# Patent

## Title
Karyotyping assay

## Abstract

This disclosure relates to methods and kits for karyotyping in which chromosomes are interrogated by amplifying loci that are not within copy number variable regions thereof.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 61/334,658, filed May 14, 2010, which is herein incorporated by reference in its entirety.


FIELD
This disclosure relates to methods and kits for karyotyping in which at least one chromosome is interrogated by amplifying one or more loci that are not within copy number variable regions (CNVR) thereof.
BACKGROUND INFORMATION
The human haploid genome is a deoxyribonucleic acid (DNA) sequence and consists of approximately three billion base pairs grouped into 23 chromosomes. However, the human genome is diploid and consists of approximately six billion base pairs grouped into 46 chromosomes. Hence, two copies of each genomic segment and two copies of each chromosome are represented in most of the human genome. The exception is the male human, which has only one copy each of chromosome X and chromosome Y. Nevertheless, variable copy numbers (i.e. not two copies) of genomic segments and chromosomes are observed in individual genomic DNA (gDNA) samples. Such copy number variable regions (CNVR) that are typically greater than one kilobase in length and generally occur at a minor frequency of equal to or greater than 1% in the population are termed “copy number variants” (CNVs; see, e.g., Feuk, et al. Nat. Rev. Genet. 7:85-97 (2006)). Copy number variation (CNV) and its mechanisms of formation, associations with phenotype, and methods of analysis have been extensively reviewed (Feuk, supra; Freeman, et al. Genome Res. 16:949-961 (2006); Sharp, et al. Annu. Rev. Genomics Hum. Genet. 7:407-442 (2006); Nature Genetics 39:S1-S54 (2007), entire issue). Currently, approximately 8600 CNVs have been identified and cover about 5-10% of the human genome (Redon, et al. Nature 444:444-454 (2006); Conrad, et al. Nature 464:704-712 (2010)). Continuing studies toward finer mapping of the CNV map and interrogating more diverse gDNA samples are tracked at the Database of Genomic Variants (http://projects.tcag.ca/variation/). Aberrations in the normal complement of 46 human chromosomes have been identified by cytogenetic analysis. Cytogenetics and its history, linking of chromosomal defects and disease, and methods of analysis have been extensively reviewed (Speicher, et al. Nat. Rev. Genet. 6:782-792 (2005); Trask, Nat. Rev. Genet. 3:769-778 (2002)). More recently, polymerase chain reaction (PCR)-based assays have been used to identify such aberrations.
There is a need in the art for methods that provide fast, accurate, easy-to-use and reliable karyotype information. Karyotype includes an analysis of chromosome number, type, shape, and banding. Currently available methods for determining karyotype and chromosome number lack accuracy due to the presence of CNVRs, are labor-intensive and do not provide for simultaneous interrogation of multiple chromosomes without requiring multiple reporting labels. Unless chromosomes are interrogated outside of CNVRs, an inaccurate karyotype or chromosome number determination may result. Accordingly, this disclosure provides such methods by using as target sequences only those targets known to be outside of CNVRs. In addition, the methods described herein provide for simultaneous interrogation of multiple chromosomes using a single, or multiple reporting labels. Using these methods, karyotypes may be rapidly and accurately determined. These and other advantages may be drawn from the description provided below.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1. Results of exemplary assay of chromosome 2.
FIG. 2. Results of exemplary assay of chromosome 3.
FIG. 3. Results of exemplary assay of chromosome 4.
FIG. 4. Results of exemplary assay of chromosome 5.
FIG. 5. Results of exemplary assay of chromosome 6.
FIG. 6. Results of exemplary assay of chromosome 20.
FIG. 7. Results of additional exemplary assay of chromosome 20.
FIG. 8. Results of exemplary assay of chromosome 22.



For FIGS. 1-8, the genomic DNA (gDNA) samples on the x-axis from left-to-right are as follows:
NA 10859_1, NA10859_2, NA 10859_3, NA 10859_4, NA 14474, NA 14476, NA 17102, NA 17103, NA 17104, NA 17105, NA 17106, NA 17107, NA 17108, NA 17109, NA 17110, NA 17111, NA 17112, NA 17113, NA 17114, NA 17115, NA 17116, NA 17117, NA 17118, NA 17119, NA 17120, NA 17121, NA 17122M NA 17123, NA 17124, NA 17125, NA 17126, NA 17127, NA 17128, NA 17129, NA 17130, NA 17131, NA 17132, NA 17134, NA 17136, NA 17137, NA 17139, NA 17140, NA 17144, NA 17147, NA 17148, NA 17149, NA 17155, NA 17188, NA 17194, NA 17201, NA 17202, NA 17203, NA 17204, NA 17205, NA 17206, NA 17207, NA 17208, NA 17209, NA 17210, NA 17211, NA 17212, NA 17213, NA 17214, NA 17215, NA 17216, NA 17217, NA 17220, NA 17221, NA 17223, NA 17225, NA 17226, NA 17227, NA 17228, NA 17230, NA 17231, NA 17232, NA 17235, NA 17237, NA 17239, NA 17240, NA 17241, NA 17242, NA 17245, NA 17247, NA 17251, NA 17253, NA 17254, NA 17255, NA 17258, NA 17259, NA 17260, NA 17261, NA 17262, NA 17263.
SUMMARY
Disclosed herein are methods for karyotype analysis and for determining the copy number of a test locus on a chromosome in a test biological sample, the test locus being located outside of a copy number variable region (CNVR) of the chromosome, by amplifying a nucleic acid sequence corresponding to the test locus to produce an amplification product, or amplicon, and quantifying the relative copy number of the chromosome in the test biological sample relative to a control biological sample. Karyotyping methods are also provided here that do not require comparison to a reference assay or reference sample. Such methods include those using virtual reference assays or virtual calibrator samples.
Embodiments of the methods disclosed herein can be performed on one or multiple samples with or without a calibrator sample or reference assay. In other embodiments, the target copy number assays can serve as the copy number reference assay herein denoted as a “virtual copy number reference assay”.
Variations of these methods, as well as reagents, kits, arrays and devices for carrying out the same are also provided.
DETAILED DESCRIPTION
This disclosure relates to methods for measuring the copy number of one, more than one, or all chromosomes of a genome using real-time amplification methods (i.e., “karyotyping”). The term “karyotype” refers to the interrogation of one, more than one, or all chromosomes of a particular biological sample, such as a nucleic acid preparation including, but not limited to, deoxyribonucleic acid (DNA, including but not limited to, genomic (gDNA) and mitochondrial DNA (mtDNA)), ribonucleic acid (RNA), proteins, cells, tissues, and organisms.
To “interrogate” one or more chromosomes means to perform an assay, including the methods described herein, to obtain information about the one or more chromosomes contained in the sample. Typically, information relating to “copy number” (CN) is desired. The term copy number generally refers to the number of amplicons resulting from polymerase chain reaction (PCR) amplification, the number of genes or regions on a chromosome, the number of chromosomes in a genome, and the like. The term “amplicon” refers to a piece of DNA formed as the product of an amplification reaction, such as PCR, ligase chain reaction (LCR) or natural gene duplication. The skilled artisan will understand that, within this disclosure, any such molecules can be referred to as target or reference loci depending on the particular assay system. For example, a genome can be queried using as the biological sample a gDNA that preferably, but not necessarily, contains at least one of each chromosome that the user desires to interrogate. In some embodiments, the gDNA sample can contain a pair of each of chromosomes 1-22 and either two X chromosomes, or an X and a Y chromosome. However, it is understood that variations and abnormalities occur such that the number or composition of chromosomes among gDNA samples can vary. In fact, the methods described herein are directed to detecting such variations and abnormalities. In addition, the user may desire to interrogate only a subset of chromosomes (i.e., one or more) present in a genome and it is therefore necessary that only those chromosomes of interest be present. Furthermore, the interrogation typically includes a reference locus (i.e., a “reference locus” or “endogenous control”) which is a locus different from the test locus and is not found within a CNVR. The copy number of the reference locus is typically, but not necessarily, known. A reference locus can reside on a chromosome of interest or on another chromosome and can be interrogated to normalize sample input or to determine relative copy number (i.e., an “endogenous control”).
The methods and assays described herein, which include the determination of the copy number of target loci, are referred to herein as “copy number assays”. The methods and assays described herein for interrogating reference loci are referred to herein as “copy number reference assays”. Typically, the reference locus is present in all samples and is used to normalize for sample input variation and determine relative copy number. However, the test locus can be present or absent. For example, a Y chromosome test assay may interrogate as “present” in a normal male sample, but “absent” in a normal female sample. Existing assay systems, such as the TaqMan® Copy Number Assays (Applied Biosystems), have been used in some instances to interrogate target and reference loci. However, interrogation of test loci within copy number variable regions (CNVRs) has been found to lead to inaccurate karyotype determinations.
An important feature of the methods described herein is that the test loci are outside of (i.e., not within) CNVRs. As described herein, a CNVR includes, but is not limited to, any type of structural variation of a target locus that can affect copy number determination including, but not limited to, duplications, deletions, insertions, repeats, substitutions, and the like. Certain variations can imitate or mimic changes in chromosomal copy number or detrimentally affect the assay by making it less reliable or accurate. Typically, a “universal” set of karyotyping assays (i.e., applicable to all samples or persons) will not target CNVRs. However, it is important to note that CNVRs may vary between individuals or populations of individuals. For instance, a duplication observed in an individual or population of individuals may not be observed in another individual or population of individuals. As such, the methods described herein may target loci that are within a CNVR in one individual or population of individuals but not within a CNVR or another individual or population of individuals.
The difference in copy number between samples is typically determined by “relative quantitation”. Relative quantitation is used to report the copy number of a target locus or chromosome on which the target locus is found in a test sample (i.e., “test locus” or “unknown locus”) relative to one or more reference loci or “calibrator samples”. With reference to the use of a calibrator sample, the term “relative to” means compared to the copy number of a target locus or chromosome on which the target locus is found in a calibrator sample. A “calibrator sample” is a biological sample, often of known karyotype (i.e., a “control gDNA”), that is interrogated before, after or simultaneously with the test sample. The number of copies in the test sample can then be determined relative to the calibrator sample. One or more reference loci or calibrator samples can be used as desired by the user. The one or more reference loci can be the same or different and can be present in the test sample(s) or calibrator sample(s). Typically, the measure of relative quantitation is reported using the term “fold change”, which refers to the amount of amplified product (which relates to the copy number) in a test sample relative to a reference or calibrator sample. Fold change can be quantified using any of several available methods, including but not limited to those described by Livak, et al. (Methods, 25:402-408 (2001)) or commercially available products such as CopyCallerTM (Applied Biosystems, see below). The methods described herein can be performed on one or more than one sample with or without a calibrator sample or reference assay. Various embodiments of such methods are described herein.
The real-time polymerase chain reaction (RT-PCR) is a conventional tool which can be used to amplify and quantify a target nucleic acid molecule, including but not limited to, DNA and RNA, in a sample. The amount of target nucleic acid can be determined as an absolute copy number or as a relative amount. Specifically, the use of RT-PCR to quantify gene expression using the comparative CT method is known to one of skill in the art (Livak, et al. (supra)). In general, the threshold cycle (CT) for a given genetic locus can be determined by arbitrarily setting a signal intensity threshold that falls within the linear range of amplification of real-time PCR data. Previous application of this calculation has been used, for example, to normalize the amplification product of a target gene to the amplification product of an endogenous control gene and then to normalize the data to a calibrator sample, such as an untreated reference sample, to determine the expression of the target gene.
Certain currently available assay systems, such as PCR-based TaqMan® systems, can be useful for relative quantification of nucleic acids amplified from a biological sample to determine gene or chromosome copy number (see, e.g., the TaqMan® assays described in the following Applied Biosystems publications: “Product Bulletin: TaqMan® Copy Number Assays and Custom TaqMan® Copy Number Assays”, “Protocol: TaqMan® Copy Number Assays” (PN 4397425 Rev. C), “Quick Reference Card: TaqMan® Copy Number Assays” (PN 4397424 Rev. B)). The TaqMan® Copy Number Assay and the TaqMan® Copy Number Reference Assay have been used in tandem to interrogate chromosomes to amplify target and control loci, respectively. Usually, the assays are carried out as a single step, “two-plex” real-time PCR assay (i.e., two separate assays within the same reaction well or container). In this type of assay, target and control loci are detected using differently labeled probes. Often, the sample interrogated by such PCR-based assays is human genomic DNA (gDNA) and, typically a minimum of two human gDNA samples are interrogated, including a test and a calibrator sample. Currently, the TaqMan® Copy Number Assay interrogates test loci, which are located throughout the genome and may reside within a copy number variable region (CNVR) and provides copy number variation (CNV) information. The TaqMan® Copy Number Reference Assay interrogates one or more reference loci which, as defined above, are found outside of CNVRs (e.g., Ribonuclease P RNA component H1 (H1RNA) gene (RPPH1) on chromosome 14, cytoband 14q11.2, also known as “RNase P”, and the telomerase reverse transcriptase (TERT) gene located on chromosome 5, cytoband 5p15.33). The TaqMan® Copy Number Reference Assay thereby serves as the endogenous control by interrogating a non-CNVR sequence of interest to normalize for sample input variation by relative quantification (RQ). For example, the TaqMan® Copy Number Assay can utilize an FAMTM dye-labeled minor groove binder (MGB) probe and unlabeled PCR primers directed at the one or more test loci in the test sample or calibrator sample, which are typically assayed separately. The TaqMan® Copy Number Reference Assay can use VIC dye-labeled TAMRATM probes directed at the one or more reference loci. By comparing the products of these reactions, the user normalizes for sample amount and determines the relative number of copies of the target loci in the sample. However, as described above, interrogation of test loci within copy number variable regions (CNVRs) has been found to lead to inaccurate karyotype determinations. Thus, it is important that the target loci are found outside of (e.g., not within) CNVRs.
The methods described herein can utilize a copy number reference assay. In certain embodiments, the target loci in the test and calibrator sample(s) of, for example, gDNA, can be the same or different. In certain embodimetns, the reference assay is used to normalize for sample input variation between samples (i.e., between multiple test samples or between the test and calibrator samples). Thus, in such assays, one or more reference loci can be amplified from the test or control samples and the copy number (i.e., fold change) is calculated as described herein as a copy number reference assay. Variations of such embodiments are also contemplated as would be understood by one of skill in the art.
The methods described herein can also be conducted using a calibrator sample that, as described above, is typically a biological sample of known karyotype, while the karyotype of the test sample is unknown. The relative difference in copy number between the calibrator sample and the test sample can be calculated as is known in the art or described herein. Typically, the “test assay” and “calibrator assay” (i.e., reference or endogenous control assay) are both used for relative quantification of all samples in order to normalize for sample input variation between samples prior to comparing the different samples themselves. The copy number of the target loci of the test samples is calculated by dividing the fold change of the target copy number in the test sample by that of the calibrator sample and then multiplying by the target copy number of the calibrator sample. This calculation is used in the analysis of the currently and commercially available TaqMan® assay systems and similar systems. The test and calibrator assays are, at least in part, directed to the same target loci, but the target loci can also be different. In some embodiments, the methods can include amplifying a target locus from outside (i.e., not within) a CNVR from a test biological sample (such as a gDNA sample in which the number of copies of a particular chromosome is not known), amplifying the same target loci from a calibrator biological sample, and calculating the copy number of the target loci in the test sample relative to the control sample. Variations of such embodiments, including those that do not use a reference assay or calibrator sample, are also contemplated as would be understood by one of skill in the art.
As discussed above, many embodiments of the methods described herein are contemplated as would be understood by one of skill in the art. Exemplary assay formats using reference or calibrator samples are illustrated in Table 1 and described below.







TABLE 1







Exemplary Karyotyping Assay Design Formats when an Unknown


Sample is Tested Relative to a Calibrator Sample










Reaction #1
Reaction #2


















No.


Detectable

Detectable




No. CN
Reactions
Plexy
Detectable
Label
Detectable
Label


Format
No. CN
Reference
(per
Per
Label (CN
(CN Ref.
Label (CN
(CN Ref.


No.
Assays
Assays
sample)
Reaction
Assay)
Assay)
Assay)
Assay)


















1
1
1
1
2
Dye #1*
Dye #2




2
1
1
2
1
Dye #1


Dye #1


3
1
1
2
1
Dye #1


Dye #2


4
1
1
2
1
DNA


DNA







Binding


Binding







Dye**


Dye


5
≧2
1
≧3
1
Dye #1


Dye #1


6
≧2
1
≧3
1
Dye #1


Dye #2


7
≧2
1
≧3
1
DNA


DNA







Binding


Binding







Dye


Dye


8
1
≧2
≧3
1
Dye #1


Dye #1


9
1
≧2
≧3
1
Dye #1


Dye #2


10
1
≧2
≧3
1
DNA


DNA







Binding


Binding







Dye


Dye


11
≧2
≧2
≧4
1
Dye #1


Dye #1


12
≧2
≧2
≧4
1
Dye #1


Dye #2


13
≧2
≧2
≧4
1
DNA


DNA







Binding


Binding







Dye


Dye


14
≧2
0
≧2
1
Dye #1

Dye #1


15
≧2
0
≧2
1
Dye #1

Dye #2


16
≧2
0
≧2
1
DNA

DNA







Binding

Binding







Dye

Dye





*Dyes are typically conjugated to primers or probes.


**DNA binding dyes are typically not conjugated to primers or probes.






NOTES


    
    
        The number of reactions is per replicate per sample. Dye #1 and Dye #2 represent different fluorophores conjugated to probes or primers of the Copy Number Assay and Copy Number Reference Assay and provide differential fluorescence as the reaction generates amplification products.
        DNA Binding Dye represents a fluorophore that binds to amplification products and provides fluorescence as the reaction generates amplification products.
        Format 1 is one 2-plex reaction (i.e. a multiplex reaction).
        Formats 2, 3, and 4 are variations of two 1-plex reactions (i.e., a single-plex reaction).
        Formats 5, 6, and 7 are variations of 1-plex reactions that combine (via average, median, etc.) CT values from multiple Copy Number Assays.
        Formats 8, 9, and 10 are variations of 1-plex reactions that combine (via average, median, etc.) CT values from multiple Copy Number Reference Assays.
        Formats 11, 12, and 13 are variations of 1-plex reactions that combine (via average, median, etc.) CT values from multiple Copy Number Assays and combine (via average, median, etc.) CT values from multiple Copy Number Reference Assays.
        Formats 14, 15, and 16 are variations of 1-plex reactions that provide multiple analyses via each Copy Number Assay, in turn, being compared to one or more of the remaining virtual Copy Number Reference Assays.
        Copy Number Assays of format 5, 6, or 7 target the same chromosome.
        Copy Number Reference Assays of format 8, 9, or 10 target the same chromosome.
        Copy Number Assays of format 11, 12, or 13 target the same chromosome.
        Copy Number Reference Assays of format 11, 12, or 13 target the same chromosome.
        Copy Number Assays of format 14, 15, or 16 target different chromosomes.
        Formats 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, and 16 may also permute and combine (via average, median, etc.) ΔCT values.
        Formats 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, and 16 may also be multiplexed (even though the multiplexed assays may not provide ΔCT value).
        (Modified) permutations of different formats are also possible (e.g. format 14 with multiple Copy Number Assays per chromosome via incorporating aspects of format 5).
        Detectable entities other than fluorophores may also be employed.
    
