WO2011027219A1 - Détection à rendement élevé de petites délétions et insertions génomiques - Google Patents

Détection à rendement élevé de petites délétions et insertions génomiques Download PDF

Info

Publication number
WO2011027219A1
WO2011027219A1 PCT/IB2010/002354 IB2010002354W WO2011027219A1 WO 2011027219 A1 WO2011027219 A1 WO 2011027219A1 IB 2010002354 W IB2010002354 W IB 2010002354W WO 2011027219 A1 WO2011027219 A1 WO 2011027219A1
Authority
WO
WIPO (PCT)
Prior art keywords
probe
ldlrex2
probes
indel
variant segment
Prior art date
Application number
PCT/IB2010/002354
Other languages
English (en)
Inventor
Marianne Stef
Diego Tejedor
Antonio Martinez
Laureano Simon
Original Assignee
Progenika Biopharma, S.A.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Progenika Biopharma, S.A. filed Critical Progenika Biopharma, S.A.
Publication of WO2011027219A1 publication Critical patent/WO2011027219A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips

Definitions

  • DNA-chips also named “micro-arrays”, “DNA-arrays” or “DNA bio- chips”, and collections of beads with attached nucleic acids
  • DNA-chips also named “micro-arrays”, “DNA-arrays” or “DNA bio- chips”, and collections of beads with attached nucleic acids
  • SNP single nucleotide polymorphism
  • the first DNA-chip was the "Southern blot" where labeled nucleic acid molecules were used to examine nucleic acid molecules attached to a solid support.
  • the support was typically a nylon membrane.
  • probes deposited on the solid surface are hybridized to cDNAs synthesized from mRNAs extracted from a given sample.
  • the cDNA has been labeled with a fluorophore. The larger the number of cDNA molecules joined to their
  • a bead set is typically coats with a number of nucleic acid probes that are labeled such that different probes can be "seen” using visualization or capture of the beads after hybridization to a target nucleic acid.
  • Gene expression DNA-chips typically also contain probes for detection of expression of control genes, often referred to as "house-keeping genes", which allow experimental results to be standardized and multiple experiments to be compared in a quantitive manner. With the DNA-chip, the levels of expression of hundreds or thousands of genes in one cell can be determined in one single experiment.
  • the cDNA of a test sample and that of a control sample can be labeled with two different fluorophores so that the same DNA-chip can be used to study differences in gene expression.
  • DNA-chips for detection of genetic polymorphisms, changes or mutations (in general, genetic variations) in the DNA sequence comprise a solid surface, typically glass, on which a high number of genetic sequences are deposited (the probes), complementary to the genetic variations to be studied.
  • the probes complementary to the genetic variations to be studied.
  • Using standard robotic printers to apply probes to the array a high density of individual probe features can be obtained, for example probe densities of 600 features per cm.sup.2 or more can be typically achieved.
  • the positioning of probes on an array is precisely controlled by the printing device (robot, inkjet printer,
  • Sub-arrays typically comprise 32 individual probe features although lower (e.g. 16) or higher (e.g. 64 or more) features can comprise each sub-array.
  • One strategy used to detect genetic variations involves hybridization to sequences which specifically recognize the normal and the mutant allele in a fragment of DNA derived from a test sample.
  • the fragment has been amplified, e.g. by using the polymerase chain reaction (PCR), and labeled e.g. with a fluorescent molecule.
  • PCR polymerase chain reaction
  • a laser can be used to detect bound labeled fragments on the chip and thus an individual who is homozygous for the normal allele can be specifically distinguished from heterozygous individuals (in the case of autosomal dominant conditions then these individuals are referred to as carriers) or those who are homozygous for the mutant allele.
  • Another strategy to detect genetic variations comprises carrying out an amplification reaction or extension reaction on the DNA-chip itself.
