and maternal well-being. Dehydroepiandrosterone (DHEA)
`is made by the fetal adrenal glands, and is metabolized in the
`placenta to estriol. The estriolenters the maternal circulation
`and is excreted by the maternal kidney in urine or by the
`maternal liver in the bile. Normal levels of estriol, measured
`in the third trimester, will give an indication of general
`well-being of the fetus. If the estriol level drops, then the
`fetus is threatened and an immediate delivery may be
`necessary. Estriol tends to be lower when Down syndrome
`is present and when there is adrenal hypoplasia with anen
`cephaly.
`0032) The Triple Screen Test
`0033. The triple screen test comprises analysis of mater
`nal serum alpha-feto-protein (MSAFP), human chorionic
`gonadotrophin (hCG), and unconjugated estriol (uB3). The
`blood test is usually performed 16-18 weeks after the last
`menstrual period. While the triple Screen test is non-inva
`Sive, abnormal test results are not indicative of a birth defect.
`Rather, the test only indicates an increased risk and Suggests
`that further testing is needed. For example, 100 out of 1,000
`women will have an abnormal result from the triple screen
`test. However, only 2-3 of the 100 women will have a fetus
`with a birth defect. This high incidence of false positives
`causes tremendous StreSS and unnecessary anxiety to the
`expectant mother.
`0034) Fetal Cells Isolated from Maternal Blood
`0035. The presence of fetal nucleated cells in maternal
`blood makes it possible to use these cells for noninvasive
`prenatal diagnosis (Walknowska, et al., Lancet 1:1119-1122,
`1969; Lo et al., Lancet 2:1363-65, 1989; Lo et al., Blood
`88:4390-95, 1996). The fetal cells can be sorted and ana
`lyzed by a variety of techniques to look for particular DNA
`sequences (Bianchi et al., Am. J. Hum. Genet. 61:822-29,
`(1997); Bianchi et al., PNAS 93:705-08, (1996)). Fluores
`cence in-situ hybridization (FISH) is one technique that can
`be applied to identify particular chromosomes of the fetal
`cells recovered from maternal blood and diagnose aneuploid
`conditions Such as trisomies and monoSomy X. Also, it has
`been reported that the number of fetal cells in maternal blood
`increases in aneuploid pregnancies.
`0036) The method of FISH uses DNA probes labeled with
`colored fluorescent tags that allow detection of Specific
`chromosomes or genes under a microScope. Using FISH,
`Subtle genetic abnormalities that cannot be detected by
`Standard karyotyping are readily identifiable. This procedure
`typically takes 24-48 hours to complete. Additionally, using
`a panel of multi-colored DNA FISH probes, abnormal
`chromosome copy numbers can be seen.
`0037. While improvements have been made for the iso
`lation and enrichment of fetal cells, it is still difficult to get
`many fetal blood cells. There may not be enough to reliably
`determine anomalies of the fetal karyotype or assay for other
`abnormalities. Furthermore, most techniques are time con
`Suming, require high-inputs of labor, and are difficult to
`implement for a high throughput fashion.
`
`0038 Fetal DNA from Maternal Blood
`0039 Fetal DNA has been detected and quantitated in
`maternal plasma and serum (Lo et al., Lancet 350:485487
`(1997); Lo et al., Am. J. hum. Genet. 62:768-775 (1998)).
`Multiple fetal cell types occur in the maternal circulation,
`including fetal granulocytes, lymphocytes, nucleated red
`blood cells, and trophoblast cells (Pertl, and Bianchi, Obstet
`rics and Gynecology 98: 483-490 (2001)). Fetal DNA can be
`detected in the Serum at the Seventh week of gestation, and
`increases with the term of the pregnancy. The fetal DNA
`present in the maternal Serum and plasma is comparable to
`the concentration of DNA obtained from fetal cell isolation
`protocols.
`0040 Circulating fetal DNA has been used to determine
`the sex of the fetus (Lo et al., Am.J. hum. Genet. 62:768-775
`(1998)). Also, fetal rhesus D genotype has been detected
`using fetal DNA. However, the diagnostic and clinical
`applications of circulating fetal DNA is limited to genes that
`are present in the fetus but not in the mother (Pertl and
`Bianchi, Obstetrics and Gynecology 98: 483-490 (2001)).
`Thus, a need Still exists for a non-invasive method that can
`determine the sequence of fetal DNA and provide definitive
`diagnosis of chromosomal abnormalities in a fetus.
`BRIEF SUMMARY OF THE INVENTION
`0041. The invention is directed to a method for detection
`of genetic disorders including mutations and chromosomal
`abnormalities. In Some embodiments, the present invention
`is used to detect mutations, and chromosomal abnormalities
`including but not limited to translocation, transversion,
`monoSomy, trisomy, and other aneuploidies, deletion, addi
`tion, amplification, fragment, translocation, and rearrange
`ment. Numerous abnormalities can be detected Simulta
`neously. The present invention also provides a non-invasive
`method to determine the sequence of fetal DNA from a
`Sample of a pregnant female. The present invention can be
`used to detect any alternation in gene Sequence as compared
`to the wild type Sequence including but not limited to point
`mutation, reading frame Shift, transition, transversion, addi
`tion, insertion, deletion, addition-deletion, frame-shift, mis
`Sense, reverse mutation, and microsatellite alteration. The
`present invention also provides a method for isolating free
`nucleic acid from a Sample containing nucleic acid. The
`present invention also provides compostions and kits.