    


In certain formats outlined in Table 1, the detectable label can be the same (e.g., formats 2, 4, 5, 7, 8, 10, 11, 13, 14, and 16). In other formats, the detectable labels can be different (e.g., Dye #1, Dye #2, and DNA binding dye as in formats 1, 3, 6, 9, 12, or 15) and are conjugated to probes or primers to provide a differential value as the reaction generates amplification products. One or more DNA binding dyes can be used to detect amplification products generated during a reaction (such as real-time PCR) in a multiplex reaction or in separate 1-plex reactions. Typically, the DNA binding dyes are not conjugated to a probe or primer. The skilled artisan will also note the following from Table 1:

    
    
        1) format 1 is a 2-plex reaction (i.e., multiplex reaction) and uses different detectable labels for each reaction;
        2) formats 2, 3, and 4 are variations of two 1-plex reactions (i.e., single-plex reaction) and use the same or different detectable labels in each reaction;
        3) formats 5, 6, and 7 are variations of 1-plex reactions that combine CT values from multiple copy number assays (via average, median, etc.), typically target the same chromosome, and can use the same or different detectable labels in each reaction;
        4) formats 8, 9, and 10 are variations of 1-plex reactions that combine CT values from multiple copy number reference assays (via average, median, etc.), target the same chromosome, and use the same or different detectable labels in each reaction;
        5) formats 11, 12, and 13 are variations of 1-plex reactions that combine CT values from multiple copy number assays (via average, median, etc.) and combine CT values from multiple copy number reference assays (via average, median, etc.), target the same chromosome, and use the same or different detectable labels in each reaction;
        6) formats 14, 15, and 16 are variations of 1-plex reactions that provide multiple analyses in which each copy number assay is compared to one or more of the remaining virtual copy number reference assays, target different chromosomes, and use the same or different detectable labels in each reaction.
    
    


Formats 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, and 16 can also permute and combine (e.g., via average, median, etc.) ΔCT values.
The ΔCT value is defined herein as the CT of the test copy number assay minus the CT of the copy number reference assay. The ΔΔCT value is defined herein as: [the CT of the test assay minus the CT of the reference assay of each test sample] minus [the CT of the test assay minus the CT of the reference assay of the calibrator sample]. In some embodiments, the ΔΔCT values can also be permuted and combined. However, because the ΔCT of the calibrator sample is subtracted from each ΔCT of the test sample, the values being permuted and combined now (ΔΔCT vs. ΔCT) are of a different scale, and track in a “parallel” manner (e.g., ΔCTs of 2, 4, 5 vs. ΔΔCTs of 1, 3, 4 where a value of 1 is subtracted from each). Formats 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, and 16 can also be multiplexed even though the multiplexed assays may not provide a ΔCT value. Modified permutations of different formats are also possible (for example, formats 5 and 14 can be combined for analysis of multiple copy number assays per chromosome).
In one embodiment, karyotyping assay format 1 of Table 1 uses a 2-plex real-time PCR reaction comprised of a copy number assay (or test assay) and a copy number reference assay wherein target loci outside of CNVRs are interrogated. Generally, the detectable labels used by each component of this karyotyping assay are different because the amplified products are contained within the same reaction. Subsequent copy number analysis by the 2−ΔΔCT method is the same as that used in the TaqMan® Copy Number Assay. In this calculation, ΔΔCT determined for each test copy number assay serves as the negative exponent of 2 in order to calculate the fold change for each test copy number assay relative to its corresponding copy number reference assay. This assay format provides for multiplexing of the copy number assay and the copy number reference assay, which minimizes the error of the ΔCT calculation and minimizes the use of reagents and consumables. This karyotyping assay format can be used to interrogate a single chromosome or multiple chromosomes. In certain embodiments, the test copy number assay provides the copy number of the target locus of the test sample, as well as the copy number of the chromosome containing the target locus. Different samples having a particular target locus known to be outside of a CNVR can be used for the reference assay. To properly normalize for sample input variation, the target locus or chromosomal copy number for the reference assay should be the same for all samples. In such embodiments, the copy number assay and copy number reference assay can target different chromosomes. Alternatively, or in addition, each chromosome can be interrogated with both a copy number assay and a copy number reference assay. Typically, but not necessarily, each chromosome is assayed separately from one another.
Karyotyping assay formats 2, 3, and 4 of Table 1 each use two single-plex real-time PCR reactions and the same, two or three different, detectable label(s). One real-time PCR reaction is the copy number assay and the other real-time PCR reaction is the copy number reference assay. The detectable label used in each assay can be the same or different. Subsequent copy number analysis by the 2−ΔΔCT method is performed as previously described hereinabove, with the exception that the replicate CT values of the copy number assay and copy number reference assay are typically, but not necessarily, averaged separately prior to calculating the average ΔCT value. These karyotyping assay designs provide the following advantages: 1) the use of copy number assay and copy number reference assay utilizing the same detectable label conjugated to the probe or primer; 2) the use of previously incompatible assays that cannot be multiplexed due to assay interaction issues or PCR bias of one assay over another; and 3) the use of a DNA binding dye in order to minimize reagent costs. It is possible to use this assay format, or modified versions thereof, to interrogate a single chromosome or multiple chromosomes. Each chromosome is interrogated with both a copy number assay and a copy number reference assay and typically, but not necessarily, each chromosome is assayed separately from one another.
In some embodiments, the karyotyping assay uses one or more copy number assays specific for the chromosome of interest, and one or more copy number reference assays. In some embodiments, the average or median CT values can be used. An average average (or “double average”) CT value is calculated by averaging the multiple average CT values derived from the replicates of each copy number assay when more than one copy number assay is used. One or more copy number assays against a chromosome or chromosomes different from the test sample can be used as controls. These control copy number assays fill the role served by copy number reference assay in karyotyping assay formats 1-4 described above. A “double” average CT value is calculated by averaging the multiple average CT values derived from the replicates of each copy number assay. The “double” average ΔCT value can then be calculated from the two “double” average CT values. Subsequent copy number analysis by the 2−ΔΔCT method is carried out as previously described herein. Karyotyping assay formats 5, 6, 7, 8, 9, 10, 11, 12, and 13 provide the following advantages: 1) allow one to use previously incompatible assays that cannot be multiplexed due to assay interaction issues or PCR bias of one assay over another; 2) allow one to use a DNA binding dye (e.g., SYBR Green I) in order to minimize reagent costs; 3) allow one to eliminate the use of a defined copy number reference assay; and 4) allow one to minimize aberrant copy number observed from a minority of copy number assays by diluting the aberrant average CT values in a “double” average CT value. Karyotyping assay formats 5, 6, 7, 8, 9, 10, 11, 12, or 13, or modified versions thereof, can be used to interrogate a single chromosome or multiple chromosomes.
In other embodiments, the copy number assays can serve as a copy number reference assay herein denoted as a “virtual copy number reference assay”. In these embodiments, a multiplicity of copy number assays are performed and each individual copy number assay is compared to one or more of the remaining copy number assays, which then serve as the copy number reference assays. For example, if ten copy number assays are performed, copy number assay no. 1 is compared to each of copy number assays nos. 2-9, copy number assay no. 2 is compared to each of copy number assays nos. 1 and 3-10, and so forth, for a possible total number of 90 different copy number calculations. For each copy number determination, any two or more of the other nine virtual copy number reference assays can be combined (e.g., via average, median, etc.) toward a total number of ten different analyses. This combining takes place at the virtual copy number reference assay CT level or ΔCT level with one copy number assay and up to nine virtual copy number reference assays. These karyotyping assay formats provide the following advantages: 1) the use of incompatible assays that cannot be multiplexed due to assay interaction issues or PCR bias of one assay over another; 2) the use of a DNA binding dye in order to minimize reagent costs; 3) elimination of a properly defined copy number reference assay; 4) confirmation of copy number using each copy number assay as a virtual copy number reference assay; and 5) for troubleshooting of a copy number assay by the use of each copy number assay as a virtual copy number reference assay.
It is also possible to further modify these karyotyping assay formats. For example, in some embodiments, at least ten different copy number analyses can be used in karyotyping assay formats 14, 15, or 16, and the ten different average ΔCT values derived from the ten different copy number analyses are then averaged toward a “double” average ΔCT value. Subsequent copy number analysis by the 2−ΔΔCT method is performed as previously described herein. This copy number analysis mirrors that of the other assays with the exception that average ΔCT values, not average CT values, are being averaged. In other embodiments, assay formats 14, 15 or 16 can be combined with assay formats 5, 6, 7, 11, 12 or 13 by averaging the average CT values from multiple copy number assays prior to calculating ΔCT values. For example, a karyotyping assay with 10 copy number assays in which five target chromosome 1 and five target chromosome 2, the replicate CT values are averaged prior to averaging the five assays targeting each chromosome. Accordingly, only two “double” average CT values exist, one for each chromosome. A variety of subsequent calculations of ΔCT are possible. These karyotyping assay formats with modified copy number analyses provide the following advantages: 1) the use of previously incompatible assays that can not be multiplexed due to assay interaction issues or PCR bias of one assay over another; 2) the use of a DNA binding dye in order to minimize reagent costs; 3) do not rely on the use of a properly defined copy number reference assay; and 4) minimize aberrant copy number results observed in some copy number assays by diluting the aberrant average ΔCT values in a “double” average ΔCT value. Such assay formats can be used to interrogate a single chromosome or multiple chromosomes. It should also be noted that the real-time PCR reactions can utilize increased multiplexing.
Karyotyping methods that use a virtual copy number reference assay (i.e., those that do not utilize either a reference assay or calibrator sample) are also provided. Situations in which a reference assay is not used include the lack of a suitable reference genomic sequence, non-identification of a control assay locus from a reference genomic sequence, assay unavailability, cost reduction, or to facilitate ease of use. Situations in which a calibrator sample is not used include unavailability of such a sample or a reference sequence, to reduce costs, or facilitate ease of use. Such assays are described below.
In the following two embodiments, the copy number assay CT values and copy number reference assay CT values for the virtual calibrator sample are calculated. The copy number assay CT values can be calculated by averaging the replicate CT for each copy number assay and then averaging these values across the multiple test samples. Alternatively, the copy number assay CT values can be calculated by averaging replicate CT values of each copy number assay, averaging these values across multiple copy number assays (those targeting the same chromosome), and averaging these values across the multiple test samples. These methods can also be applied to the copy number reference assay CT values. Depending upon the number of copy number assays and copy number reference assays used, up to four formats can be used to perform this calculation. Furthermore, the copy number assays and the copy number reference assays for the test samples can be treated separately or in combination as outlined in Table 1.
In the first of these embodiments, a “virtual calibrator sample” can be used in lieu of an actual calibrator sample (i.e., virtual calibrator assay no. 1). Typically, in these karyotyping assays, one or more copy number assays targeting one or more test chromosomes, one or more copy number reference assays targeting one or more reference chromosomes, and two or more test samples (e.g., gDNA) are utilized. In these assays, the copy number of the reference chromosome may or may not be previously known. An average CT value is calculated from the replicate CT values of copy number assay. If more than one copy number assay is used, the average CT values of each copy number assay are averaged. This calculation is performed for each of the two or more test samples, and for the one or more reference assay(s). The average CT values or “double” average CT values can then be further averaged across the two or more test samples. This “double” or “triple” average CT value is representative of a “virtual” calibrator sample. If appropriate, other calculations, such as the median, can be used in lieu of the average. The calculated CT values from the copy number assay(s) and copy number reference assay(s) can then be used to calculate the ΔCT value for each of the test samples. Subsequent copy number analysis by the 2−ΔΔCT method (see below) is similar to that as previously described herein. More accurate results will typically result when the test samples are normalized to the same concentration prior to chromosomal copy number analysis and when additional reference assays are utilized. Even in the presence of equal numbers of target loci, different reference assays may provide different CT values due to differences in assay performance. Therefore, the use of additional reference assays can provide a more accurate CT representation of each test gDNA sample. Likewise, the use of additional test samples can provide a more accurate CT representation of the “virtual” calibrator sample.
In another of these embodiments, a modified “virtual calibrator sample” (i.e., virtual calibrator assay no. 2) can be used in lieu of an actual calibrator sample as described in the immediately preceding paragraph. However, after averaging replicate CT values, the average CT values from one copy number reference assay can be averaged across the multiple test samples. This “double” average CT value represents the CT value from the virtual calibrator sample. If multiple copy number reference assays are used, average CT values from replicates can be averaged across multiple copy number reference assays and then averaged across multiple test samples toward a “triple” average CT value representing the virtual calibrator sample. Multiple copy number assays can be treated separately or combined.
In yet other embodiments, multiple copy number assays with virtual calibrator assays are used, for example, multiple copy number assays targeting one or more test loci on a chromosome (virtual calibrator assay no. 1 targeting the test sample); multiple copy number assays targeting one or more test locus on a reference chromosome (virtual calibrator assay no. 2 targeting the virtual calibrator sample); multiple copy number reference assays targeting a reference chromosome (virtual calibrator assay no. 3, copy number reference assay targeting the test sample and the virtual calibrator sample). In these embodiments, the copy number of the reference chromosome may or may not be previously known. The copy number of the targeted chromosome(s) in virtual calibrator assays no. 2 and no. 3 need not be the same. In these embodiments, a calibrator sample is not required. “Double” average CT values are calculated from each of the three assay groups. The “double” average CT values from virtual calibrator assays no. 1 and no. 3 are then used to calculate the ΔCT value for the test sample. If appropriate, other calculations, such as the median, can be used in lieu of the average. The “double” average CT values from virtual calibrator assays no. 2 and no. 3 are used to calculate the ΔCT value for the “virtual” calibrator sample. Subsequent copy number analysis by the 2−ΔΔCT method is similar to that previously described herein. It is noted that the use of additional test and reference assays can provide a more accurate CT representation of the test gDNA sample and “virtual” calibrator gDNA sample.
Yet another embodiment is similar to that described in the immediately preceding paragraph except that virtual calibrator assay #3 is omitted. Because the test sample and “virtual” calibrator sample are derived from the same biological sample, there is no need to normalize for input variation by using one or more reference assays. Therefore, the reference assays can be removed from the workflow. Upon calculating the “double” average CT values from virtual calibrator assays no. 1 and no. 2, the “double” average CT value from virtual calibrator assay no. 2, representing the “virtual” calibrator sample, is subtracted from the “double” average CT value from assay group no. 1, representing the test sample, to calculate a ΔCT value. If appropriate, other calculations, such as the median, can be used in lieu of the average. Because the reference assays are not used, calculation of relative quantity via the equation of 2−ΔΔCT is replaced with calculation of relative quantity via the equation of the 2−ΔCT. Subsequent copy number analysis is similar to that previously described herein.
As described above, data generated using the karyotyping methods described herein (with or without reference or calibrator samples) can be analyzed using any of several well-known methods or algorithms. As an example, relative copy number can be calculated using assay format 1 (of Table 1) as shown in Table 2:







TABLE 2







Karyotyping Assay Format 1 Performed Without Replicates












Test or
Cali-




Unknown
brator


Step
Description
Sample
Sample













1
Determine copy number of calibrator sample
TBD
2


2
Collect CT value from copy number assay
23
20


3
Collect CT value from copy number reference
23
21



assay




4
Calculate ΔCT value
0
−1



(CT from CN Assay − CT from CN Ref Assay)




5
Calculate ΔΔCT value 
1
0



(ΔCT from each sample − ΔCT from calibrator





sample)




6
Calculate relative quantity (RQ = 2−ΔΔCT)
0.5
1


7
Calculate copy number
1
2



(CN = RQ × CN of calibrator sample)