  • differential hybridization based methods there are a number of methods for analyzing hybridization data for genotyping. For example, one can analyze an increase in hybridization level, wherein the hybridization level of complementary probes to the normal and mutant alleles are compared. One can also analyze a decrease in hybridization level, wherein differences in the sequence between a control sample and a test sample can be identified by a fall in the hybridization level of the totally complementary oligonucleotides with a reference sequence. A complete loss is produced in mutant homozygous individuals while there is only 50% loss in heterozygotes.
  • oligonucleotide In DNA-chips for examining all the bases of a sequence of "n" nucleotides (“oligonucleotide”) of length in both strands, a minimum of “2n” oligonucleotides that overlap with the previous oligonucleotide in all the sequence except in the nucleotide are necessary. Typically the size of the oligonucleotides is about 25 nucleotides. The increased number of oligonucleotides used to reconstruct the sequence reduces errors derived from fluctuation of the hybridization level. However, the exact change in sequence cannot be identified with this method; sequencing is later necessary in order to identify the mutation.
  • a mutation specific primer is fixed on the slide and after an extension reaction with fluorescent dideoxynucleotides, the image of the DNA-chip is captured with a scanner.
  • the primer extension strategy two oligonucleotides are designed for detection of the wild type and mutant sequences respectively.
  • the extension reaction is subsequently carried out with one fluorescently labeled nucleotide and the remaining nucleotides unlabelled.
  • the starting material can be either an RNA sample or a DNA product amplified by PCR.
  • Tag arrays strategy an extension reaction is carried out in solution with specific primers, which carry a determined 5' sequence or "tag".
  • specific primers which carry a determined 5' sequence or "tag”.
  • DNA-chips with oligonucleotides complementary to these sequences or "tags” allows the capture of the resultant products of the extension. Examples of this include the high density DNA-chip “Flex-flex” (Affymetrix).
  • Small duplications, insertions and deletions are genetic variations that are quite difficult to automatically detect in resequencing assays because they are not automatically identified by the software analyzing the data.
  • Small duplications, insertions and deletions that are generally less than or equal tolO nucleotides are called indels. Only known indels can be detected because specific probes can be designed for their detection. In addition, novel indels are difficult to predict. In many diseases, indels represent from 10 to 40% of point mutations, and not detecting them can dramatically lead to the underestimation of the mutation rate and also the perceived mutation detection rate.
  • the present invention provides methods for the designing and using probe sets that allow detection of single nucleotide polymorphisms, small deletions and insertions of a small number of nucleotides referred to herein as "indels".
  • the methods are based on a novel design system that allows rapid detection and characterization of the indels in any selected nucleic acid sequence.
  • the invention provides a method of designing a library of probes for detecting at least one indel variation in a genetic variant segment, the method comprising the steps of (a) selecting a nucleic acid variant segment of N nucleotides long, wherein N can be any length between 25 and 5000; (b) selecting N number of probe sets each designed to hybridize to one of the N number of nucleotides in the nucleic acid variant segment; and (c) selecting at least one probe subset for the probe set, wherein the probe subset comprises at least two different probes forming a pair of probes, one designed to specifically hybridize to a normal/wild-type or a control sequence and one designed to specifically hybridize to a sequence with the at least one indel variation.
  • the method further comprises a step of manufacturing the probe sets selected/designing through steps (a), (b) and (c).
  • the method further comprises performing at least one of the steps
  • Such computer implemented system can be performed by setting forth an algorithm with the novel selection rules set forth in the method and allowing a computer to automatically select the probes using input parameters for the desired length of the probe, the type of an indel, the desired target variant sequence and so forth.
  • the method further comprises a step of attaching the probe on a solid surface.
  • the at least one pair of probes consist of a normal/control probe and a variant probe, both of which interrogate the about same region on the genetic variant segment, wherein the both probes forming the probe sub-set have the same sequence length and are of the same type of nucleic acids.