`0042. In one aspect, the invention is directed to methods
`for detecting chromosomal abnormalities. In one embodi
`ment, the present invention is directed to a method for
`detecting chromosomal abnormalities, Said method compris
`ing quantitating the relative amount of the alleles at a
`heterozygous locus of interest, where the heterozygous locus
`of interest was previously identified by determining the
`Sequence of alleles at a locus of interest from template DNA,
`wherein Said relative amount is expressed as a ratio, and
`wherein Said ratio indicates the presence or absence of a
`chromosomal abnormality.
`0043. In some embodiments, determining the sequence
`includes using a method that is allele Specific PCR, mass
`Spectrometry, hybridization, primer extension, fluorescence
`resonance energy transfer (FRET), Sequencing, Sanger
`dideoxy Sequencing, DNA microarray, GeneCHIP arrayS,
`HuSNP arrays, CodeLink Arrays, BeadArray Technology,
`Mass ARRAY, MassEXTEND, SNP-IT, TaqMan, Invader
`Strand Assay, Southern blot, slot blot, dot blot, or MALDI
`TOF mass spectrometry.
`
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`US 2004/O137470 A1
`
`Jul. 15, 2004
`
`0044) In some embodiments, template DNA is obtained
`from human, non-human, mammal, reptile, cattle, cat, dog,
`goat, Swine, pig, monkey, ape, gorilla, bull, cow, bear, horse,
`sheep, poultry, mouse, rat, fish, dolphin, whale, or Shark. In
`an embodiment, the template DNA is obtained from a human
`Source. In a preferred embodiment, the template DNA is
`obtained from a pregnant human female. In Some embodi
`ments, the template DNA is obtained from a sample that is
`a cell, fetal cell, tissue, blood, Serum, plasma, Saliva, urine,
`tear, vaginal Secretion, Sweat, umbilical cord blood, chori
`onic Villi, amniotic fluid, embryonic tissue, an embryo, a
`two-celled embryo, a four-celled embryo, an eight celled
`embryo, a 16-celled embryo, a 32-celled embryo, a 64-celled
`embryo, a 128-celled embryo, a 256-celled embryo, a 512
`celled embryo, a 1024-celled embryo, lymph fluid, cere
`broSpinal fluid, mucosa Secretion, peritoneal fluid, asciitic
`fluid, fecal matter, or body exudates. In these embodiments,
`the Sample may be mixed with an agent that inhibits cell
`lysis to inhibit the lysis of cells, if cells are present, where
`the agent is a membrane Stabilizer, a cross-linker, or a cell
`lysis inhibitor. In Some of these embodiments, agent is a cell
`lysis inhibitor, and may be glutaraldehyde, derivatives of
`glutaraldehyde, formaldehyde, formalin, or derivatives of
`formaldehyde. In Some of these embodiments the Sample is
`blood and in one embodiment the sample is blood from a
`pregnant female, e.g., a human female. In the latter embodi
`ment, the fetus may be at a gestational age Selected from the
`group consisting of 0-44-8, 8-12, 12-16, 16-20, 20-24,
`24-28, 28-32, 32-36, 36-40, 40-44, 44-48, 48-52, or more
`than 52 weeks. In Some of these embodiments, the template
`DNA may be obtained from plasma or from serum from the
`blood. In these embodiments, the template DNA may
`include a mixture of maternal DNA and fetal DNA, and in
`one embodiment, prior to determining the Sequence of
`alleles of a locus of interest from template DNA, maternal
`DNA is Sequenced to identify a homozygous locus of
`interest, and the homozygous locus of interest is the locus of
`interest analyzed in the template DNA. In another embodi
`ment, maternal DNA is Sequenced to identify a heterozygous
`locus of interest, and the heterozygous locus of interest is the
`locus of interest analyzed in the template DNA.
`0.045. In embodiments, alleles of multiple loci of interest
`are Sequenced and their relative amounts quantitated and
`expressed as a ratio. In one embodiment, the Sequence of
`alleles of one to tens to hundreds to thousands of loci of
`interest on a Single chromosome on template DNA is deter
`mined. In another embodiment, the Sequence of alleles of
`one to tens to hundreds to thousands of loci of interest on
`multiple chromosomes is determined.
`0046. In an embodiment, the locus of interest is suspected
`of containing a single nucleotide polymorphism or mutation.
`The method can be used for determining Sequences of
`multiple loci of interest concurrently. The template DNA can
`comprise multiple loci from a Single chromosome. The
`template DNA can comprise multiple loci from different
`chromosomes. The loci of interest on template DNA can be
`amplified in one reaction. Alternatively, each of the loci of
`interest on template DNA can be amplified in a Separate
`reaction. The amplified DNA can be pooled together prior to
`digestion of the amplified DNA. Each of the labeled DNA
`containing a locus of interest can be separated prior to
`determining the Sequence of the locus of interest. In one
`
`embodiment, at least one of the loci of interest is Suspected
`of containing a single nucleotide polymorphism or a muta
`tion.
`0047 There is no limitation as to the chromosomes that
`can be compared. The ratio for the alleles at a heterozygous
`locus of interest on any chromosome can be compared to the
`ratio for the alleles at a heterozygous locus of interest on any
`other chromosome. In another embodiment, the ratio of
`alleles at a heterozygous locus of interest on a chromosome
`is compared to the ratio of alleles at a heterozygous locus of
`interest on two, three, four or more than four chromosomes.
`In another embodiment, the ratio of alleles at multiple loci
`of interest on a chromosome is compared to the ratio of
`alleles at multiple loci of interest on two, three, four, or more
`than four chromosomes. In embodiments, the ratio for
`alleles at heterozygous loci of interest on a chromosome are
`Summed and compared to the ratio for alleles at heterozy
`gous loci of interest on a different chromosome, where a
`difference in ratioS indicates the presence of a chromosomal
`abnormality. In Some of these embodiments, the chromo
`Somes that are compared are human chromos