As understood by those of skill in the art, a one cycle difference is theoretically a two-fold difference in quantity of the target sample. The skilled artisan can derive from step 2 that the Test or Unknown Sample (CT value=23) can have eight-fold fewer copies than the Calibrator Sample (CT value=20). However, because the copy number reference assay targets the same number of copies in all samples, the test sample (CT value=23) actually contains four-fold less sample input the calibrator sample (CT value=21). Because of the normalization of sample input variation via the copy number reference assay, it can be determined that the test sample has two-fold fewer copies than the calibrator sample (i.e., 4-fold variation is accounted for as sample input variation from the initial 8-fold variation) indicating that the actual copy number variation between the test and calibrator samples is two-fold.
The data generated using the methods described herein can also be analyzed using CopyCallerTM software available from Applied Biosystems. CopyCallerTM software is based on the 2−ΔΔCT method described by Livak, et al. (supra). In one method, the user is required to select one sample as the calibrator sample and to identify the copy number thereof. For example, if the target loci in the test sample (such as gDNA) is detected using VICTM dye and the target loci in the calibrator sample is detected using FAMTM dye, the ΔCT value is calculated for each reaction well by subtracting the VICTM CT value from the FAMTM CT value. The calculation of the ΔCT value normalizes for sample input variation between wells. Because multiple replicates are typically assayed, the ΔCT values from each replicate for each sample are averaged toward an average ΔCT value for each sample. The ΔΔCT value is calculated for each sample by subtracting the average ΔCT value of the calibrator sample from the average ΔCT value of each test sample. The calculation of the ΔΔCT value provides for determining the copy number of a test sample relative to the calibrator sample of known copy number. The ΔΔCT values are converted to a relative quantity (RQ) by the equation of RQ=2−ΔΔCT. The RQ value of each sample is then multiplied by the copy number of the calibrator sample to obtain the copy number determination of each sample.
Another method of copy number analysis by CopyCallerTM utilizes proprietary algorithms for determining copy number without selection of a calibrator sample. The user is only required to identify the most common copy number to be provided by the samples. Both methods of copy number analysis by CopyCallerTM employ outlier removal strategies for all CT and ΔCT values. CopyCallerTM can also provide confidence and absolute Z-score values for the predicted copy number values. It is also noted that copy number analysis methods other than the 2−ΔΔCT method can be utilized (e.g., Cikos, et al. Anal Biochem. 384:1-10 (2009)). Other methods of analysis can also be used, as would be understood by one of skill in the art.
While many of the embodiments described herein relate to PCR, other amplification or product detection methods or reagents can also be utilized. For example, such methods or reagents may include any of several methods that can be used to amplify the target nucleic acid from the sample. The term “amplifying” which typically refers to an “exponential” increase in target nucleic acid is used herein to describe both linear and exponential increases in the numbers of a select target sequence of nucleic acid. The term “amplification reaction mixture” refers to an aqueous solution comprising the various reagents used to amplify a target nucleic acid. These include enzymes, including, but not limited to, polymerases (such as DNA polymerase, RNA polymerase and reverse transcriptase), aqueous buffers, salts, amplification primers, target nucleic acid, and nucleoside triphosphates. Depending upon the context, the mixture can be either a complete or incomplete amplification reaction mixture. The method used to amplify the target nucleic acid can be any method available to one of skill in the art. Any in vitro means for multiplying or amplifying the copies of a target sequence of nucleic acid can be utilized. These include linear, logarithmic, or any other amplification method. Exemplary methods include polymerase chain reaction (PCR; see, e.g., U.S. Pat. Nos. 4,683,202; 4,683,195; 4,965,188; and 5,035,996), isothermal procedures (using one or more RNA polymerases (see, e.g., WO 2006/081222), strand displacement (see, e.g., U.S. Pat. No. RE39,007), partial destruction of primer molecules (see, e.g., WO 2006/087574), ligase chain reaction (LCR) (see, e.g., Wu, et al. Genomics 4:560-569 (1989) and Barany, Proc. Natl. Acad. Sci. USA 88:189-193 (1991)), Qβ RNA replicase systems, RNA transcription-based systems (e.g., TAS, 3SR), rolling circle amplification (RCA) (see, e.g., U.S. Pat. No. 5,854,033; U.S. Pub. No. 2004/0265897; Lizardi, et al. Nat. Genet. 19: 225-232 (1998); and Barter, et al. Nucleic Acid Res. 26:5073-5078 (1998)), and strand displacement amplification (SDA) (Little, et al. Clin. Chem. 45:777-784 (1999)), among others. Many systems are suitable for use in amplifying target nucleic acids and are contemplated herein as would be understood by one of skill in the art.
Any of several methods can be used to detect amplified target nucleic acids using primers or probes. Many different reagents, systems, or detectable labels can be used in the methods described herein. These include, for example, TaqMan® systems, detectable nucleic acid intercalating agents, such as ethidium bromide and SYBR dyes, detectable label-quencher systems (e.g., FRET, salicylate/DTPA ligand systems (see, e.g., Oser et al. Angew. Chem. Int. Ed. Engl. 29:1167-1169 (1990)), displacement hybridization, homologous probes, assays described in EP 070685), molecular beacons (e.g., NASBA), Scorpion, locked nucleic acid (LNA) bases (Singh, et al. Chem. Commun. 4:455-456 (1998)), peptide nucleic acid (PNA) probes (Pellestor, et al. Eur. J. Hum. Gen. 12:694-700 (2004)), Eclipse probes (Afonina, et al. Biotechniques 32:940-949 (2002)), light-up probes (Svanvik, et al. Anal. Biochem. 281:26-35 (2000)), molecular beacons (Tyagi, et al. Nat. Biotechnol. 14:303-308 (1996)), tripartite molecular beacons (Nutiu, Nucleic Acids Res. 30:E94 (2002)), QuantiProbes (www.qiagen.com), HyBeacons (French, et al. Mol. Cell. Probes 15:363-374 (2001)), displacement probes (Li, et al. Nucleic Acids Res. 30:E5 (2002)), HybProbes (Cardullo, et al. Proc. Natl. Acad. Sci. USA 85:8790-8794 (1988)), MGB Alert® probes (www.nanogen.com), Q-PNA (Fiandaca, et al. Genome Res. 11:609-613 (2001)), Plexor (www.Promega.com), LUX primers (Nazarenko, et al. Nucleic Acids Res. 30:E37 (2002)), Scorpion primers (Whitcombe, et al. Nat. Biotechnol. 17:804-807 (1999)), AmpliFluor® (Sunrise) primers (Nazarenko, et al. Nucleic Acids Res. 25:2516-2521 (1997)), DzyNA primers (Todd, et al. Clin. Chem. 46:625-630 (2000)), and the like. In each of these assays, the generation of amplification products can be monitored while the reaction is in progress. An apparatus for detecting the signal generated by the detectable label can be used to detect, measure, and quantify the signal before, during, or after amplification. The particular type of signal may dictate the choice of detection method. For example, in some embodiments, fluorescent dyes are used to label probes or amplified products. The probes bind to single-stranded or double-stranded amplified products, or the dyes intercalate into the double-stranded amplified products, and consequently, the resulting fluorescence increases as the amount of amplified product increases. In some embodiments, the Tm is ascertained by observing a fluorescence decrease as the double-stranded amplified product dissociates and the intercalating dye is released therefrom. The amount of fluorescence can be quantitated using standard equipment such as a spectra-fluorometer, for example. The use of other methods or reagents is also contemplated herein as would be understood by one of skill in the art.
One exemplary method for amplifying and detecting target nucleic acids is the TaqMan® system described above (see, e.g., U.S. Pat. Nos. 4,889,818; 5,079,352; 5,210,015; 5,436,134; 5,487,972; 5,658,751; 5,538,848; 5,618,711; 5,677,152; 5,723,591; 5,773,258; 5,789,224; 5,801,155; 5,804,375; 5,876,930; 5,994,056; 6,030,787; 6,084,102; 6,127,155; 6,171,785; 6,214,979; 6,258,569; 6,814,934; 6,821,727; 7,141,377; and 7,445,900). As described herein and elsewhere, TaqMan® assays are typically carried out by performing nucleic acid amplification on a target polynucleotide using a nucleic acid polymerase having 5′-to-3′ nuclease activity, a primer capable of hybridizing to said target polynucleotide, and an oligonucleotide probe capable of hybridizing to said target polynucleotide 3′ relative to said primer. The oligonucleotide probe typically includes a detectable label (e.g., a fluorescent reporter molecule) and a quencher molecule capable of quenching the fluorescence of said reporter molecule. The oligonucleotide probe typically exists in at least one single-stranded conformation when unhybridized where said quencher molecule quenches the fluorescence of said reporter molecule. When hybridized to a target nucleic acid, the probe exhibits at least one other conformation in which the fluorescence of the reporter molecule is unquenched. Typically, the detectable label and quencher molecule are part of a single probe. As amplification proceeds, the polymerase digests the probe to separate the detectable label from the quencher molecule. The detectable label (e.g., fluorescence) is monitored during the reaction, where detection of the label corresponds to the occurrence of nucleic acid amplification (e.g., the higher the signal the greater the amount of amplification). Variations of TaqMan® assays (e.g., LNATM spiked TaqMan® assay) are known in the art and would be suitable for use in the methods described herein.
Another exemplary system utilizes double-stranded probes in displacement hybridization methods (see, e.g., Morrison, et al. Anal. Biochem. 183:231-244 (1989); and Li, et al., (supra)). In such methods, the probe typically includes two complementary oligonucleotides of different lengths where one includes a detectable label and the other includes a quencher molecule. When not bound to a target nucleic acid, the quencher suppresses the signal from the detectable label. The probe becomes detectable upon displacement hybridization with a target nucleic acid. Multiple probes can be used, each containing different detectable labels, such that multiple target nucleic acids may be queried in a single reaction.
Additional exemplary methods for amplifying and detecting target nucleic acids involve “molecular beacons”, which are single-stranded hairpin shaped oligonucleotide probes. In the presence of the target sequence, the probe unfolds, binds and emits a signal (e.g., fluoresces). A molecular beacon typically includes at least four components: 1) the “loop”, an 18-30 nucleotide region which is complementary to the target sequence; 2) two 5-7 nucleotide “stems” found on either end of the loop and being complementary to one another; 3) at the 5′ end, a detectable label; and 4) at the 3′ end, a quencher dye that prevents the detectable label from emitting a signal when the probe is in the closed loop shape (i.e., not bound to a target nucleic acid). Thus, in the presence of a complementary target, the “stem” portion of the beacon separates out resulting in the probe hybridizing to the target. Other types of molecular beacons are also known and may be suitable for use in the methods described herein. Molecular beacons can be used in a variety of assay systems. One such system is nucleic acid sequence-based amplification (NASBA®), a single step isothermal process for amplifying RNA to double stranded DNA without temperature cycling. A NASBA® reaction typically requires avian myeloblastosis virus (AMV), reverse transcriptase (RT), T7 RNA polymerase, RNase H, and two oligonucleotide primers. After amplification, the amplified target nucleic acid can be detected using a molecular beacon. Other uses for molecular beacons are known in the art and would be suitable for use in the methods described herein.
The Scorpion system is another exemplary assay format that can be used in the methods described herein. Scorpion primers are bi-functional molecules in which a primer is covalently linked to the probe, along with a detectable label (e.g., a fluorophore) and a quencher. In the presence of a target nucleic acid, the detectable label and the quencher separate which leads to an increase in signal emitted from the detectable label. Typically, a primer used in the amplification reaction includes a probe element at the 5′ end along with a “PCR blocker” element (e.g., HEG monomer) at the start of the hairpin loop. The probe typically includes a self-complementary stem sequence with a detectable label at one end and a quencher at the other. In the initial amplification cycles (e.g., PCR), the primer hybridizes to the target and extension occurs due to the action of the polymerase. The Scorpion system can be used to examine and identify point mutations using multiple probes that have different tags to distinguish between the probes. Using PCR as an example, after one extension cycle is complete, the newly synthesized target region will be attached to the same strand as the probe. Following the second cycle of denaturation and annealing, the probe and the target hybridize. The hairpin sequence then hybridizes to a part of the newly produced PCR product. This results in the separation of the detectable label from the quencher and causes emission of the signal. Other uses for molecular beacons are known in the art and would be suitable for use in the methods described herein.
One or more detectable labels or quenching agents are typically attached to a primer or probe. The detectable label can emit a signal when free or when bound to the target nucleic acid. The detectable label can also emit a signal when in proximity to another detectable label. Detectable labels can also be used with quencher molecules such that the signal is only detectable when not in sufficiently close proximity to the quencher molecule. For instance, in some embodiments, the assay system can cause the detectable label to be liberated from the quenching molecule. Any of several detectable labels can be used to label the primers and probes used in the methods described herein.
As mentioned above, in some embodiments the detectable label can be attached to a probe, which can be incorporated into a primer, or may otherwise bind to amplified target nucleic acid (e.g., a detectable nucleic acid binding agent such as an intercalating or non-intercalating dye). When using more than one detectable label, each should differ in their spectral properties such that the labels can be distinguished from each other, or such that together the detectable labels emit a signal that is not emitted by either detectable label alone. Exemplary detectable labels include, for instance, a fluorescent dye or fluorphore (e.g., a chemical group that can be excited by light to emit fluorescence or phosphorescence), “acceptor dyes” capable of quenching a fluorescent signal from a fluorescent donor dye, and the like. Suitable detectable labels may include, for example, fluorosceins (e.g., 5-carboxy-2,7-dichlorofluorescein; 5-Carboxyfluorescein (5-FAM); 5-HAT (Hydroxy Tryptamine); 6-HAT; 6-JOE; 6-carboxyfluorescein (6-FAM); FITC); Alexa fluors (e.g., 350, 405, 430, 488, 500, 514, 532, 546, 555, 568, 594, 610, 633, 635, 647, 660, 680, 700, 750); BODIPY fluorophores (e.g., 492/515, 493/503, 500/510, 505/515, 530/550, 542/563, 558/568, 564/570, 576/589, 581/591, 630/650-X, 650/665-X, 665/676, FL, FL ATP, FI-Ceramide, R6G SE, TMR, TMR-X conjugate, TMR-X, SE, TR, TR ATP, TR-X SE), coumarins (e.g., 7-amino-4-methylcoumarin, AMC, AMCA, AMCA-S, AMCA-X, ABQ, CPM methylcoumarin, coumarin phalloidin, hydroxycoumarin, CMFDA, methoxycoumarin), calcein, calcein AM, calcein blue, calcium dyes (e.g., calcium crimson, calcium green, calcium orange, calcofluor white), Cascade Blue, Cascade Yellow; CyTM dyes (e.g., 3, 3.18, 3.5, 5, 5.18, 5.5, 7), cyan GFP, cyclic AMP Fluorosensor (FiCRhR), fluorescent proteins (e.g., green fluorescent protein (e.g., GFP. EGFP), blue fluorescent protein (e.g., BFP, EBFP, EBFP2, Azurite, mKalama1), cyan fluorescent protein (e.g., ECFP, Cerulean, CyPet), yellow fluorescent protein (e.g., YFP, Citrine, Venus, YPet), FRET donor/acceptor pairs (e.g., fluorescein/tetramethylrhodamine, IAEDANS/fluorescein, EDANS/dabcyl, fluorescein/fluorescein, BODIPY FL/BODIPY FL, Fluorescein/QSY7 and QSY9), LysoTracker and LysoSensor (e.g., LysoTracker Blue DND-22, LysoTracker Blue-White DPX, LysoTracker Yellow HCK-123, LysoTracker Green DND-26, LysoTracker Red DND-99, LysoSensor Blue DND-167, LysoSensor Green DND-189, LysoSensor Green DND-153, LysoSensor Yellow/Blue DND-160, LysoSensor Yellow/Blue 10,000 MW dextran), Oregon Green (e.g., 488, 488-X, 500, 514); rhodamines (e.g., 110, 123, B, B 200, BB, BG, B extra, 5-carboxytetramethylrhodamine (5-TAMRA), 5 GLD, 6-Carboxyrhodamine 6G, Lissamine, Lissamine Rhodamine B, Phallicidine, Phalloidine, Red, Rhod-2,5-ROX (carboxy-X-rhodamine), Sulphorhodamine B can C, Sulphorhodamine G Extra, Tetramethylrhodamine (TRITC), WT), Texas Red, Texas Red-X, VIC and other labels described in, e.g., US Pub. No. 2009/0197254), among others as would be known to those of skill in the art.
The amplified target nucleic acid can also be detected using a detectable nucleic acid binding agent (see, e.g., Table 1) which can be, for example, an intercalating agent or a non-intercalating agent. As used herein, an intercalating agent is an agent or moiety capable of non-covalent insertion between stacked base pairs of a double-stranded nucleic acid molecule. A non-intercalating agent is one that does not insert into the double-stranded nucleic acid molecule. The nucleic acid binding agent can produce a detectable signal directly or indirectly. The signal can be detectable directly using, for example, fluorescence or absorbance, or indirectly using, for example, any moiety or ligand that is detectably affected by its proximity to double-stranded nucleic acid is suitable, for example a substituted label moiety or binding ligand attached to the nucleic acid binding agent. It is typically necessary for the nucleic acid binding agent to produce a detectable signal when bound to a double-stranded nucleic acid that is distinguishable from the signal produced when that same agent is in solution or bound to a single-stranded nucleic acid. For example, intercalating agents, such as ethidium bromide and SYBR dyes, fluoresce more intensely when intercalated into double-stranded DNA than when bound to single-stranded DNA, RNA, or in solution (see, e.g., U.S. Pat. Nos. 5,994,056; 6,171,785; and 6,814,934). Similarly, actinomycin D fluoresces red when bound to single-stranded nucleic acids, and green when bound to double-stranded nucleic acids. And in another example, the photoreactive psoralen 4-aminomethyle-4-5′,8-trimethylpsoralen (AMT) has been reported to exhibit decreased absorption at long wavelengths and fluorescence upon intercalation into double-stranded DNA (Johnston et al. Photochem. Photobiol. 33:785-791 (1981). For example, U.S. Pat. No. 4,257,774 describes the direct binding of fluorescent intercalators to DNA (e.g., ethidium salts, daunomycin, mepacrine and acridine orange, 4′,6-diamidino-α-phenylindole). Non-intercalating agents (e.g., minor groove binders such as Hoechst 33258, distamycin, netropsin) may also be suitable for use. For example, Hoechst 33258 (Searle, et al. Nucleic Acids Res. 18:3753-3762 (1990)) exhibits altered fluorescence with an increasing amount of target. Exemplary detectable DNA binding agents may include, for example, acridine derivatives (e.g., acridine homodimer, acridine orange, acridine yellow, 9-amino-6-chloro-2-methoxyacridine (ACMA), proflavin), actinomycins (e.g., actinomycin D (Jain, et al. J. Mol. Biol. 68:1-10 (1972), 7-amino-actinomycin D (7-AAD)), anthramycin, auramine, azure B, BOBOTM-1, BOBOTM-3, BO-PROTM-1, BO-PROTM-3, chromomycin (e.g., A3), crystal violet, cyanine dyes, DAPI (Kapuściński, et al. Nucleic Acids Res. 6:3519-3534 (1979)), 4′,6-diamidino-2-phenylindole (DAPI), daunomycin, distamycin (e.g., distamycin D), dyes described in U.S. Pat. No. 7,387,887, ellipticine, ethidium salts (e.g., ethidium bromide, ethidium homdimer-1, ethidium homdimer-2, dihydroethidium (also known as hydroethidine), ethidium monoazide), fluorcoumanin, fluorescent intercalators as described in U.S. Pat. No. 4,257,774, GelStar®(Cambrex Bio Science Rockland Inc., Rockland, Me.), hexidium iodide, Hoechst 33258 (Searle, et al., (supra)), Hoechst 33342, Hoechst 34580, homidium, hydroxystilbamidine, JO-JO-1, JO-PROTM-1, LDS 751, LOLO-1, LO-PROTM-1, malachite green, mepacrine (e.g., orange), mithramycin, netropsin, the Nissl substance, 4′,6-diamidino-α-phenylindole, proflavine, POPOTM-1, POPOTM-3, PO-PROTM-1, propidium iodide, ruthenium polypyridyls, Sevron dyes (e.g., Brilliant Red 2B, Brilliant Red 4G, Brilliant Red B, Orange, Yellow L), SYBR 101, SYBR 102, SYBER 103, SYBR® Gold, SYBR® Green I (U.S. Pat. Nos. 5,436,134 and 5,658,751), SYBR® Green II, SYTOX® Blue, SYTOX® Green, SYTOX® Orange, SYTO® 1, SYTO® 11, SYTO® 13, SYTO® 14, SYTO® 15, SYTO® 16, SYTO® 17, SYTO® 18, SYTO® 20, SYTO® 21, SYTO® 22, SYTO® 23, SYTO® 24, SYTO® 25, SYTO® 40, SYTO® 43, SYTO® 44, SYTO® 45, SYTO® 59, SYTO® 60, SYTO® 61, SYTO® 62, SYTO® 63, SYTO® 64, SYTO® 80, SYTO® 81, SYTO® 82, SYTO® 83, SYTO® 84, SYTO® 85, thiazole orange (Aldrich Chemical Co., Milwaukee, Wis.), TO-PRO-1, TO-PRO-3, TO-PRO-5, TOTO-1, TOTO-2, TOTOTM-3, YO-PRO®-1, YO-PRO®-3, YOYO-1, and YOYO®-3 (Molecular Probes, Inc., Eugene, Oreg.), among others. SYBR® Green I (see, e.g., U.S. Pat. Nos. 5,436,134; 5,658,751; and/or 6,569,927), for example, has been used to monitor a PCR reaction by amplifying the target sequence in the presence of the dye, exciting the biological sample with light at a wavelength absorbed by the dye and detecting the emission therefrom; and, determining a melting profile of the amplified target sequence. The presence of amplified products and, therefore, the target sequence in the sample, can thereafter be determined by, for example, performing a melting curve analysis (i.e., non-linear least squares regression of the sum of multiple gaussians). It is to be understood that the use of the SYBR® Green dye is presented as an example and that many such dyes may be used in the methods described herein. Other nucleic acid binding agents can also be suitable as would be understood by one of skill in the art.
The nature of the test and control amplified products can vary depending on the type of assay system that is utilized. For example, the product of PCR amplification of DNA is typically referred to as an “amplicon”. In contrast, the Ligation Chain Reaction product is instead referred to as “LCR product” or “ligation product”. In PCR, primers are utilized but in other methods, such as LCR ligation probes, can be utilized. It is known that both PCR and LCR functions through exponential amplification or linear amplification. It is important to note, however, that regardless of the kind of method used to perform amplification or detection of target loci, any target loci should exhibit similar “amplification efficiency” to one another (e.g., within about 10 percent). This means that within a particular reaction, the target loci being compared should amplify to approximately the same extent (e.g., within about 10 percent) under similar reaction conditions. It is understood in the art that amplification efficiency may be affected by, for example, sample quality (e.g., purity, presence of reaction inhibitors, and the like) or sequence (e.g., G/C content, mismatches, and the like). As used herein, amplification efficiency refers to any product that may be quantified to determine copy number (e.g., a PCR amplicon, an LCR ligation product, or similar product). Variations in amplification due to factors other than copy number may thereby be limited.
For example, when using PCR, it may be optimal to amplify amplicons of a similar size (e.g., within about ten percent of the length of any other amplicon) and amplify with similar amplification efficiency (e.g., within about ten percent of one another). PCR-related amplification efficiencies can be tested as described by Livak, et al. (supra), or as is otherwise known in the art. Briefly, Livak, et al. teach that, as the ΔCT varies with template dilution, amplifications can be performed over a particular range (e.g., a 100-fold range) and the ΔCT calculated at each point. A log of dilution versus ΔCT is made and if the absolute value of the slope is close to zero, the efficiencies are similar. For PCR-based assays, the resultant amplicons are typically about 70 to 125 nucleotides in length, and can be less than or equal to about 110 nucleotides in length. The amplification efficiency is typically controlled by amplifying amplicons of similar G/C content, length, or melting temperature. The primers used in the test and control assays are also typically of similar character (e.g., G/C content, length, melting temperature). Variations of these parameters can also be used, as would be understood by the skilled artisan.
The methods described herein typically require a target locus to be present on the one or more chromosomes being studied (exceptions include, for example, the absence of a target locus such as when the Y chromosome is assayed in a normal (i.e., XX) female sample). Pre-existing bioinformatics algorithms can be used to generate “qualified” pre-characterized genome targets and to design assays against those targets. Qualified genome targets may include, for example, genes, exons, introns, intergenic regions, junctions thereof, evolutionarily conserved regions (“cold” spots not prone to change), or other regions within the genome. In certain embodiments, multiple target loci can be interrogated on a chromosome. This can be used to control for unanticipated abnormalities in regions containing target loci. It is possible that such regions can include a duplication, deletion, or other abnormality that may result in an inaccurate karyotype determination. By targeting multiple target loci of a chromosome, the user can control for such unanticipated abnormalities. Thus, in certain embodiments, one, more than one, at least two, or two or more loci are targeted from at least one chromosome or arm thereof, but optionally include more than one arm, to be interrogated. In certain embodiments, three, four, five, six, seven, eight, nine, 10 or more loci may be interrogated. In one embodiment, four loci are interrogated on each chromosome. In some embodiments, it may also be important to separate the target loci by a particular approximate number of nucleotides which can, in some embodiments, be calculated as is known in the art to further insure against interrogating a CNVR. It may also be beneficial to separate each target loci from any other target loci by approximately the same number of nucleotides (e.g., along the arm of a chromosome). Many suitable targets exist on each chromosome.
Exemplary targets (which can serve as qualified targets) and their corresponding PCR primers that can be used to interrogate human chromosomes are shown in Table 3. The probes that can be used in combination with particular primers or primer pairs, and the sequences amplified using particular primer pairs are shown in Table 4. One or more of such target loci can be amplified from a biological sample (e.g., gDNA). It should be understood that non-CNVR targets and corresponding probes or primers other than those listed in Tables 3 and 4 can be used in the methods disclosed herein. It is noted that chromosomes of other organisms may be similarly interrogated but that different target loci are used.