  • the normal (N) or control (C) probe comprises the normal sequence (which is the wild type sequence) or a known SNP polymorphism (which is the control sequence) of genetic variant segment and the variant probe comprises an indel-containing variant sequence of genetic variant segment
  • the at least two different probes has the difference between them located in position -4, -3, -2, -1, 0, +1, +2, +3 or +4 position of the probe, wherein the position 0 is the center nucleotide of the probe when the probe has an odd number of nucleotides; and wherein the position -1 and +1 are the two central nucleotides of the probe when the probe has an even number of nucleotides.
  • probes are longer than 25 nt, e. g. 40 or 50 nt long, or
  • the position of the indel variation can be between -25 and +25 position (inclusive) from the center of the probe, e. g. -25, -24, -23, -22, -21, -20, -19, -18, -17, -16, -15, -14, -13, -12, -11, -10, -9,-8, - 7, -6, -5, -4, -3, -2, -1, 0, +1, +2, +3, +4, +5, +6, +7, +8, +9, +10, +11, +12, +13, +14, +15, +16, +17, +18, +20, +21, +22, +23, +24 or +25.
  • the probes are DNA, RNA or PNA. Pairs of probes that are of the same type of nucleic acid means that the probe members constituting the pair of probes are all DNA, RNA or PNA.
  • the probe members constituting the pair of probes cannot be a mixture of one probe that is a DNA and the other probe is a RNA, a mixture of one probes that is a RNA and the other probe is a PNA, or a mixture of one probes that is a DNA and the other probe is a PNA. This ensures that the differences in the intensity are due to the presence of an indel and not due to the differences in binding affinity of the types of nucleic acid.
  • the indel variation is a deletion, an insertion or a duplication of nucleotide.
  • the deletion, insertion or duplication can be 1-10, 1-20, 1-30, 1-40 or 1-50 nucleotides long. In some embodiments, the indel is 1-10 nucleotides long.
  • the deletion can be up to 50 nt, e. g. 11, 12, 13, 14, 15, 16, 17, 18,
  • the insertion can be up to 50 nt, e. g. 11, 12, 13, 14, 15, 16, 17,
  • the duplication can be up to 50 nt, e. g. 11, 12, 13, 14, 15, 16, 17,
  • the deletion is selected from a group consisting of one, two, three, four, five, six, seven, eight, nine, and ten nucleotides. In some embodiments, deletion of 1-10, 1-15, 1-
  • the insertion is selected from a group consisting of one, two, three, four, five, six, seven, eight, nine, and ten nucleotides.
  • the duplication is selected from a group consisting of one, two, three, four, five, six, seven, eight, nine, and ten nucleotides. In some embodiments, insertion of 1-10, 1- 15, 1-20, 1-50 nucleotides can be detected.
  • the probes of the at least one probe sub-set is complementary to the sense strand of the genetic variant segment.
  • probes of the at least one probe sub-set is complementary to the anti-sense strand of the genetic variant segment.
  • the method comprising a step of selecting one set of probe sets and as many probe sets as the length or number of nt (or base pairs) in the variant segment under interrogation, wherein each probe set comprises a single probe sub-set, wherein each probe sub-set comprises at least a pair of probes consisting of one control (C) or normal (N) /wild type probe and one variation probe (V).
  • C control
  • N normal
  • V variation probe
  • each probe set comprises at least two probe sub-sets, wherein each probe sub-set comprises at least one pair of probes, wherein each pair of probes consists of one control or normal or control probe and one variation probe, wherein the probes of each pair of probes making up the probe sub-set differ from each other in terms of length, indel variation location inside the probe and strand of interrogation of the variant segment.
  • more than two types of indels are designed to be detected, further comprising a step of designing as many probe sub-sets as there are indels that need to be detected, and wherein each pair of probes within a probe sub-set is designed to differ from each other in terms of length, interrogating position inside the probe and strand of interrogation of the variant segment.
  • the invention also provides a library of probes prepared by the methods described above.
  • the probes are selected from the probes set forth in Table 1, SEQ
  • the probes consist of, or consist essentially of SEQ ID NOS: 1-
  • the invention also provides for methods of using or use of the library of probes designed/selected according to any one of methods described above.
  • the invention also provides a DNA-chip comprising the library of probes set forth above and use of the DNA chips, and well as a collection of microbeads comprising the library of probes and uses thereof.
  • Figure 1A shows a pair of probes consisting of a control or normal/wild type probe and a variation probe (SEQ ID NOS: 4607 and 4608, respectively in the order of appearance); the pair of probes is used to investigate an indel located at nucleotide 16 which is the nucleotide of interest in a genetic variant segment.
  • the indel here is a deletion of one base (G in position 16, illustrated in box) (Dell).
  • Figure IB shows a pair of probes consisting of a control or normal/wild type probe and a variation probe (SEQ ID NOS: 4609 and 4610, respectively in the order of appearance); the pair of probes is used to investigate an indel consisting of a deletion of four nucleotides located at nucleotide 16 which is the nucleotide of interest in a genetic variant segment.
  • the indel here is a deletion of the nucleotide 16 as well as the three nucleotides adjacent in 3' (boxed) (Del4).
  • Figure 2 shows the possible differences in various pairs of probes making up a indel probe sub-set: differences in probe length, 21 and 25 nucleotides long; differences in strand interrogated by the probes, the sense or anti-sense strand; and differences in the position of the indel location within the probes such as at the 11 nucleotide (0 position) in a 21 nucleotide long probe or at the 13 nucleotide (0 position) in a 25 nucleotide long probe.
  • Figure 3 shows different pairs of probes making up a probe sub-set and a different probe sub-set make up a probe set (SEQ ID NOS: 4611-4615, 4612, 4616, 4614, 4617-4621, 4618, 4622, 4620 and 4623, respectively in the order of appearance).
  • Figure 4A shows an embodiment of a library of probes on a solid support for detecting a single indel.
  • the library has N probe sets for interrogating a variant segment having a length of N basepair long, and each probe set comprises of one probe sub-set which comprises a pair of probes consisting of one control or normal/wild type probe (C) and one variation probe (V).
  • Figure 4B shows an embodiment of a library of probes on a solid support for detecting a single indel.
  • the library has N probe sets for interrogating a variant segment having a length of N basepair long, and each probe set comprises of one probe sub-set which comprises several pair of probes, each of which consists of one control or normal/wild type probe (C) and one variation probe (V).
  • Figure 4C shows an embodiment of a library of probes on a solid support for detecting four distinct indels.
  • the library has N probe sets for interrogating a variant segment having a length of N basepair long, and each probe set comprises of four probe sub-sets, one probe sub-set for each distinct indel, each probe sub-set comprises 32 pair of probes, each of which consists of one control or normal/wild type probe (C) and one variation probe (V).
  • Figure 5 shows one embodiment of the indel detection and analysis method.
  • Figure 6 shows one embodiment of the indel detection and analysis method.
  • Figure 7 shows one embodiment of the indel detection and analysis method.
  • Figure 8 illustrates three probes having 25, 23 and 20 nucleotide bases respectively showing the -3, -2, -1, 0 , +1, +2, +3 positions of the indel within the probes.
  • Figure 9 shows a schematic presentation of an embodiment of replicate probe features of the pair of probes comprising the probe sub-sets on a flat solid support.
  • Figure 10 shows is a block diagram showing an exemplary system for detecting an indel type of genetic variation.
  • Figure 11 shows an exemplary set of instructions on a computer readable storage medium for use with the systems described herein.
  • the present invention relates to an in vitro method of detecting genetic variations in an individual, specifically variations (e.g. duplications, insertions or deletions or loss of a small number of nucleotides collectively referred to as "indels") in a sequence segment in the genome.
  • variations e.g. duplications, insertions or deletions or loss of a small number of nucleotides collectively referred to as "indels"
  • the inventors have developed a sensitive, specific and reproducible computer implemented method for simultaneously detecting and characterizing indel variations in a genome. The method does not require prior knowledge of any indel in the segment. Therefore, novel indels can be discovered by the method described herein.
  • the inventors also developed methods for designing oligonucleotide probe sets for carrying out the method of detecting indel variations.