TABLE 3







Exemplary Targets and PCR Primers

















SEQ

SEQ


Chromosome

Gene

ID

ID


location
Cytoband
Symbol
Forward Primer
NO
Reverse Primer
NO
















1: 36669968-
1p34.3
C1orf102
CCAGGGCTGCCTAT
1
GCTGATCCGGCAGAC
2


36670038


TGACTT

ACT



 


1: 19882469-
1p36.13
TMCO4
CCCACGGCCTTCAG
3
GGGCTCTGCACACCC
4


19882569


GAT

A



 


1: 176197341-
1q25.2
SEC16B
AACCCACCAGCCTG
5
TGAGCTCAGTCAGTA
6


176197427


AACTG

CATCAGAGAT



 


1: 203386468-
1q32.1
DSTYK
CACAATGCTGCCTG
7
TGCTTTGTTGGCAGC
8


203386559


ACATCAT

TGGATA



 


2: 51062327-
2p16.3
NRXN1
TGACAGCTTTTGGC
9
CCTGTCACTGGGAGG
10


51062418


TCAGAAATTAGA

AAATCCATA



 


2: 27426789-
2p23.3
GTF3C2
CACGATGAGGGTTG
11
GATTCTTGAGCCAGC
12


27426880


AGGGAAAA

AGCTGTA



 


2: 141646278-
2q22.1
LRP1B
GCCAGGATGCTCCA
13
AAGGATTATTCTGAC
14


141646366


TGTAGTA

GGTTCATGTTGT



 


2: 166773406-
2q24.3
SCN9A
AGGTGAAAAAAGTA
15
TTGATCAAAGCTTAT
16


166773515


CTTATGAGGATGAT

GGCTCTATCAACT






GAAT





 


3: 71541265-
3p14.1
FOXP1
AAACAGGAGAATGA
17
CCATAGACAGATTAG
18


71541358


ATGAATGAATATGC

CCTGCTTCTT






T





 


3: 51489723-
3p21.2
VPRBP
GCCATCCTCCTTTT
19
TGGGTTTGGTGCCTC
20


51489813


TCTCATCCT

ACA



 


3: 98908486-
3q11.2
EPHA6
TGGCATGTGAGATG
21
AAAGGGTACCACTAG
22


98908579


TGTTCAAGAAA

AGAGGCA



 


3: 142551967-
3q23
ZBTB38
TCCCTCAAGTTTAT
23
AGAGCAAGACGTCCA
24


142552067


TCAGTCTCCTTATG

AATTGAAGTA






T





 


4: 46733909-
4p12
GABRB1
CATTTCCAAGTACA
25
CCTCAGTGGTGCAAA
26


46734018


GTAACTCCACAGTA

AACAGTT



 


4: 6931464-
4p16.1
KIAA0232
CCCAAGCACCTGTA
27
CCTCAAACATCTGCC
28


6931545


GCATCATC

TTCATGTGT



 


4: 100651173-
4q23
C4orf17
GGAATGTTACCACG
29
CAGCTGTCTTCATCC
30


100651274


TTGCTAAGC

AGTTTCTCA



 


4: 146906234-
4q31.22
ZNF827
TTTCTGGGAGCCAT
31
CAGCGAATCAAATAG
32


146906321


CTGAATCATC

CCCCTCTT



 


5: 26772993-
5p14.1
LOC100131678
GGTATGACCTGGAC
33
AGGAGGGAGATAAAA
34


26773084


ACATTAAGCA

ATAATGAGAAATGTA








AGC



 


5: 13791807-
5p15.2
DNAH5
GCAAGCCACAGGAA
35
TGTACAATTTGACTT
36


13791884


GAGGAA

GGTGCCAGAA



 


5: 108773400-
5q21.3
PJA2
GCGGAACAAGCCAG
37
GAGGTTCGAGCGCTG
38


108773485


ACTGAA

TTCT



 


5: 122258322-
5q23.2
SNX24
GTTTTAGAAGGAAG
39
AGAATGTGCAGTCAA
40


122258422


AGGGCTAGGAA

CACTCAGAA



 


6: 12970453-
6p24.1
PHACTR1
GTATAGGCAGCGAC
41
AGATCTCCACAGGAC
42


12970529


AGCACTT

TCACCAA



 


6: 3672898-
6p25.2
C6orf145
GAAAACCAAAGCAA
43
GGGAGATGGGAGTAA
44


3672977


CAAGGTGAGT

GTTCCAAAC



 


6: 74590195-
6q13
CD109
GGGTTGCAGGGATG
45
TGCATTCATTCTGAT
46


74590286


GTGTA

CTTCCACACA



 


6: 112680120-
6q21
LAMA4
GGGATCTGTAACTT
47
GCAGCACAGCAAAGT
48


112680214


GACCAAGGT

GTTTCAG



 


7: 47936016-
7p12.3
PKD1L1
GATGGTATCTCCCA
49
CAATTTGCAGCACAA
50


47936124


CAAGTCACTAC

GGAGCTA



 


7: 40801751-
7p14.1
C7orf10
CTGTCTGATTGTTA
51
GTCACAACTTGTTCT
52


40801837


AGAGGGCTTGT

TGGGAGTTTG



 


7: 99515052-
7q22.1
ZNF3
GATAGATGGCCTGG
53
GCCTTCTAGGGACTG
54


99515140


CAGTAAGAAC

ACTTCAA



 


7: 134591172-
7q33
STRA8
TGAACCTTTGACAC
55
TGACAGCAAATATTA
56


134591254


CTTCCCAAA

CCGAAGGTGAT



 


8: 12902405-
8p22
C8orf79
GATCTCCTCTTCTG
57
TGTGATGACAAAAAC
58


12902494


TGCCTACATC

ATAAATGAGACTGAG








T



 


8: 10106677-
8p23.1
MSRA
GTTCTCTCTGGCCG
59
CACTTCTGCAGAGGC
60


10106775


TCAATATCTT

TGGAA



 


8: 61648551-
8q12.1
RAB2A
GGGACCTGGGTTTC
61
ACCCAGAGTCGAGTG
62


61648656


CTGATACT

GACAAT



 


8: 75439579-
8q21.11
GDAP1
TGGAGAGAACCCCA
63
CTAAAGCAATGTGTG
64


75439660


GGCTTTAT

TGATTCATAAGCA



 


9: 26949592-
9p21.2
IFT74
AGATGGGATCAAGG
65
GGGCACATAAACTTC
66


26949694


GTAAATCAGAGT

TCTACATCCA



 


9: 408149-
9p24.3
DOCK8
TGGCCATAGCAGGG
67
ACCAGACACATGTGC
68


408232


AATAATTTCA

ACCAA



 


9: 78498063-
9q21.13
PRUNE2
GGATGGGCAATTTT
69
ATCTCTTAAGCACCT
70


78498161


AGGTAATCTCCAA

ACCCTGGT



 


9: 100023301-
9q22.33
TBC1D2
CCAGTGCCTTGTGC
71
CCTCCCTGAGCTCAC
72


100023371


AGGAT

AAGATT



 


10: 17011452-
10p13
CUBN
AAATAGAATATGAG
73
TCAAGTTGTAGTCAG
74


17011533


ACATGAGTAAATAT

TGCCACAAA






GCCCTTTT





 


10: 5401127-
10p15.1
UCN3
CAAAAGCTACAAGC
75
ACACATATGTACAGA
76


5401221


CAGAGATACGA

GAGACCAGGAA



 


10: 63195641-
10q21.2
C10orf107
AAGACAATGTGCAG
77
GGCCTCTTCCTGTAT
78


63195741


CAAAAGATAGC

TTGCAGTTT



 


10: 121323685-
10826.11
LOC100133264
CAAATTTTACCCAC
79
GCTTAAGTTGTATGT
80


121323788

TIAL1
ACAGCCTGAAA

GTGCAGAGTT



 


11: 46730822-
11p11.2
CKAP5
AAAACAAGTAGGGC
81
TGCCCTTTCAACTGC
82


46730911


ACTGGAAGAA

TGATTCTAAT



 


11: 6200897-
11p15.4
FAM160A2
CATTGCAGAACTCC
83
CTGTCTGCGACGGGA
84


6200970


AGGGAACT

AGA



 


11: 62241048-
11q12.3
HNRNPUL2
CCCCGGCTTCGGTT
85
AGTACAAGGAGGAGG
86


62241147


CT

CAAGGAA



 


11: 130999952-
11q25
NTM
GGAATCCAGGAGGT
87
CAGCCAACTAAACTG
88


131000023


GGTGATG

TATGCTCTGT



 


12: 27346341-
12p11.23
STK38L
AAGAAATTAGAAGT
89
GGGTGCTTTTGTACA
90


27346426


GGCCATGGAAGA

GTAATTATAGGT



 


12: 16036914-
12p12.3
DERA
CAGGTCAATCTACT
91
CCGCTACTGAAAGAT
92


16037009


GCTAAGGGATT

AAAGCCACTT



 


12: 42605583-
12q12
TMEM117
AGGCTGGTGGAGGC
93
GAAACCCTGGGAAAC
94


42605676


TAGT

CATACCAT



 


12: 92044805-
12q22
LOC643339
CTTGGGATGTTTTA
95
GGGAAGGGACCTGAG
96


92044882


TAAGTGTCTGTCTG

GATAGG






T





 


13: 19660225-
13q12.11
GJB2
TTGACATGAGGCCA
97
GCACCTAACAACATT
98


19660311


TTTGCTATCA

GTAGCCTCAA



 


13: 38442357-
13q13.3
STOML3
AGATCTGGGACAAG
99
GCTAATGTCAACGAT
100


38442447


GTCTGTGT

GTCCATCAAG



 


13: 95040142-
13q32.1
DZIP1
ACTTCTCCAGTTGA
101
GGTAATGGAGACTCA
102


95040222


AAGGGTATCCA

TGAATGACACT



 


13: 114069879-
13q34
UPF3A
GCCTAGAATATCCT
103
TGCTTCCAGTCTTGG
104


114069968


GCAGTGGTAGA

CATCTTTT



 


14: 24173173-
14q12
GZMB
GGAGGAAGGCCAGC
105
ACAACAGCAGCTCCA
106


24173244


AGAAG

ACCA



 


14: 34648789-
14q13.2
PPP2R3C
CAGCCTTTTCACCA
107
ACCAGACACCACCTA
108


34648863


ACCTTCAAA

TGATTGGA



 


14: 72506044-
14q24.2
ZFYVE1
GATAGGCGCACCAG
109
CAGTGGCCAAACACG
110


72506141


GAATGA

AATTAAAGT



 


14: 104621601-
14q32.33

CATCCACTCACCAC
111
GGCATGAGGCTTGGA
112


104621710


TGTATCCA

AGCA



 


15: 33605950-
15q14
ATPBD4
GAGCACCCACTGTG
113
ACACCAGCAGAATTA
114


33606033


TACGA

TGCCATACTT



 


15: 43667323-
15q21.1
PLDN
GGGATGGATTTACC
115
AGCTGACCAAGCATT
116


43667413


AGAAAGATGATCAG

TCATGAGT



 


15: 77533137-
15q25.1
KIAA1024
ACTCAGGCTTACCA
117
TGCATGGCAAAGGAA
118


77533242


TATTTGTTTGTACT

ATCCCA






T





 


15: 86971227-
15q26.1
AEN
AGGTGATAAATAGC
119
CCATACCTGAACGTG
120


86971330


TTACATTTTAGAGT

ATGCCA






TTGCT





 


16: 31240261-
16p11.2
ITGAM
TGATGGTTTTCTGG
121
CCACTTCCCTGGGAT
122


31240341


TGTCCCTTTAG

TGAACT



 


16: 4814659-
16p13.3
N-PAC
GCCCAAGAGCCTGA
123
CCGGGCCATCGATGA
124


4814737


GTTCT

AGAG



 


16: 56871110-
16q21
CCDC113
CCATCGTAAGGCTT
125
GGCTACTTCCCAGCA
126


56871184


GGAATCGAA

AGGTAAT



 


16: 85328817-
16q24.1
LOC729979
GGTCCAGCCCTTCT
127
ACCAGCACTCACCGC
128


85328925


CAACAC

TAAA



 


17: 8680592-
17p13.1
PIK3R6
TCCTGGTAGGGATA
129
GTCTGGTTTGCAGGG
130


8680671


CAGCTCATT

ACACT



 


17: 854262-
17p13.3
ABR
GGCAGCCGATGGTC
131
GGCCTTGGGCAGAAC
132


854342


AGTA

AGTAAATA



 


17: 45884445-
17q21.33
ACSF2
CCCTCTGCTGGCAC
133
GCCCAGGTGGGACTG
134


45884521


CTTTAAG

AGA



 


17: 77723894-
17q25.3
CCDC57
GCTTAGAGCCTCCA
135
AGACTTTCCATCCAG
136


77723990


TTTCTTTCCT

TGAGATCCA



 


18: 13731517-
18p11.21
RNMT
GATTGACAAATTTC
137
TCAGCCTGCTCATAA
138


13731609


GTGACCCACAA

GACTCAAATG



 


18: 201348-
18p11.32
USP14
TCCAGAGCTTTAGA
139
GTGGTCTGCTTCTGT
140


201426


GGAAGACACAT

CCTCTTTT



 


18: 57674840-
18q21.33
RNF152
TGCTGTCCTTACTC
141
GCCTAAGTTTGCAGA
142


57674938


ATTCCACATC

CCCTCAA



 


18: 74964920-
18q23
ATP9B
GTGGTCCATATGGT
143
AAAGGGACTCTTCCG
144


74964997


CCCTTCTTAAA

GTTCTTG



 


19: 17313717-
19p13.11
GTPBP3
CCGTTCGACCCTTG
145
ACTACGAAACAAATA
146


17313790


ATGCT

TACTGAGATCCTCCT



 


19: 14507730-
19p13.12
GPSN2
GGTGACTCACTGGG
147
GAGTGCCTACAGCAA
148


14507818


CAGATTC

AGAAATGG



 


19: 47551898-
19q13.2
MEGF8
GCTCTGCCGATGTC
149
CAGTGTGGGCATTGC
150


47551977


CTCA

AGTTC



 


19: 62959915-
19q13.43
ZNF776
CAGTACTGGTGAGG
151
TCCAGGCTTATAACG
152


62960014


GAAATCTGTT

TAGGACACTA



 


20: 13418983-
20p12.1
TASP1
CCACTTCCCTCAAT
153
GGGTTTTGCCAACTT
154


13419079


CATGTGACA

TTTCTGGATT



 


20: 2311101-
20p13
TGM6
GTGCGAAGGTTAGT
155
GAGAGGAACCCAGCA
156


2311185


TTCTGAGGAA

AGTTAGTT



 


20: 34441093-
20q11.23
DLGAP4
CCCATGGGAGATGC
157
GGTCTCCTGGTTCCA
158


34441175


TCTTGA

TCTCTTC



 


20: 42805126-
20q13.12
LOC100128040
GCCCTGTCCCTGCT
159
AGGTGCCCCTTAGCC
160


42805219


TCTG

AGTA



 


21: 27228739-
21q21.3
ADAMTS5
CAGGCAGCTTCTTG
161
GTGTCCCGGCATGGA
162


27228824


GTCAGA

TGT



 


21: 32606313-
21q22.11
MRAP
AGCATCCTATGCCT
163
CAGTAAGCAAGCAGG
164


32606415


TTGACAAAGA

CTAGGT



 


21: 32989371-
21q22.11
SYNJ1
GTTCTGATTTCTAC
165
GGAATCAGTCTTTGC
166


32989464


TCCTCCACACA

ATTTGCATCT



 


21: 38009196-
21q22.13
KCNJ6
CTGATGTGTCTTGG
167
CCATGGATCAGGACG
168


38009283


CAGGTCAT

TCGAAAG



 


22: 15964337-
22q11.1
IL17RA
CCTTACCCATCCCA
169
GTAGAGTGAGTGTGA
170


15964432


ACTAGCCTTA

CGTTGGAT



 


22: 29154514-
22q12.2
MTP18
CCTCCTGATCATAC
171
CACATTCCCTGGCAG
172


29154594


TCTGGTACCT

GAGTAG



 


22: 34452693-
22q12.3
APOL5
CACATGAGGCTTTC
173
CCTTGATGCCCTGGA
174


34452785


GGAGGAATAA

TAGCTTTTAC



 


22: 48978363-
22q13.33
TRABD
CCATCCCTCTCCAC
175
GTCTGGGAACTCGCC
176


48978457


AGCAA

AATCAT



 


X: 50512491-
Xp11.22
SHROOM4
GGAAATTGTCAGGT
177
CCCATTTCATGTCCT
178


50512567


CAGCTCAGT

CTCAGAAGAA



 


X: 24141606-
Xp22.11
ZFX
TGCTTTAGGCAGGT
179
GGGCCAAGAAGGTAC
180


24141705


GTGAACT

AGGAATTT



 


X: 109581619-
Xq22.3
RGAG1
CCCCATGGTCAACA
181
CATCACCCCAGAGGC
182


109581695


CAAAATGTAG

TGTT



 


X: 123351536-
Xq25
ODZ1
GGTCAGAAAGTTAC
183
CCAGTGTCTGCAGTG
184


123351615


CAGGACTTGT

GACAAAA



 


Y: 7271634-
Yp11.2
PRKY
GTGATCTGAATGAT
185
ACACGGATAGCCAGA
186


7271743


GTTGAACAAGCA

CAATGAAATAC



 


Y: 5423177-
Yp11.2
PCDH11Y
TCCTGCAAAATGAC
187
ACATGTCAGGCCTTT
188


5423278


TTGAGTTGGTA

TTATTTTGTAGC



 


Y: 20211350-
Yq11.222
CYorf15A
CTTCCAGGGAAATT
189
GTTTCCCTGTTGAGA
190


20211455


CACCTCTTCT

ATGTTTCCAT



 


Y: 21330672-
Yq11.223
RPS4Y2
GAAAGGGAAGAGCA
191
GGCCTCAAAGTCCCA
192


21330747


CACAGTCT

CACAA
















TABLE 4







Exemplary Amplified Sequences

















Primer









Pair

Probe
Amplified
Amplicon


Chromosome

Gene
SEQ ID
Probe
SEQ ID
Sequence
SEQ ID


location
Cytoband
Symbol
NO)
Sequence
NO
(amplicon)
NO





1: 36669968-
1p34.3
C1orf102
1/2
TCTTCCAA
193
CCAGGGCTG
289


36670038



GACCTGCA

CCTATTGAC







CATCCG

TTACTCGGA









TGTGCAGGT









CTTGGAAGA









AGATGAGGA









GTGTCTGCC









GGATCAGC



 


1: 19882469-
1p36.13
TMCO4
3/4
CAGGAGAC
194
CCCACGGCC
290


19882569



AGTGAGTG

TTCAGGATG







AGCACC

GCATCCATC









TGCTTGGCA









TAGTCCAGG









TGGCCGCTG









ACCTGCAGG









AGACAGTGA









GTGAGCACC









ACCGTGGGT









GTGCAGAGC









CC



 


1: 176197341-
1q25.2
SEC16B
5/6
CCCCAGCT
195
AACCCACCA
291


176197427



TCAGCAGC

GCCTGAACT







TTG

GGACTCCAA









GCTGCTGAA









GCTGGGGGA









TCATCCCGC









TCCGGGGCA









TCTCTGATG









TACTGACTG









AGCTCA



 


1: 203386468-
1q32.1
DSTYK
7/8
CCGTGCCA
196
CACAATGCT
292


203386559



AGATCACT

GCCTGACAT







GACTTA

CATGGCCTC









TGGCTTGCA









GAATCCTAA









GTCAGTGAT









CTTGGCACG









GTTCTGCTT









ATCCAGCTG









CCAACAAAG









CA



 


2: 51062327-
2p16.3
NRXN1
 9/10
AACCACCA
197
TGACAGCTT
293


51062418



ACAAAGGG

TTGGCTCAG







ATTCT

AAATTAGAA









AATGAAATG









ATAACCACC









AACAAAGGG









ATTCTGCAG









CTGAGTATG









GATTTCCTC









CCAGTGACA









GG



 


2: 27426789-
2p23.3
GTF3C2
11/12
ACCTTCCC
198
CACGATGAG
294


27426880



AGTTTGAT

GGTTGAGGG







AAGCAC

AAAAACAGT









TGGTAGGAA









CAAGTGCTT









ATCAAACTG









GGAAGGTGC









TCTTATTTA









CAGCTGCTG









GCTCAAGAA









TC



 


2: 141646278-
2q22.1
LRP1B
13/14
CTGGCCTG
199
GCCAGGATG
295


141646366



TGTGTACT

CTCCATGTA







TCT

GTATTGCAT









TATAACATG









GTCTCAGCT









GGCCTGTGT









GTACTTCTA









CAACATGAA









CCGTCAGAA









TAATCCTT



 


2: 166773406-
2q24.3
SCN9A
15/16
ACCCATGC
200
AGGTGAAAA
296


166773515



CTCTTTCT

AAGTACTTA







AATAAC

TGAGGATGA









TGAATAGTT









GGAAAAACT









AGTAAAATA









GGGTTGGTT









ATTAGAAAG









AGGCATGGG









TAGTTGATA









GAGCCATAA









GCTTTGATC









AA



 


3: 71541265-
3p14.1
FOXP1
17/18
CCTGCCTA
201
AAACAGGAG
297


71541358



CCAAAGAG

AATGAATGA







GATACA

ATGAATATG









CTAATACAA









CCACCTCTG









TATCCTCTT









TGGTAGGCA









GGAGGCAAG









AAGCAGGCT









AATCTGTCT









ATGG



 


3: 51489723-
3p21.2
VPRBP
19/20
TCCCAGCC
202
GCCATCCTC
298


51489813



CACTGTAA

CTTTTTCTC







ATG

ATCCTGTGG









GGCTACTTA









TGATGTGAT









GCCATTTAC









AGTGGGCTG









GGATTACAG









GTGTGAGGC









ACCAAACCC









A



 


3: 98908486-
3q11.2
EPHA6
21/22
AATGCCAC
203
TGGCATGTG
299


98908579



CAGTGACC

AGATGTGTT







TTCAG

CAAGAAACT









CAAGACTAA









GGGAATGAA









TGAATGAAT









GCCACCAGT









GACCTTCAG









TGCCTCTCT









AGTGGTACC









CTTT



 


3: 142551967-
3q23
ZBTB38
23/24
TCTTCTTT
204
TCCCTCAAG
300


142552067



GCTCCTGA

TTTATTCAG







GACCTC

TCTCCTTAT









GTAATCAGT









AATTCTATC









AAATCCTCT









TCTTTGCTC









CTGAGACCT









CACCTACTT









CAATTTGGA









CGTCTTGCT









CT



 


4: 46733909-
4p12
GABRB1
25/26
ATAGGTGT
205
CATTTCCAA
301


46734018



CACTGTAA

GTACAGTAA







AGCAAC

CTCCACAGT









ACTATCCTG









TTGCTTTAC









AGGGACACC









TATGCCTTT









TTTATTCAG









AATAAAGAA









CATTGCAAA









CTGTTTTTG









CACCACTGA









GG



 


4: 6931464-
4p16.1
KIAA0232
27/28
TCCTGCTG
206
CCCAAGCAC
302


6931545



CCCACTGA

CTGTAGCAT







CCTG

CATCGTCCA









CGTCCTGCT









GCCCACTGA









CCTGTCCGG









CTCCACACA









TGAAGGCAG









ATGTTTGAG









G



 


4: 100651173-
4q23
C4orf17
29/30
CCACACAG
207
GGAATGTTA
303


100651274



GCTCCTTG

CCACGTTGC







GAGTAA

TAAGCTATG









TAACATATC









TTAACAACC









AGGGAGCCA









CACAGGCTC









CTTGGAGTA









AGAGTGTGA









GAAACTGGA









TGAAGACAG









CTG



 


4: 146906234-
4q31.22
ZNF827
31/32
TTGCTGTT
208
TTTCTGGGA
304


146906321



GGCTGAAT

GCCATCTGA







CACT

ATCATCTTT









GCTGTTGGC









TGAATCACT









CAGAGCTGA









GAGGGAGGA









TGAAGAGGG









GCTATTTGA









TTCGCTG



 


5: 26772993-
5p14.1
LOC100131678
33/34
AATGGAGA
209
GGTATGACC
305


26773084



AGGGAAAA

TGGACACAT







TTAC

TAAGCATTC









GTAATTTTC









CCTTCTCCA









TTACTAGAT









ACAGTGCTT









ACATTTCTC









ATTATTTTT









ATCTCCCTC









CT



 


5: 13791807-
5p15.2
DNAH5
35/36
CAGGGAGA
210
GCAAGCCAC
306


13791884



CAAAACAG

AGGAAGAGG







AAATAT

AAGCCCAAA









GGCAGGGAG









ACAAAACAG









AAATATGCT









TCTGGCACC









AAGTCAAAT









TGTACA



 


5: 108773400-
5q21.3
PJA2
37/38
CCGGAGCC
211
GCGGAACAA
307


108773485



GCTGCACA

GCCAGACTG







T

AAAAAAAAA









AAAAAAAAC









CCTCACCGA









AATGTGCAG









CGGCTCCGG









AGCGAGAAC









AGCGCTCGA









ACCTC



 


5: 122258322-
5q23.2
SNX24
39/40
CTAGGTAC
212
GTTTTAGAA
308


122258422



TGCGCCAC

GGAAGAGGG







TTTT

CTAGGAAGA









AAAGTGGCG









CAGTACCTA









GTAGGTAAG









TATAATCTG









GATGCTCCC









AGTAATTCT









GAGTGTTGA









CTGCACATT









CT



 


6: 12970453-
6p24.1
PHACTR1
41/42
ATGCAACT
213
GTATAGGCA
309


12970529



CATGCTGA

GCGACAGCA







ATTTA

CTTGTAAAT









TCAGCATGA









GTTGCATGG









TTGGCCAAT









GTTGGTGAG









TCCTGTGGA









GATCT



 


6: 3672898-
6p25.2
C6orf145
43/44
CCTCAAGC
214
GAAAACCAA
310


3672977



TCCGACCC

AGCAACAAG







CTCCT

GTGAGTCCT









CAGGAGGGG









TCGGAGCTT









GAGGTTTTG









GAGTTTGGA









ACTTACTCC









CATCTCCC



 


6: 74590195-
6q13
CD109
45/46
CATGTATA
215
GGGTTGCAG
311


74590286



GCTGCATA

GGATGGTGT







GATTTC

ACAACAGGT









CCTAGCATG









TATAGCTGC









ATAGATTTC









TTCACCTGA









TCTTTGTGT









GGAAGATCA









GAATGAATG









CA



 


6: 112680120-
6q21
LAMA4
47/48
CAGTCTGA
216
GGGATCTGT
312


112680214



TGGTCCCA

AACTTGACC







AGTTGA

AAGGTCAAA









GAGCTTGAA









ATTTCAACT









TGGGACCAT









CAGACTGAA









AACCTGCAA









TCTGAAACA









CTTTGCTGT









GCTGC



 


7: 47936016-
7p12.3
PKD1L1
49/50
CCACCCTT
217
GATGGTATC
313


47936124



TCTACATT

TCCCACAAG







TCTCC

TCACTACTT









CCTGTGTTT









TTGCGAAAA









GCTCCCCGT









GAGGGTGGG









TGCCACCCT









TTCTACATT









TCTCCCTAG









CTCCTTGTG









CTGCAAATT









G



 


7: 40801751-
7p14.1
C7orf10
51/52
TCCTTGCC
218
CTGTCTGAT
314


40801837



AACTAGAA

TGTTAAGAG







ACTATG

GGCTTGTAT









TCTCTTGAA









AATCATAGT









TTCTAGTTG









GCAAGGAGC









AAACTCCCA









AGAACAAGT









TGTGAC



 


7: 99515052-
7q22.1
ZNF3
53/54
AAAGAATC
219
GATAGATGG
315


99515140



AGGCAGGT

CCTGGCAGT







AAAGCT

AAGAACAAG









ACACGGAAA









GCTTTACCT









GCCTGATTC









TTTCCTTCC









TTCTTTGAA









GTCAGTCCC









TAGAAGGC



 


7: 134591172-
7q33
STRA8
55/56
ACGCTGGG
220
TGAACCTTT
316


134591254



CTATTTCA

GACACCTTC







TCATCT

CCAAAACGC









TGGGCTATT









TCATCATCT









TCTACAGTC









TTCATCACC









TTCGGTAAT









ATTTGCTGT









CA



 


8: 12902405-
8p22
C8orf79
57/58
CTTCCCCC
221
GATCTCCTC
317


12902494



AGCAAAGT

TTCTGTGCC







TAGTTG

TACATCAAC









TTCCCCCAG









CAAAGTTAG









TTGTATCTT









TGTCTACTC









AGTCTCATT









TATGTTTTT









GTCATCACA



 


8: 10106677-
8p23.1
MSRA
59/60
CCACGGTC
222
GTTCTCTCT
318


10106775



CACTCTGT

GGCCGTCAA







CCACGT

TATCTTAAT









GAAAGTGAC









ATTCCGTTG









GCCACGGTC









CACTCTGTC









CACGTGGAG









GGCCGGGTT









CCAGCCTCT









GCAGAAGTG



 


8: 61648551-
8q12.1
RAB2A
61/62
ACAAGGCA
223
GGGACCTGG
319


61648656



AGACAGAG

GTTTCCTGA







ATGTAC

TACTTCCTA









TGTGTCACA









GTTTTCCCT









TAAATGATA









ACCGTACAT









CTCTGTCTT









GCCTTGTCC









TTGAATTGT









CCACTCGAC









TCTGGGT



 


8: 75439579-
8q21.11
GDAP1
63/64
CTTTGACC
224
TGGAGAGAA
320


75439660



TCAGTGTT

CCCCAGGCT







AATTTT

TTATATGTA









TACTTTGAC









CTCAGTGTT









AATTTTAAA









TGCTTATGA









ATCACACAC









ATTGCTTTA









G



 


9: 26949592-
9p21.2
IFT74
65/66
CTCCAGTC
225
AGATGGGAT
321


26949694



TCAACAGC

CAAGGGTAA







CATTCC

ATCAGAGTA









AGATTGATC









TTGAATGAG









AGAAGGAAT









GGCTGTTGA









GACTGGAGG









GCAGGATGG









ATGTAGAGA









AGTTTATGT









GCCC



 