  • the method is also useful for the development of products for commercial, fast and reliable genotyping methods to detect known indels.
  • products such as kits
  • Such products can be used for, e.g., diagnostic and prognostic purposes and for the purposes of identifying individuals susceptible for, e.g., side effects associated with known indels or drug responsive individuals, wherein the drug response is associated with a known indel.
  • the method is the detection of mutations responsible of the illness Hypercholesterolemia Familiar.
  • the method is achieved with the use of a specially designed library of probes together with a computation algorithm for analysis of the data obtained from the library of probes.
  • the invention provides methods and products for determining the presence of insertions or deletions of a small number of nucleotides (indels) in a genetic segment of interest, also named genetic variant segment, such as an exon, an intron or a promoter, in the target nucleic acid (NA) sample.
  • a genetic segment of interest also named genetic variant segment, such as an exon, an intron or a promoter
  • NA target nucleic acid
  • the inventors showed that by using a specifically designed library of probes in hybridization experiments with a target NA sample to be genotyped, any indels harbored within a target NA sample can be detected with accuracy. Further, in the event that the indels are unknown, the type of indels found in the target NA sample can also be characterized.
  • embodiments of the invention can therefore provide considerable efficiency in terms of savings in time and cost when compared to other methods of detecting and characterizing indels, for example, full NA sequencing of the genetic variant segment.
  • the method is unique in that it is based on a combination of (1) use of a solid support based array, such as NA-chips/microbeads genotyping strategy with some distinct modifications in the probe selection and array design, and (2) a sequential computation system (algorithm) amenable for electronically processing and interpreting the data generated by the genotyping strategy (based on a selection of the probes to be included in the computation of the genotype).
  • a sequential computation system amenable for electronically processing and interpreting the data generated by the genotyping strategy (based on a selection of the probes to be included in the computation of the genotype).
  • This combination of genotyping strategy and a sequential computation system guarantees high level of specificity, sensitivity and reproducibility of results.
  • This method is versatile because any solid support, such as, chips or microbeads that are coated with the selected unique probes can be used, for example, in clinical genetic diagnosis.
  • the method is versatile for processing and interpreting of the data and it can be performed manually or by using a computer that is programmed to carry out the
  • One specific advantage of the method is the availability of a library of probes for every nucleotide position of any genetic segment of interest. It is not necessary to have prior knowledge of the indels, for example, whether the indel variation is a deletion or an insertion, or the number of nucleotides (nt) deleted or inserted.
  • the library of probes is specially designed to contain all probes that will detect all possible permutations of indels in a genetic variant segment.
  • the genetic variant segment can be e.g., having a length of N nt, wherein N can be, for example, 100-2000 or 50-5000 nt long. In some embodiments shorter than 50, such as 10-15, 25-50 nucleic acid fragments can be analyzed.
  • fragments that are larger such as about 50 or 100 to 2000, to 3000, to
  • Another specific advantage is the fact that direct identification of indel changes can be achieved, while previously used methods only detect the possible presence of an indel, without actually identifying the exact genetic change (i.e. the identity of the sequence variation).
  • NA nucleic acid
  • RNA RNA and polymers thereof in either single- or double-stranded form.
  • the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides.
  • base pairs bp
  • nucleotides nt are used to refer to the building block bases.
  • peptide-nucleic acid refers to any synthetic nucleic acid analog (deoxyribonucleic acid (DNA) mimics with a pseudopeptide backbone) which can hybridize to form double-stranded structures with DNA in a similar fashion as naturally occurring nucleic acids.
  • PNA is an extremely good structural mimic of DNA (or of ribonucleic acid (RNA)), and PNA oligomers are able to form very stable duplex structures with Watson-Crick complementary DNA and RNA (or PNA) oligomers, and they can also bind to targets in duplex DNA by helix invasion.
  • Other type of complementary base pairing such as the Hoogsteen pairing is possible too.
  • PNA may be an oligomer, linked polymer or chimeric oligomer.