9: 408149-
9p24.3
DOCK8
67/68
CCAAGGAA
226
TGGCCATAG
322


408232



GACAGCAC

CAGGGAATA







TATTC

ATTTCAATT









TGAAAACAA









GTGGAATAG









TGCTGTCTT









CCTTGGTNT









GTTGGTGCA









CATGTGTCT









GGT



 


9: 78498063-
9q21.13
PRUNE2
69/70
CTGCTGAG
227
GGATGGGCA
323


78498161



TAATTCAC

ATTTTAGGT







TTTCCC

AATCTCCAA









TTGACCTAA









CTCTAATGG









AATGGGAAA









GTGAATTAC









TCAGCAGAT









GACCACCAG









GGTAGGTGC









TTAAGAGAT



 


9: 100023301-
9q22.33
TBC1D2
71/72
CCTGCCCA
228
CCAGTGCCT
324


100023371



GGAGCTAG

TGTGCAGGA







TG

TCTTCACTA









GCTCCTGGG









CAGGGAGAG









GGAAGAATC









TTGTGAGCT









CAGGGAGG



 


10: 17011452-
10p13
CUBN
73/74
ACAACCAG
229
AAATAGAAT
325


17011533



CCACATGG

ATGAGACAT







ATTC

GAGTAAATA









TGCCCTTTT









ATACAACCA









GCCACATGG









ATTCTTTGT









GGCACTGAC









TACAACTTG









A



 


10: 5401127-
10p15.1
UCN3
75/76
CTGAGCAA
230
CAAAAGCTA
326


5401221



GCATTTGA

CAAGCCAGA







TCCTGC

GATACGATA









CAACAAGGA









CATTGCTCT









GCAGGATCA









AATGCTTGC









TCAGATTTC









CTGGTCTCT









CTGTACATA









TGTGT



 


10: 63195641-
10q21.2
C10orf107
77/78
CTTCACAG
231
AAGACAATG
327


63195741



ACCGAGAT

TGCAGCAAA







AAACG

AGATAGCTC









CATCATAAC









CACGTTTTT









TATGATTGT









CTTCACAGA









CCGAGATAA









ACGAAAAAC









TGCAAATAC









AGGAAGAGG









CC



 


10: 121323685-
10q26.11
LOC100133264
79/80
TCGGTCTT
232
CAAATTTTA
328


121323788

TIAL1

CTGCATCT

CCCACACAG







TCC

CCTGAAAAA









TACCTTGAA









AGCAAACCT









CGGTCTTCT









GCATCTTCC









AATTGATTC









CTTTACAAA









CTCTGCACA









CATACAACT









TAAGC



 


11: 46730822-
11p11.2
CKAP5
81/82
ACCCAACA
233
AAAACAAGT
329


46730911



CAACAGCA

AGGGCACTG







TTAAGT

GAAGAAAAA









CCCAACACA









ACAGCATTA









AGTTTCAAA









CCTGCATTC









CAATTAGAA









TCAGCAGTT









GAAAGGGCA



 


11: 6200897-
11p15.4
FAM160A2
83/84
CAAGGGCT
234
CATTGCAGA
330


6200970



GGCACTCC

ACTCCAGGG







CA

AACTCATGA









AGAGTGCAA









GGGCTGGCA









CTCCCAGCC









AGTCTTCCC









GTCGCAGAC









AG



 


11: 62241048-
11q12.3
HNRNPUL2
85/86
TTTCGGTT
235
CCCCGGCTT
331


62241147



GTTTCGGC

CGGTTCTGC







GATTTG

CGGTTACGC









TTGTTTCGG









TTGTTTCGG









CGATTTGTC









CGCTTCTCG









GAGGGGGGC









AGAAGCTTC









CTTGCCTCC









TCCTTGTAC









T



 


11: 130999952-
11q25
NTM
87/88
ATCAGGCA
236
GGAATCCAG
332


131000023



GCCAGGAT

GAGGTGGTG







TT

ATGATCAGG









CAGCCAGGA









TTTCTGTCT









CCACAGAGC









ATACAGTTT









AGTTGGCTG



 


12: 27346341-
12p11.23
STK38L
89/90
ACCTCTTC
237
AAGAAATTA
333


27346426



ATCTGCTA

GAAGTGGCC







ATCCTTC

ATGGAAGAA









GAAGGATTA









GCAGATGAA









GAGGTAATG









TAATTACCT









ATAATTACT









GTACAAAAG









CACCC



 


12: 16036914-
12p12.3
DERA
91/92
CAGCCCAG
238
CAGGTCAAT
334


16037009



CAAATGCA

CTACTGCTA







CACAT

AGGGATTTC









AGCCCAGCA









AATGCACAC









ATTAAGAAT









AATGCCAGA









ATGTAGAAA









AGTGGCTTT









ATCTTTCAG









TAGCGG



 


12: 42605583-
12q12
TMEM117
93/94
CTGCGGGC
239
AGGCTGGTG
335


42605676



TTTAGGAC

GAGGCTAGT







TCCA

GCTCCGCCA









CAGCTGCGG









GCTTTAGGA









CTCCACCTC









GTCAGTCAT









CCATGCCAA









TGGTATGGT









TTCCCAGGG









TTTC



 


12: 92044805-
12q22
LOC643339
95/96
ACTTCCTA
240
CTTGGGATG
336


92044882



TGACAGCC

TTTTATAAG







AATCAC

TGTCTGTCT









GTACTTCCT









ATGACAGCC









AATCACATC









CAACCTATC









CTCAGGTCC









CTTCCC



 


13: 19660225-
13q12.11
GJB2
97/98
AAGCCATC
241
TTGACATGA
337


19660311



ACTAGGAA

GGCCATTTG







CTTCT

CTATCATAA









GCCATCACT









AGGAACTTC









TAGTCTGTC









TCACTCGAT









TGAGGCTAC









AATGTTGTT









AGGTGC



 


13: 38442357-
13q13.3
STOML3
 99/100
TTGAGCCA
242
AGATCTGGG
338


38442447



GCAGAAAT

ACAAGGTCT







GTT

GTGTCCCTA









AGACATTTC









TCAGAGTGG









TTTGAGCCA









GCAGAAATG









TTGCTTGAT









GGACATCGT









TGACATTAG









C



 


13: 95040142-
13q32.1
DZIP1
101/102
CAGATCAG
243
ACTTCTCCA
339


95040222



TGCAGTGT

GTTGAAAGG







TTCTCA

GTATCCATT









TGAGAAACA









CTGCACTGA









TCTGGAATA









TAGTGTCAT









TCATGAGTC









TCCATTACC



 


13: 114069879-
13q34
UPF3A
103/104
TTGCTCCA
244
GCCTAGAAT
340


114069968



TTCCAGAA

ATCCTGCAG







GATAGC

TGGTAGAGT









TTGCTCCAT









TCCAGAAGA









TAGCCAAAA









AGAAGCTGA









GAAAAAAAG









ATGCCAAGA









CTGGAAGCA



 


14: 24173173-
14q12
GZMB
105/106
CTGAGAAG
245
GGAGGAAGG
341


24173244



ATGCAACC

CCAGCAGAA







AATCCT

GCAGGATTG









GTTGCATCT









TCTCAGGAA









GGCTGCCCT









GGTTGGAGC









TGCTGTTGT



 


14: 34648789-
14q13.2
PPP2R3C
107/108
AAGCGATG
246
CAGCCTTTT
342


34648863



ATCAATTA

CACCAACCT







CGAAAACT

TCAAAAAGT









TTTCGTAAT









TGATCATCG









CTTCCTCTC









CAATCATAG









GTGGTGTCT









GGT



 


14: 72506044-
14q24.2
ZFYVE1
109/110
CCGCCATA
247
GATAGGCGC
343


72506141



TACTTCCC

ACCAGGAAT







TAAAGCT

GACCGCCAT









ATACTTCCC









TAAAGCTCA









ACCCACCCA









CCAGTTCAG









TTAAGAATT









ATACTTTAA









TTCGTGTTT









GGCCACTG



 


14: 104621601-
14q32.33

111/112
TCTCCTCC
248
CATCCACTC
344


104621710



CTTTGTTT

ACCACTGTA







TCCC

TCCATCCAC









CTCTCCTCC









CTTTGTTTT









CCCTACAAG









CCCCACGTC









CTGGGGGGC









TGACTCCAA









CTGGGGGTG









CTGCTTCCA









AGCCTCATG









CC



 


15: 33605950-
15q14
ATPBD4
113/114
ACGTGGCT
249
GAGCACCCA
345


33606033



CAGCACTG

CTGTGTACG







TATAC

AGTACACAA









AGTGACCAC









GTGGCTCAG









CACTGTATA









CAAATAAGT









ATGGCATAA









TTCTGCTGG









TGT



 


15: 43667323-
15q21.1
PLDN
115/116
ACGTCACC
250
GGGATGGAT
346


43667413



TCTCTGAA

TTACCAGAA







TTAT

AGATGATCA









GCTTATAAT









TCAGAGAGG









TGACGTATC









CTATAATAT









TGACCACTC









ATGAAATGC









TTGGTCAGC









T



 


15: 77533137-
15q25.1
KIAA1024
117/118
CTGGGCCT
251
ACTCAGGCT
347


77533242



TGGTTTTC

TACCATATT







CA

TGTTTGTAC









TTCTTTTAT









TCACTTCAG









GAGACACTG









GGCCTTGGT









TTTCCAAAT









AGGGTTTTT









GACCTGGGA









TTTCCTTTG









CCATGCA



 


15: 86971227-
15q26.1
AEN
119/120
CTGTGGGC
252
AGGTGATAA
348


86971330



TTTACAAA

ATAGCTTAC







TTTTA

ATTTTAGAG









TTTGCTTTC









TGTTATAAA









AGTTGTACG









CATTGATAT









AAAATTTGT









AAAGCCCAC









AGTGGCATC









ACGTTCAGG









TATGG



 


16: 31240261-
16p11.2
ITGAM
121/122
ACTGAGAG
253
TGATGGTTT
349


31240341



TCAAGGCA

TCTGGTGTC







ATCAT

CCTTTAGGT









CCCAGCCAG









TACTGAGAG









TCAAGGCAA









TCATGGAGT









TCAATCCCA









GGGAAGTGG



 


16: 4814659-
16p13.3
N-PAC
123/124
ACCATGTC
254
GCCCAAGAG
350


4814737



CTCCTGTG

CCTGAGTTC







TCCGTC

TCCTGAGAC









GGACACAGG









AGGACATGG









TGAGATGAG









AAGCTCCTC









TTCATCGAT









GGCCCGG



 


16: 56871110-
16q21
CCDC113
125/126
CAACTGCT
255
CCATCGTAA
351


56871184



CATTGGTT

GGCTTGGAA







ATTTTC

TCGAATGAA









AATAACCAA









TGAGCAGTT









GCAGGCAGA









TTACCTTGC









TGGGAAGTA









GCC



 


16: 85328817-
16q24.1
LOC729979
127/128
AAGCCCTT
256
GGTCCAGCC
352


85328925



GAGCCATC

CTTCTCAAC







TTT

ACNGGAAAG









CCCTTGAGC









CATCTTTGA









TTTGTGTGT









TTTGATCTA









ATTGCACTA









CTGCTTGCA









ATGCTTGTT









TTTAGCGGT









GAGTGCTGG









T



 


17: 8680592-
17p13.1
PIK3R6
129/130
TTCGGCAA
257
TCCTGGTAG
353


8680671



TGACCATC

GGATACAGC







CTTTG

TCATTCGTC









AAGTTCTGT









TCGGCAATG









ACCATCCTT









TGGTACAGT









GTCCCTGCA









AACCAGAC



 


17: 854262-
17p13.3
ABR
131/132
CCTCTTGG
258
GGCAGCCGA
354


854342



GCATGTCT

TGGTCAGTA







TTCCT

CTTCCTTCC









TCTTGGGCA









TGTCTTTCC









TCCGTGCAC









AGAGTATTT









ACTGTTCTG









CCCAAGGCC



 


17: 45884445-
17q21.33
ACSF2
133/134
CAGATAGG
259
CCCTCTGCT
355


45884521



AGCCTTGA

GGCACCTTT







AGAAACA

AAGGTGGGG









CTGTGCTTT









GTTTCTTCA









AGGCTCCTA









TCTGGTCTC









AGTCCCACC









TGGGC



 


17: 77723894-
17q25.3
CCDC57
135/136
TTGCGAAA
260
GCTTAGAGC
356


77723990



CGCGATTG

CTCCATTTC







CCCA

TTTCCTCAT









CTGGGCAAT









CGCGTTTCG









CAAGCTCGT









GTTCTGCTC









TCGGAGCCG









CTGGATCTC









ACTGGATGG









AAAGTCT



 


18: 13731517-
18p11.21
RNMT
137/138
ATGACAGA
261
GATTGACAA
357


13731609



CAAACTGA

ATTTCGTGA







CAACTGC

CCCACAAAT









GTGTTTTGA









CATCTGCAG









TTGTCAGTT









TGTCTGTCA









TTACTCATT









TGAGTCTTA









TGAGCAGGC









TGA



 


18: 201348-
18p11.32
USP14
139/140
CAAATGAG
262
TCCAGAGCT
358


201426



GTGAAACA

TTAGAGGAA







TAAACCC

GACACATAG









GTGGGTTTA









TGTTTCACC









TCATTTGGA









ACAAAAGAG









GACAGAAGC









AGACCAC



 


18: 57674840-
18q21.33
RNF152
141/142
AAGATTGC
263
TGCTGTCCT
359


57674938



TCGACCAC

TACTCATTC







CCCTCC

CACATCCTT









ACTAGAGGT









GAGGGGTTG









GGGGAGGGG









TGGTCGAGC









AATCTTTTG









TACTTTTGA









GGGTCTGCA









AACTTAGGC



 


18: 74964920-
18q23
ATP9B
143/144
TAGTGGTG
264
GTGGTCCAT
360


74964997



AGAACACC

ATGGTCCCT







CATCTTC

TCTTAAAGA









AGATGGGTG









TTCTCACCA









CTATTTACA









GCCAAGAAC









CGGAAGAGT









CCCTTT



 


19: 17313717-
19p13.11
GTPBP3
145/146
CCAACCCG
265
CCGTTCGAC
361


17313790



GATGCCCC

CCTTGATGC









TGGGGCATC









CGGGTTGGG









ATGGAGATA









GGAGGATCT









CAGTATATT









TGTTTCGTA









GT



 


19: 14507730-
19p13.12
GPSN2
147/148
CAGGACAG
266
GGTGACTCA
362


14507818



AAGGGACT

CTGGGCAGA







CCACC

TTCTCCTGG









TGGAGTCCC









TTCTGTCCT









GGCTGTAGC









TTTGTACTT









AGGCCATTT









CTTTGCTGT









AGGCACTC



 


19: 47551898-
19q13.2
MEGF8
149/150
CACTGCCG
267
GCTCTGCCG
363


47551977



CATGGCTC

ATGTCCTCA







T

GGGCTGGGC









TGGCCCACA









CTGCCGCAT









GGCTCTGTG









TCCTGAGAA









CTGCAATGC









CCACACTG



 


19: 62959915-
19q13.43
ZNF776
151/152
CAAGTCCT
268
CAGTACTGG
364


62960014



ACAGTGCA

TGAGGGAAA







TTCATG

TCTGTTCTC









AAGTCCTAC









AGTGCATTC









ATGTGTCCT









GAAGTCCAG









AATTCTGTA









GGATAGTGT









CCTACGTTA









TAAGCCTGG









A



 


20: 13418983-
20p12.1
TASP1
153/154
CAGTGAAG
269
CCACTTCCC
365


13419079



CCACCTTT

TCAATCATG







AAATCA

TGACACCAC









TTCAGTGAA









GCCACCTTT









AAATCATCT









GTTTTTGAA









TTTGTCTGG









AATCCAGAA









AAAGTTGGC









AAAACCC



 


20: 2311101-
20p13
TGM6
155/156
AATGGGCA
270
GTGCGAAGG
366


2311185



TCATCATC

TTAGTTTCT







AACTTT

GAGGAAGGA









AAAAAGTTG









ATGATGATG









CCCATTGTC









AGGTCTGTA









ACTAACTTG









CTGGGTTCC









TCTC



 


20: 34441093-
20q11.23
DLGAP4
157/158
CCCACCAC
271
CCCATGGGA
367


34441175



CAGAATAG

GATGCTCTT







TCTTT

GAGAAAGAC









TATTCTGGT









GGTGGGTGC









GGGATCCTG









TCAGGGGGA









AGAGATGGA









ACCAGGAGA









CC



 


20: 42805126-
20q13.12
LOC100128040
159/160
CTGTGTGC
272
GCCCTGTCC
368


42805219



AGATGTCG

CTGCTTCTG







AAAAT

GAAAAAGAA









TTTTCGACA









TCTGCACAC









AGACAGTTG









TGAAAAAGG









AGGAGAAGC









AGCTACTGG









CTAAGGGGC









ACCT



 


21: 27228739-
21q21.3
ADAMTS5
161/162
CATCTGGC
273
CAGGCAGCT
369


27228824



CCTGGCGT

TCTTGGTCA







ACCA

GACAGACCA









TCTGGCCCT









GGCGTACCA









CAGCACACC









ACAGGCGAG









CACAGACAT









CCATGCCGG









GACAC



 


21: 32606313-
21q22.11
MRAP
163/164
CCAGGTGT
274
AGCATCCTA
370


32606415



TGCTGAGT

TGCCTTTGA







TTAT

CAAAGATTG









CAGTGGCCC









CTCGAGTGC









AGAGGTCAT









CCCAGGTGT









TGCTGAGTT









TATTGAGCA









CACCTAGCC









TGCTTGCTT









ACTG



 


21: 32989371-
21q22.11
SYNJ1
165/166
CACTATGG
275
GTTCTGATT
371


32989464



CGTGAATT

TCTACTCCT







GTG

CCACACATA









AGACGTAAT









AACCAGTCA









TCACAATTC









ACGCCATAG









TGTTTGAGA









TGCAAATGC









AAAGACTGA









TTCC



 


21: 38009196-
21q22.13
KCNJ6
167/168
CCATTCAC
276
CTGATGTGT
372


38009283



CAGCCAAA

CTTGGCAGG







GTT

TCATCCCTG









GCCTGCTTA









GGCAACTTT









GGCTGGTGA









ATGGCCACT









GGGCTTTCG









ACGTCCTGA









TCCATGG



 