  • Methods for the chemical synthesis and assembly of PNAs are well known in the art and are described in U. S. Patents Nos: 5,539,082, 5,527,675, 5,623,049, 5,714,331, 5,736,336, 5,773,571, and 5,786,571.
  • Uses of the PNA technology are also well known in the art; see U. S. patents Nos. 6,265,166, 6,596,486, and 6,949,343. These references are hereby incorporated by reference in their entirety.
  • nucleic acid refers to the member probes being all DNA, all RNA, or all PNA, and there is no mixture of RNA, DNA and/or PNA in a pair of probes.
  • nucleic acid also include PNA.
  • complementary base pair refers to A:T and G:C in DNA
  • RNA in RNA.
  • Most DNA consists of sequences of nucleotide only four nitrogenous bases: base or base adenine (A), thymine (T), guanine (G), and cytosine (C) or pseudocytosine (J).
  • the pairing is based on the Watson-Crick pairing or the Hoogsteen pairing. Together these bases form the genetic alphabet, and long ordered sequences of them contain, in coded form, much of the information present in genes.
  • Most RNA also consists of sequences of only four bases. However, in RNA, thymine is replaced by uridine (U).
  • the term "indels” refers to small duplications, deletions and/or insertions which involve anywhere between one to ten nucleotides (nt) and in other embodiments, the indels are duplications, deletions and/or insertions involving up to 50 nt.
  • the indel is a one nt deletion indel. In another embodiment, the indel is a two nt deletion indel.
  • the indel is a three nt deletion indel, a four nt deletion indel, a five nt deletion indel, a six nt deletion indel, a seven nt deletion indel, an eight nt deletion indel, a nine nt deletion indel, or a ten nt deletion indel.
  • the indel is a one nt insertion indel, a two nt insertion indel, a three nt insertion indel, a four nt insertion indel, a five nt insertion indel, a six nt insertion indel, a seven nt insertion indel, an eight nt insertion indel, a nine nt insertion indel, or a ten nt insertion indel.
  • the duplication, deletion or insertion can be up to 50 nt, e. g.
  • the phrase "genetic variant segment” refers to a segment or region of nucleic acid (NA) wherein there may be or are commonly known sequence variations within a population of an animal species, including humans. Such allelic variations may be silent or causal, or disease or disease-risk causing mutations.
  • a “genetic variant segment” refers to or included a NA segment or NA region where there is a likelihood of sequence variations within a population of an animal species, e.g., a region prone to spontaneous mutations, a region of known genetic instability, and/or a region associated with a disease or disorder that is known to be linked to mutations in the gene.
  • the NA can be DNA or RNA.
  • the NA is typically a genomic DNA, but in some embodiments it can also be a primary transcript or fragments thereof or a messenger RNA or fragments thereof.
  • the sequence variation or genetic variant present in the "genetic variant segment” is an indel.
  • the phrase "genetic non-variant segment” or “non-variant segment” refers to a segment or region of nucleic acid (NA) wherein the sequence is constant within a population of animal species, meaning that it is know that there is no allelic variation in the population in this region. While the “genetic non-variant segments” or “non-variant segment” do not have allelic variations among individuals in a population, they can have known mutations that result in very obvious and distinct phenotypes. Two normal individuals who are of the same gender and do not exhibit any of the obvious and distinct phenotypes (e.g. Down syndrome) that are associated with known mutations at these "genetic non-variant segments" would have identical "genetic non-variant segments”.
  • NA nucleic acid
  • Genetic non-variant segments function as the reference/control segments in the present invention in the analysis of indels. Mutations in non-variant segments can be selected from known disease -causing regions, such the DSCR1 locus on chromosome 21, the PLP locus and F9 locus on chromosome X, or any other region, which results in an unmistakable phenotype, wherein an absence of a phenotype, such as a Down syndrome, indicates that this region does not have variations in the subject, such as a human individual or an animal, whose nucleic acid is to be analyzed or in the sample from an individual or an animal whose sample is used as a control. A skilled artisan can easily select these regions based on these criteria and common knowledge of genetic diseases.