22: 15964337-
22q11.1
IL17RA
169/170
CCCAGACC
277
CCTTACCCA
373


15964432



AGAAGAGT

TCACACTCA







TCCAC

GCCTTACCC









ATCCTCGCC









TCTCTCCTC









AGCCCAGAC









CAGAAGAGT









TCCACCAGC









GATCCAACG









TCACACTCA









CTCTAC



 


22: 29154514-
22q12.2
MTP18
171/172
CTGTGCAT
278
CCTCCTGAT
374


29154594



CGGCCTCC

CATACTCTG







TGC

GTACCTGGC









CTGTGCATC









GGCCTCCTG









CTTCATGTC









AACCTCCTA









CTCCTGCCA









GGGAATGTG



 


22: 34452693-
22q12.3
APOL5
173/174
CCAGCAGC
279
CACATGAGG
375


34452785



CTCGATTT

CTTTCGGAG







CAGAC

GAATAAATT









GGTCTGAAA









TCGAGGCTG









CTGGCTTTT









GTGTTAATA









ANTGTGTAA









AAGCTATCC









AGGGCATCA









AGG



 


22: 48978363-
22q13.33
TRABD
175/176
CTGCTTGC
280
CCATCCCTC
376


48978457



AGCGTTCC

TCCACAGCA







ACGTC

AGGATGACG









TGGAACGCT









GCAAGCAGA









AGGACCTAC









TGGAGCAGA









TGATGGCCG









AGATGATTG









GCGAGTTCC









CAGAC



 


X: 50512491-
Xp11.22
SHROOM4
177/178
ACAGGCCC
281
GGAAATTGT
377


50512567



CATTAATT

CAGGTCAGC







TATG

TCAGTGCCT









ACAGGCCCC









ATTAATTTA









TGTCTCCTT









CTTCTGAGA









GGACATGAA









ATGGG



 


X: 24141606-
Xp22.11
ZFX
179/180
CCAAATGC
282
TGCTTTAGG
378


24141705



CAGTCAAA

CAGGTGTGA







GTCA

ACTCCAGCC









CAAATGCCA









GTCAAAGTC









AAGGCATGG









GTTTTCCTA









GCCTATCTT









ANAGGAAAT









TCCTGTACC









TTCTTGGCC









C



 


X: 109581619-
Xq22.3
RGAG1
181/182
CACTGGCG
283
CCCCATGGT
379


109581695



GATTAGAC

CAACACAAA







ATCATT

ATGTAGACT









CTGAAATGA









TGTCTAATC









CGCCAGTGA









GAGCAACAG









CCTCTGGGG









TGATG



 


X: 123351536-
Xq25
ODZ1
183/184
ACCTTCCT
284
GGTCAGAAA
380


123351615



CACCCAGA

GTTACCAGG







ATAA

ACTTGTCTT









GATACCTTA









TTCTGGGTG









AGGAAGGTC









TTATTTTTG









TCCACTGCA









GACACTGG



 


Y: 7271634-
Yp11.2
PRKY
185/186
ACTCTCCT
285
GTGATCTGA
381


7271743



CCCTTTGC

ATGATGTTG







TTCTC

AACAAGCAT









TATCAAAGA









ATTCCACGA









TGAGAAGCA









AAGGGAGGA









GAGTGGAAC









TTTTGAAAA









CCTGTATTT









CATTGTCTG









GCTATCCGT









GT



 


Y: 5423177-
Yp11.2
PCDH11Y
187/188
CAGGGCCA
286
TCCTGCAAA
382


5423278



AGTAGTAA

ATGACTTGA







AGACCT

GTTGGTAAA









GTAGAAAGT









TTTATGACT









ACAAATTTC









AGGGCCAAG









TAGTAAAGA









CCTGCTACA









AAATAAAAA









GGCCTGACA









TGT



 


Y: 20211350-
Yq11.222
CYorf15A
189/190
AACTATAC
287
CTTCCAGGG
383


20211455



GATTTGAA

AAATTCACC







ACAAAATT

TCTTCTATA







C

GAAGAGTTT









GTTTTGAAC









TATACGATT









TGAAACAAA









ATTCTTTTT









TTGGAGACT









ATGGAAACA









TTCTCAACA









GGGAAAC



 


Y: 21330672-
Yq11.223
RPS4Y2
191/192
CCGATATT
288
GAAAGGGAA
384


21330747



GCTGGACC

GAGCACACA







ACCTCT

GTCTGTGTT









AAGAGGTGG









TCCAGCAAT









ATCGGCAGG









GCTTGTGTG









GGACTTTGA









GGCC









Any of the exemplary loci shown in Table 3, or any other suitable target loci that are outside of a CNVR, can be used in the karyotyping methods described herein. Such target loci can also be combined with target loci that fall within a CNVR, if desired. Particular loci can be targeted on each chromosome and any such target loci can be combined with any other target loci to form a subset, or screening panel. For instance, any of SEQ ID NOS. 1-192 can be used to interrogate chromosomes 1-22, X or Y as shown below: SEQ ID NOS. 1, 2, 3, 4, 5, 6, 7, 8 (chromosome 1); SEQ ID NOS. 9, 10, 11, 12, 13, 14, 15, 16 (chromosome 2); SEQ ID NOS.: 17, 18, 19, 20, 21, 22, 23, 24 (chromosome 3); SEQ ID NOS. 25, 26, 27, 28, 29, 30, 31, 32 (chromosome 4), 33, 34, 35, 36, 37, 38, 39, 40 (chromosome 5); SEQ ID NOS. 41, 42, 43, 44, 45, 46, 47, 48 (chromosome 6); SEQ ID NOS. 49, 50, 51, 52, 53, 54, 55, 56 (chromosome 7); SEQ ID NOS. 57, 58, 59, 60, 61, 62, 63, 64 (chromosome 8); SEQ ID NOS. 65, 66, 67, 68, 69, 70, 71, 72 (chromosome 9); SEQ ID NOS. 73, 74, 75, 76, 77, 78, 79, 80 (chromosome 10); SEQ ID NOS. 81, 82, 83, 84, 85, 86, 87, 88 (chromosome 11); SEQ ID NOS. 89, 90, 91, 92, 93, 94, 95, 96 (chromosome 12); SEQ ID NOS. 97, 98, 99, 100, 101, 102, 103, 104 (chromosome 13); SEQ ID NOS. 105, 106, 107, 108, 109, 110, 111, 112 (chromosome 14); SEQ ID NOS. 113, 114, 115, 116, 117, 118, 119, 120 (chromosome 15); SEQ ID NOS. 121, 122, 123, 124, 125, 126, 127, 128 (chromosome 16); SEQ ID NOS. 129, 130, 131, 132, 133, 134, 135, 136 (chromosome 17); SEQ ID NOS. 137, 138, 139, 140, 141, 142, 143, 144 (chromosome 18); SEQ ID NOS. 145, 146, 147, 148, 149, 150, 151, 152 (chromosome 19); SEQ ID NOS. 153, 154, 155, 156, 157, 158, 159, 160 (chromosome 20); SEQ ID NOS. 161, 162, 163, 164, 165, 166, 167, 168 (chromosome 21); SEQ ID NOS. 169, 170, 171, 172, 173, 174, 175, 176 (chromosome 22); SEQ ID NOS. 177, 178, 179, 180, 181, 182, 183, 184 (X chromosome); or SEQ ID NOS. 185, 186, 187, 188, 189, 190, 191, 192 (Y chromosome). Typically, the primers are used in pairs as set forth in Tables 3 and 4. It is understood that other primers and primer pairs not listed in Tables 3 and 4 can be used to interrogate chromosomes or parts of chromosomes 1-22, X and Y. The probes (e.g., SEQ ID NOS. 193-288) are typically used to detect their corresponding amplified sequence (e.g., SEQ ID NOS. 289-384) (Table 4). When multiple chromosomes are interrogated, any of SEQ ID NOS. 1-192 can be used to interrogate their respective chromosomes. Any one or more of SEQ ID NOS. 1-192 can be used with any other one or more of SEQ ID NOS. 1-192 to perform such assays. For instance, any one or more of SEQ ID NOS. 1-8 can be used with any one or more of SEQ ID NOS. 9-16 to interrogate chromosomes 1 and 2, respectively. Other similar subsets of SEQ ID NOS. 1-192 can also be suitable for use, as would be contemplated by one of skill in the art. At least one additional locus can also be interrogated as a control (e.g., “control gene”). Such additional loci can be, for example, the aforementioned RNase P (chromosome 14, cytoband 14q11.2) or TERT (chromosome 5, cytoband 5p15.33).
In certain embodiments, the karyotyping methods described herein are used to simultaneously interrogate multiple chromosomes. For example, using a human gDNA sample, more than one chromosome (i.e., chromosomes 1-22, X and Y) can be simultaneously interrogated in the same or separate assays. Furthermore, a subset of the total complement of chromosomes can be interrogated in the same or separate assays. Individual chromosomes can be targeted by amplifying loci specific to such chromosomes that are outside of CNVRs. For example, chromosome 1 can be interrogated along with any one or more of chromosomes 2-22, X or Y; chromosome 2 can be interrogated along with any one or more of chromosomes 1 and 3-22, X or Y; chromosome 3 can be interrogated along with any one or more of chromosomes 1-2, 3-22, X or Y; chromosome 4 can be interrogated along with any one or more of chromosomes 1-3, 5-22, X or Y; chromosome 5 can be interrogated along with any one or more of chromosomes 1-4, 6-22, X or Y; chromosome 6 can be interrogated along with any one or more of chromosomes 1-5, 7-22, X or Y; chromosome 7 can be interrogated along with any one or more of chromosomes 1-6, 8-22, X or Y; chromosome 8 can be interrogated along with any one or more of chromosomes 1-7, 9-22, X or Y; chromosome 9 can be interrogated along with any one or more of chromosomes 1-8, 10-22, X or Y; chromosome 10 can be interrogated along with any one or more of chromosomes 1-9, 11-22, X or Y; chromosome 11 can be interrogated along with any one or more of chromosomes 1-10, 12-22, X or Y; chromosome 12 can be interrogated along with any one or more of chromosomes 1-11, 13-22, X or Y; chromosome 13 can be interrogated along with any one or more of chromosomes 1-12, 14-22, X or Y; chromosome 14 can be interrogated along with any one or more of chromosomes 1-13, 15-22, X or Y; chromosome 15 can be interrogated along with any one or more of chromosomes 1-14, 16-22, X or Y; chromosome 16 can be interrogated along with any one or more of chromosomes 1-15, 17-22, X or Y; chromosome 17 can be interrogated along with any one or more of chromosomes 1-16, 18-22, X or Y; chromosome 18 can be interrogated along with any one or more of chromosomes 1-17, 19-22, X or Y; chromosome 19 can be interrogated along with any one or more of chromosomes 1-18, 20-22, X or Y; chromosome 20 can be interrogated along with any one or more of chromosomes 1-19, 21, 22, X or Y; chromosome 21 can be interrogated along with any one or more of chromosomes 1-20, 22, X or Y; chromosome 22 can be interrogated along with any one or more of chromosomes 1-21, X or Y; X chromosome can be interrogated along with any one or more of chromosomes 1-22 or Y; Y chromosome can be interrogated along with any one or more of chromosomes 1-22 or X. Other subsets of chromosomes can also be interrogated as would be understood by one of skill in the art. For PCR-based assays, any of the primer sets shown in Tables 3 or 4 can be used as may any other suitable primer set.
Described herein are karyotyping methods for determining the copy number of one or more chromosomes in a test biological sample (such as a test gDNA). In certain embodiments, the methods can be used to determine the karyotype of a test genome. The karyotype information typically relates to one or more chromosomes of a genome.
Such methods described herein can be used in prenatal diagnostic assays to screen for and detect chromosomal abnormalities in a fetus or embryo, such as Down's Syndrome (Trisomy 21), Edward's Syndrome (Trisomy 18) and Patau Syndrome (Trisomy 13). These assays can be performed on biological samples such as fetal cells or cell-free fetal DNA in maternal blood, amniotic fluid, trophoblast cells, chorionic villus samples and percutaneous umbilical cord blood. Furthermore, the methods disclosed herein can also be performed on embryos used in in vitro fertilization (IVF) wherein blastocyst cells or cells biopsied from the trophectoderm are analyzed from embryos that are 3 to 6 days post-fertilization, in order to determine if they should be implanted in utero.
The methods described herein can be useful in, for example, stem cell analysis, quality control assays of cell cultures, analysis of samples of limited quantity (e.g. single cell, formalin-fixed paraffin-embedded (FFPE)), comparisons between cell or tissue types, comparisons between diseased or non-diseased tissues, detection of chromosomal polymorphisms, and the like. The methods disclosed herein can be used to compare chromosome copy number between, for example, cell types (e.g., lymphocyte, epithelial cell), tissue types (e.g., neural tissue, skeletal muscle tissue), disease states (e.g., cancerous, non-cancerous), or types of organisms (e.g. human, mouse, plant, fruit fly). Other uses for the methods described herein will be apparent to one of skill in the art.
The methods described herein can be used to detect chromosomal abnormalities. Exemplary abnormalities include, but are not limited to, deletion, duplication, translocation, mosaicism, aneuploidy (e.g., nullisomy, disomy, trisomy and tetrasomy), inversion, ring formation, isochromosome formation, or chromosomal instability syndromes. Pathologies have been associated with abnormalities in particular human chromosomes including, for example, chromosome 1 (e.g., acute lymphoblastic leukemia, acute megakaryoblastic leukemia, alveolar rhabdomyosarcoma), chromosome 2 (e.g., anaplastic large cell lymphoma, alveolar rhabdomyosarcoma), chromosome 3, chromosome 4 (e.g., Wolf-Hirschhorn syndrome, acute lymphoblastic leukemia), chromosome 5 (e.g., Cri du chat, also known as Chromosome 5q deletion syndrome, anaplastic large cell lymphoma), chromosome 6, chromosome 7 (e.g., Williams syndrome), chromosome 8 (e.g., Burkitt's lymphoma, acute lymphoblastic leukemia, acute myeloblastic leukemia with maturation), chromosome 9 (e.g., Trisomy 9, Warkany syndrome 2, acute lymphoblastic leukemia, Philadelphia chromosome), chromosome 10, chromosome 11 (e.g., Jacobsen syndrome, mantle cell lymphoma, multiple myeloma, acute lymphoblastic leukemia, Ewing's sarcoma, desmoplastic small round cell tumor), chromosome 12 (e.g., acute lymphoblastic leukemia, myxoid liposarcoma), chromosome 13 (e.g., Patau syndrome, alveolar rhabdomyosarcoma, breast and ovarian cancers, deafness, Wilson's disease), chromosome 14 (e.g., Burkitt's lymphoma, follicular lymphoma, acute lymphoblastic leukemia), chromosome 15 (e.g., Angelman syndrome, Prader-Willi syndrome, acute promyelocytic leukemia, Marfan syndrome, Tay-Sach's disease), chromosome 16 (e.g., Trisomy 16, myxoid liposarcoma, polycystic kidney disease, α-thalassemia), chromosome 17 (e.g., Miller-Dieker syndrome, Smith-Magenis syndrome, acute promyelocytic leukemia, dermatofibrosarcoma protuberans, Charcot-Marie-Tooth disease), chromosome 18 (e.g., Edwards syndrome, Burkitt's lymphoma, follicular lymphoma, mantle cell lymphoma, multiple myeloma, synovial sarcoma, Nieman-Pick disease, pancreatic cancer), chromosome 19 (e.g., acute lymphoblastic leukemia), chromosome 20, chromosome 21 (e.g., Down syndrome, acute lymphoblastic leukemia, acute myeloblastic leukemia with maturation), chromosome 22 (e.g., Di George's syndrome, Trisomy 22, acute lymphoblastic leukemia, Philadelphia chromosome, acute megakaryoblastic leukemia, Ewing's sarcoma, dermatofibrosarcoma protuberans, desmoplastic small round cell tumor), X chromosome (e.g., Fragile X syndrome, Turner syndrome, Triple X syndrome, Klinefelter's syndrome, synovial sarcoma, mixed gonadal dysgenesis, XX gonadal dysgenesis, uniparental disomy, Duchenne muscular dystrophy, X-linked diseases, hemophilia, adrenoleukodystrophy, Hunter's disease), or Y chromosome (e.g., Klinefelter's syndrome, uniparental disomy, acute myeloid leukemia). Any of these disorders, or any other disorders that may be detected by karyotyping as would be known by the skilled artisan, can be studied using the methods described herein.
Kits for determining the copy number of multiple chromosomes are also provided. For use with a PCR-based assay, the kit can include at least a set of primers for amplifying at least one test locus on a chromosome (e.g., any one or more of SEQ ID NOS. 1-192) or corresponding probes (e.g., any one or more of SEQ ID NOS. 193-288). The kit can also include samples of pre-determined amplified sequences such as those listed in Table 4 (e.g., any one or more of SEQ ID NOS. 289-384), which can optionally be affixed to a solid support (e.g. a membrane, glass slide, multi-well plate) for use as a control reaction. The kit can also optionally include stock solutions, buffers, enzymes, detectable labels or reagents required for detection, tubes, membranes, and the like that can be used to complete the amplification reaction. In some embodiments, multiple primer sets are included. The kits can also comprise the reagents required to use a reference (e.g., primers and probes for amplifying or detecting a reference sequence such as RNase P or TERT) or calibrator sample (i.e., a control genome of known karyotype, a chromosome sample, or the like). In some embodiments, the kits can contain multiple sets of primers for amplifying multiple target loci on a single chromosome or among two or more chromosomes (e.g., any combination of primers sets and probes shown in Tables 3 and 4, or any primer/probe sets that target loci outside of CNVRs). In one embodiment, a multi-well plate contains within its wells an array of replicate wells for use in carrying out assays. For example, a 384-well array containing four replicate wells for each of chromosomes 1-22, X and Y comprising primer/probe sets targeting loci outside of CNVRs for each of the chromosomes, is provided, optionally including reagents such as stock solutions, buffers, enzymes such as polymerases, and detectable labels. Other embodiments of particular systems and kits are also contemplated which would be understood by one of skill in the art. In addition, the arrays can be customized depending on the target samples being analyzed, for example, arrays comprising one, two, three or more chromosomes comprising one, two, or more than two loci to be analyzed per chromosome. Furthermore, the number of replicate wells or samples per chromosome per array can vary (e.g., one, two, three, four, or more than four).
All references cited within this disclosure are hereby incorporated by reference in their entirety. Certain embodiments are further described in the following examples. These embodiments are provided as examples only and are not intended to limit the scope of the claims in any way.
EXAMPLES
A. Karyotyping of Chromosomes 2, 3, 4, 5, 6, 20 and 22
The primers and probes used to interrogate the targets of interest described below were designed using masked genomic DNA sequences (the human genome assembly B36) by an Applied Biosytems proprietary TaqMan® Copy Number Assay Design Pipeline. The existing pre-designed TaqMan® copy number assay protocols were utilized. TaqMan® copy number assays targeting chromosomal regions located outside of copy number variable regions (CNVR) on each of the 24 chromosomes were designed (the data shown below, however, relates only to chromosomes 2, 3, 4, 5, 6, 20 and 22.) As described above, to cover all 24 chromosomes on a 384 well plate or TaqMan® array card (TLDA), four assays can be selected for each chromosome, using two assays on each arm of each chromosome whenever possible. The assays were run as duplex TaqMan® real-time PCR reactions. A FAMTM dye-based assay was used to detect test target loci and the VIC® dye-based assay was used for the reference locus RNase P (PN 4316844 from Applied Biosystems). Each assay utilized 10 ng gDNA, 1× TaqMan® probe/primer mix (900 nM of each primer, 250 nM of each probe) in 1× TaqMan® Genotyping Master Mix in a 10 μl reaction (run in quadruplicate). PCR reactions were incubated in an Applied Biosystems 7900HT SDS instrument for 10 minutes at 95° C., followed by 40 cycles of 15 seconds at 95° C. and 60 seconds at 60° C. Real-time data was collected by the SDS 2.3 software. The relative quantification analysis was performed to calculate estimated copy number of each sample for the gene of interest by CopyCallerTM software.
These methods were used to interrogate loci on chromosomes 2, 3, 4, 5, 6, 20 and 22 of human gDNA samples (a set of 92 African American and Caucasian gDNA samples from Coriell Institute) using the following primer sets and probes:












TABLE 5






SEQ ID







NO.