  • the "genetic non-variant segments” can be DNA or RNA.
  • the NA can be genomic DNA, a primary transcript or fragments thereof or a messenger RNA or fragments thereof.
  • the non-variant segment selected for analysis of human samples is derived from the human chromosome 21.
  • the non-variant segment is derived from the Down Syndrome Critical Region 1 (DSCR1) on chromosome 21.
  • the gene DSCR1 is also called RCAN1 for Regulator of Calcineurin 1.
  • DSCR1/RCAN1 is located at position 21q22.1-q22.2; chromosome 21 : 34,810,654-34,909,252 (SEQ ID NO: 4576) with respect to human genome assembly 18 March 2006 (GENBANKTM accession number for its mRNA: NM_004414.5, SEQ ID NO: 4577). It is involved in the development of the phenotype of the Down syndrome.
  • This gene part of this gene or the region of the chromosome 21 wherein this gene is located can be used as the non- variant segment for the normalization in human samples in the presently claimed methods.
  • the term "known genotype” when used in reference to control data of the genetic variant segment means that the type of indels, the number of nt involved, and the position or location, the bases or nt sequence(s) of the indel in the genetic variant segment are known, for example, one nt insertion at position K is the genetic variant in the segment.
  • the term "known genotype” when used in reference to control data means a SNP that is known, for example, it can be either a T nucleotide or a C nucleotide.
  • "known genotype” when used in reference to normal or wild type genotype data of the genetic variant segment which is the normal genotype in the population, meaning no indels at all in the genetic variant segment and the wild-type sequences are known.
  • test nucleic acid refers to a nucleic acid (NA) sequence wherein the indels within the sequence is unknown.
  • a test nucleic acid (tNA) refers to a NA sequence wherein the indel within the sequence is of interest to the investigator and the tNA therefore is being studied, regardless of whether the indel is known or not. For example, the investigator would like to verify that the indicated indel in the tNA is accurate and valid.
  • a “test nucleic acid (tNA) sample” refers to a NA sample comprising at least one tNA.
  • a control nucleic acid refers to a nucleic acid (NA) sequence wherein the indel within the sequence is known.
  • a cNA is a NA sequence that is normal/wild type and has no known indel within the sequence.
  • a cNA is a NA sequence that has a SNP that is known, for example, it can be either a T or a C, within the control sequence.
  • a control NA can be used in parallel with a tNA in the methods described herein for the detection and analysis of the indel in the tNA.
  • a "control nucleic acid (cNA)" sample refers to a NA sample comprising at least one cNA.
  • target nucleic acids refers to the nucleic acids that are to be hybridized to the probes immobilized on solid support(s) described herein.
  • Target NAs can comprise both the control nucleic acid and the test nucleic acid.
  • target NAs can be detectably labeled or fragmented to smaller segments of nucleic acid sequences.
  • the term "probe” refers to a short sequence of NA, typically consisting between 15nt-50nt, including all of the whole integers between 15-50, wherein the short sequence is complementary to a small portion of a genetic variant segment or complementary to a small portion of a non-variant segment (the control segment) that is under interrogation such that the probe can hybridize to the segment by complementary base pairing.
  • probes that are 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleic acids long.
  • probes can be a DNA, RNA, peptide nucleic acid (PNA) or hybrids thereof. Modifications to the backbone of the NA are encompassed within the definition.
  • the probe is a DNA-probe.
  • the probe is an RNA-probe.
  • the probe is a PNA-probe. Probes are preferably single-stranded probes, but double-stranded or partially double-stranded probes can also be used.
  • the term "variation affecting one nucleotide” refers to any of a plurality of insertions or deletions affecting one nt of interest, exclusively, and/or one nt of interest together with an addition of one to nine nt located contiguously at its Y side.
  • the additional nt can be one and up to 50 nt, e. g. 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 and 50 nt.
  • a deletion of only the nt of interest is an example of such a variation, i.e., affecting one nt.