SEQ ID





(primer

NO.
Amplified sequence
SEQ ID NO.


Chromosome
pairs)
Probe
(probe)
(amplicon)
(amplicon)







 2
 9/10
AACCACCA
197
TGACAGCTTTTGGCTCAGAAAT
293




ACAAAGGG

TAGAAAATGAAATGATAACCAC





ATTCT

CAACAAAGGGATTCTGCAGCTG







AGTATGGATTTCCTCCCAGTGA







CAGG



 



11/12
ACCTTCCC
198
CACGATGAGGGTTGAGGGAAAA
294




AGTTTGAT

ACAGTTGGTAGGAACAAGTGCT





AAGCAC

TATCAAACTGGGAAGGTGCTCT







TATTTACAGCTGCTGGCTCAAG







AATC



 



13/14
CTGGCCTG
199
GCCAGGATGCTCCATGTAGTAT
295




TGTGTACT

TGCATTATAACATGGTCTCAGC





TCT

TGGCCTGTGTGTACTTCTACAA







CATGAACCGTCAGAATAATCCT







T



 



15/16
ACCCATGC
200
AGGTGAAAAAAGTACTTATGAG
296




CTCTTTCT

GATGATGAATAGTTGGAAAAAC





AATAAC

TAGTAAAATAGGGTTGGTTATT







AGAAAGAGGCATGGGTAGTTGA







TAGAGCCATAAGCTTTGATCAA



 


 3
17/18
CCTGCCTA
201
AAACAGGAGAATGAATGAATGA
297




CCAAAGAG

ATATGCTAATACAACCACCTCT





GATACA

GTATCCTCTTTGGTAGGCAGGA







GGCAAGAAGCAGGCTAATCTGT







CTATGG



 



19/20
TCCCAGCC
202
GCCATCCTCCTTTTTCTCATCC
298




CACTGTAA

TGTGGGGCTACTTATGATGTGA





ATG

TGCCATTTACAGTGGGCTGGGA







TTACAGGTGTGAGGCACCAAAC







CCA



 



21/22
AATGCCAC
203
TGGCATGTGAGATGTGTTCAAG
299




CAGTGACC

AAACTCAAGACTAAGGGAATGA





TTCAG

ATGAATGAATGCCACCAGTGAC







CTTCAGTGCCTCTCTAGTGGTA







CCCTTT



 



23/24
TCTTCTTT
204
TCCCTCAAGTTTATTCAGTCTC
300




GCTCCTGA

CTTATGTAATCAGTAATTCTAT





GACCTC

CAAATCCTCTTCTTTGCTCCTG







AGACCTCACCTACTTCAATTTG







GACGTCTTGCTCT



 


 4
25/26
ATAGGTGT
205
CATTTCCAAGTACAGTAACTCC
301




CCCTGTAA

ACAGTACTATCCTGTTGCTTTA





AGCAAC

CAGGGACACCTATGCCTTTTTT







CTTCAGAATAAAGAACATTGCA







AACTGTTTTTGCACCACTGAGG



 



27/28
TCCTGCTG
206
CCCAAGCACCTGTAGCATCATC
302




CCCACTGA

GTCCACGTCCTGCTGCCCACTG





CCTG

ACCTGTCCGGCTCCACACATGA







AGGCAGATGTTTGAGG



 



29/30
CCACACAG
207
GGAATGTTACCACGTTGCTAAG
303




GCTCCTTG

CTATGTAACATATCTTAACAAC





GAGTAA

CAGGGAGCCACACAGGCTCCTT







GGAGTAAGAGTGTGAGAAACTG







GATGAAGACAGCTG



 



31/32
TTGCTGTT
208
TTTCTGGGAGCCATCTGAATCA
304




GGCTGAAT

TCTTTGCTGTTGGCTGAATCAC





CACT

TCAGAGCTGAGAGGGAGGATGA







AGAGGGGCTATTTGATTCGCTG



 


 5
33/34
AATGGAGA
209
GGTATGACCTGGACACATTAAG
305




AGGGAAAA

CATTCGTAATTTTCCCTTCTCC





TTAC

ATTACTAGATACAGTGCTTACA







TTTCTCATTATTTTTATCTCCC







TCCT



 



35/36
CAGGGAGA
210
GCAAGCCACAGGAAGAGGAAGC
306




CAAAACAG

CCAAAGGCAGGGAGACAAAACA





AAATAT

GAAATATGCTTCTGGCACCAAG







TCAAATTGTACA



 



37/38
CCGGAGCC
211
GCGGAACAAGCCAGACTGAAAA
307




GCTGCACA

AAAAAAAAAAAAACCCTCACCG





T

AAATGTGCAGCGGCTCCGGAGC







GAGAACAGCGCTCGAACCTC



 



39/40
CTAGGTAC
212
GTTTTAGAAGGAAGAGGGCTAG
308




TGCGCCAC

GAAGAAAAGTGGCGCAGTACCT





TTTT

AGTAGGTAAGTATAATCTGGAT







GCTCCCAGTAATTCTGAGTGTT







GACTGCACATTCT



 


 6
41/42
ATGCAACT
213
GTATAGGCAGCGACAGCACTTG
309




CATGCTGA

TAAATTCAGCATGAGTTGCATG





ATTTA

GTTGGCCAATGTTGGTGAGTCC







TGTGGAGATCT



 



43/44
CCTCAAGC
214
GAAAACCAAAGCAACAAGGTGA
310




TCCGACCC

GTCCTCAGGAGGGGTCGGAGCT





CTCCT

TGAGGTTTTGGAGTTTGGAACT







TACTCCCATCTCCC



 



45/46
CATGTATA
215
GGGTTGCAGGGATGGTGTACAA
311




GCTGCATA

CAGGTCCTAGCATGTATAGCTG





GATTTC

CATAGATTTCTTCACCTGATCT







TTGTGTGGAAGATCAGAATGAA







TGCA



 



47/48
CAGTCTGA
216
GGGATCTGTAACTTGACCAAGG
312




TGGTCCCA

TCAAAGAGCTTGAAATTTCAAC





AGTTGA

TTGGGACCATCAGACTGAAAAC







CTGCAATCTGAAACACTTTGCT







GTGCTGC



 


20
153/154
CAGTGAAG
269
CCACTTCCCTCAATCATGTGAC
365




CCACCTTT

ACCACTTCAGTGAAGCCACCTT





AAATCA

TAAATCATCTGTTTTTGAATTT







GTCTGGAATCCAGAAAAAGTTG







GCAAAACCC



 



155/156
AATGGGCA
270
GTGCGAAGGTTAGTTTCTGAGG
366




TCATCATC

AAGGAAAAAAGTTGATGATGAT





AACTTT

GCCCATTGTCAGGTCTGTAACT







AACTTGCTGGGTTCCTCTC



 



157/158
CCCACCAC
271
CCCATGGGAGATGCTCTTGAGA
367




CAGAATAG

AAGACTATTCTGGTGGTGGGTG





TCTTT

CGGGATCCTGTCAGGGGGAAGA







GATGGAACCAGGAGACC



 



159/160
CTGTGTGC
272
GCCCTGTCCCTGCTTCTGGAAA
368




AGATGTCG

AAGAATTTTCGACATCTGCACA





AAAAT

CAGACAGTTGTGAAAAAGGAGG







AGAAGCAGCTACTGGCTAAGGG







GCACCT



 


22
169/170
CCCAGACC
277
CCTTACCCATCCCAACTAGCCT
373




AGAAGAGT

TACCCATCCTCGCCTCTCTCCT





TCCAC

CAGCCCAGACCAGAAGAGTTCC







ACCAGCGATCCAACGTCACACT







CACTCTAC



 



171/172
CTGTGCAT
278
CCTCCTGATCATACTCTGGTAC
374




CGGCCTCC

CTGGCCTGTGCATCGGCCTCCT





TGC

GCTTCATGTCAACCTCCTACTC







CTGCCAGGGAATGTG



 



173/174
CCAGCAGC
279
CACATGAGGCTTTCGGAGGAAT
375




CTCGATTT

AAATTGGTCTGAAATCGAGGCT





CAGAC

GCTGGCTTTTGTGTTAATAANT







GTGTAAAAGCTATCCAGGGCAT







CAAGG



 



175/176
CTGCTTGC
280
CCATCCCTCTCCACAGCAAGGA
376




AGCGTTCC

TGACGTGGAACGCTGCAAGCAG





ACGTC

AAGGACCTACTGGAGCAGATGA







TGGCCGAGATGATTGGCGAGTT







CCCAGAC









The resultant data is shown in FIG. 1 (chromosome 2), FIG. 2 (chromosome 3), FIG. 3 (chromosome 4), FIG. 4 (chromosome 5), FIG. 5 (chromosome 6), FIG. 6 (chromosome 20), FIG. 7 (chromosome 20), and FIG. 8 (chromosome 22). As shown therein, the methods described herein (e.g., interrogating target loci located outside CNVRs) has been used to accurately karyotype various chromosomes found within the human genome.
B. Detection of Disease Pathology by Karyotype Analysis
Many forms of cancer have been associated with various chromosomal abnormalities (see discussion hereinabove). Detection of diseases or susceptibility to diseases is critical for proper diagnosis or treatment. For example, certain breast and ovarian cancers are associated with abnormalities in chromosome 13. Primers and probes useful for interrogation of non-CNVR loci of chromosome 13 can be used to determine whether a patient has or is susceptible to having a breast or ovarian cancer.
The primers and probes used to interrogate the chromosome 13 target loci are designed using masked genomic DNA sequences (the human genome assembly B36) by an Applied Biosystems proprietary TaqMan® Copy Number Assay Design Pipeline and interrogated using copy number assays of format 1 and using virtual calibrator assay no. 1. Test samples are obtained from normal and tumor cell and tissue. As described above, four assays are selected for chromosome 13, using two assays on each arm of the chromosome. The assays are run as duplex TaqMan® real-time PCR reactions. A FAMTM dye-based assay is used to detect test target loci and the VIC® dye-based assay is used for the reference locus RNase P (PN 4316844 from Applied Biosystems) in assay format 1. Each assay uses 10 ng gDNA, 1× TaqMan® probe/primer mix (900 nM of each primer, 250 nM of each probe) in 1× TaqMan® Genotyping Master Mix in a 10 μl reaction (run in quadruplicate). PCR reactions are incubated in an Applied Biosystems 7900HT SDS instrument for 10 minutes at 95° C., followed by 40 cycles of 15 seconds at 95° C. and 60 seconds at 60° C. Real-time data is collected by the SDS 2.3 software. The relative quantification analysis is performed as described herein above to calculate estimated copy number of each sample for the gene of interest by CopyCallerTM software.
Identification of abnormal chromosomal copy number and karyotype of chromosome 13 indicates the presence or susceptibility to the breast or ovarian cancer associated with chromosome 13.
While certain embodiments have been described in terms of the preferred embodiments, it is understood that variations and modifications will occur to those skilled in the art. Therefore, it is intended that the appended claims cover all such equivalent variations that come within the scope of the following claims.

## Claims

1. A method for determining the copy number of at least one test locus on at least one chromosome in a test sample, the method comprising:
interrogating at least one test locus on at least one chromosome in the test sample with at least one primer and at least one probe, wherein the interrogating includes quantifying the copy number of at least one test locus, and wherein the at least one test locus is located on a region of the chromosome that is not within a copy number variable region (CNVR) on the chromosome; and
determining the copy number of at least one chromosome in the test sample on which at least one of the interrogated test loci is located.

2. The method of claim 1, wherein the determining of the copy number of at least one chromosome in the test sample comprises determining the copy number of more than one chromosome in the test sample.

3. The method of claim 1, wherein the copy number of more than one test loci in the test sample is quantified.

4. The method of claim 1, further comprising quantifying the copy number of the interrogated test locus relative to the copy number of at least one of the reference loci of the calibrator sample, wherein the calibrator sample is a virtual calibrator sample derived by a method comprising the steps of:
(a) interrogating one or more test loci in each test sample using one or more copy number assays and calculating an average CT for each of said copy number assays;
(b) interrogating one or more reference loci in each test sample using one or more copy number reference assays and calculating an average CT from each of said copy number reference assays;
(c) averaging the copy number assay average CT values of (a);
(d) averaging the copy number reference assay average CT values of (b);
(e) calculating the relative quantity using values of (c) and (d); and
(f) determining the copy number for each test locus by multiplying the relative quantity by the copy number of the calibrator sample.

5. The method of claim 1, wherein the test sample comprises genomic DNA.

6. The method of claim 1, wherein at least one of the interrogated test loci and at least one of the reference loci correspond to the same location on a chromosome.

7. The method of claim 1, wherein at least one of the interrogated test loci and at least one of the reference loci correspond to different locations on the chromosome.

8. The method of claim 1, further comprising the steps of:
(a) amplifying from a first genomic DNA sample at least one or more test loci from at least one chromosome and at least one or more reference loci from at least one chromosome;
(b) amplifying from a second test genomic DNA sample at least one or more target loci from at least one chromosome and at least one or more reference loci from at least one chromosome;
(c) calculating the average CT values for each of amplifications (a) and (b);
(d) calculating the average of the average CT values for each of amplifications (a) and (b); and
(e) calculating the relative copy number from the calculations in (d),
wherein the one or more test loci from (a) and (b) are located on a region of the chromosome that is not within a copy number variable region on the chromosome.

9. The method of claim 1, further comprising the steps of:
(a) amplifying from a first genomic DNA sample one or more target loci on a chromosome;
(b) amplifying from a second genomic DNA sample one or more target loci on a chromosome;
(c) amplifying from the genomic DNA sample one or more reference loci on a chromosome that is different from the target loci in (a) and (b);
(d) calculating the average CT values for each of amplifications (a), (b), and (c);
(e) calculating the average of the average CT values for each of amplifications (a), (b), and (c);
(f) calculating the ΔCT value from the average calculated in (e) for amplifications (a) and (c);
(g) calculating the relative copy number from the calculations in (f) in the genomic DNA sample,
wherein the target loci are located on a region of the chromosome that is not within a copy number variable region on the chromosome.

10. The method of claim 1, further comprising the steps of:
(a) amplifying from a genomic DNA sample one or more test loci on a chromosome;
(b) performing a second amplification from the genomic DNA sample one or more test loci on a chromosome;
(c) calculating the average CT values for each of amplifications (a) and (b);
(d) calculating the average of the average CT values for each of amplifications (a) and (b);
(e) calculating the ΔCT value by subtracting the average calculated in (d) for amplification (b) from the average calculated in (d) for amplification (a); and
(f) calculating the relative copy number from the calculations in (e) in the genomic DNA sample,
wherein the target loci are located on a region of the chromosome that is not within a copy number variable region on the chromosome.

11. The method of claim 1, wherein the at least one test locus is amplified using the polymerase chain reaction to produce amplicons.

12. The method of claim 11, wherein the amplification efficiency of each amplicon amplified from the test genome is within about ten percent of any other amplicon.

13. The method of claim 12, wherein at least two target loci are amplified from each arm of each chromosome being interrogated.

14. The method of claim 13, wherein at least four target loci are amplified from each chromosome being assayed.

15. The method of claim 11, wherein multiple target loci are amplified and each is from any other target loci on the arm by approximately the same number of nucleotides.

16. The method of claim 11, wherein the amplicons are less than or equal to about 110 nucleotides in length.

17. The method of claim 16, wherein the amplicons are about 70 to 110 nucleotides in length.

18. The method of claim 11, wherein primers utilized in the polymerase chain reaction are specific for regions outside of a copy number variable region.

19. A method for determining the copy number of at least one test locus on at least one chromosome in a test sample, the method comprising: interrogating at least one test locus on at least one chromosome in the test sample with at least one primer and at least one probe; wherein the interrogating includes quantifying the copy number of at least one test locus, and wherein the at least one test locus is located on a region of the chromosome that is not within a copy number variable region (CNVR) on the chromosome, wherein primers utilized in the polymerase chain reaction are specific for regions outside of a copy number variable region and wherein the primers are selected from the group consisting of SEQ ID NOS. 1-192.

20. The method of claim 19, further comprising diagnosing a disease resulting from a chromosomal abnormality based on the copy number of the at least on test locus.

21. The method of claim 2, further comprising diagnosing a disease resulting from a chromosomal abnormality based on the copy number of the at least one test locus.