  • a deletion of the nt of interest together with one contiguous Y nucleotide is another example of such a variation affecting one nt.
  • Del3 a deletion of the nt of interest together with two contiguous Y nt
  • Del4 a deletion of the nt of interest together with three contiguous Y nt
  • Del5, a deletion of the nt of interest together with four contiguous Y nt
  • Del6, a deletion of the nt of interest together with five contiguous Y nt
  • Del7 a deletion of the nt of interest together with six contiguous Y nt
  • Del8 a deletion of the nt of interest together with seven contiguous Y nt
  • Del9 a deletion of the nt of interest together with eight contiguous Y nt
  • DellO and a deletion of the nt of interest together with nine contiguous Y nt.
  • a duplication of only the nt of interest is another example of a variation affecting one nt.
  • nucleotide (two-nt duplication, or Dup2) is another example of such a variation affecting one nt.
  • Other examples include Dup3, a duplication of the nt of interest together with two contiguous Y nt; Dup4, a duplication of the nt of interest together with three contiguous Y nt; Dup5, a duplication of the nt of interest together with four contiguous Y nt; Dup6, a duplication of the nt of interest together with five contiguous Y nt; Dup7, a duplication of the nt of interest together with six contiguous Y nt; Dup8, a duplication of the nt of interest together with seven contiguous Y nt; Dup9, a duplication of the nt of interest together with eight contiguous Y nt; DuplO, and a duplication of the nt of interest together with nine contiguous Y nt.
  • the Fig. 1 shows graphically an example of Dell and Del4 variations affecting one nt, at position 16, of the wild type sequence.
  • the duplications are insertions, where the sequences of the inserted nt are different from that of the nt of interest.
  • the term "indel probe sub-set” or "probe sub-set” refers to a collection of probes interrogating one kind of indel variation affecting one nt.
  • a first probe sub-set investigates the presence of a single nt insertion at K position in variant segment
  • a second probe sub-set investigates the presence of a two- nt insertion at K position
  • a third probe sub-set investigates the presence of a three-nt insertion at K position
  • a fourth probe sub-set investigates the presence of a single nt deletion at K position
  • a fifth probe sub-set investigates the presence of a two-nt delenios at K position
  • a sixth probe sub-set investigates the presence of a three-nt deletion at K position
  • a seventh probe sub-set investigates the presence of a single nt duplication at K position
  • a probe sub-set investigates the presence of a two-nt duplication at K position
  • an "indel probe sub-set” or “probe sub-set” comprises at least one pair of probes, wherein the pair of probes consist of one control or normal/wild type probe and one variation probe.
  • one control or normal/wild type probe and one variation probe making up a pair of probes that have the same length and sequence except for the indel variation, be it an insertion, deletion or duplication.
  • probes can be designed with (1) different lengths, at (2) different positions in the probes wherein the indel variation to be detected are located; and (3) to hybridize to the sense strand solely, to the antisense strand solely, or to both strands.
  • a given indel probe sub-set can comprise probes of two different lengths, with sense and antisense probes of both lengths, and with two positions by which the variations will be detected in the probes of one length and three positions by which the variations will be detected in the probes of the other length.
  • All indels probe sub-sets should include the normal (i.e., wild type) probes matching the variation probes in terms of length, strand and position of the variation in the probe.
  • Fig. 2 shows an example of ten variations designed for the variation probes making up the pairs of probes that would make up an indel probe sub-set.
  • the variation probes are of two different lengths (21 nt and 25 nt long). Variation probes of both lengths are designed for either the sense or antisense strands or both. Probes which are 21 nt-long carry the variation-detecting nt at positions 9 and 11, while probes which are 25-nt long carry the variation-detecting nt at positions 11, 13 and 15. In this example, positions of variation- detecting nt are the same for the sense and antisense probes, but do not necessarily have to be so in all embodiments.
  • the indel probe sub-set includes the normal (i.e., wild type) probes that match the 10 variation probes.
  • the indel probe sub-set includes the control probes that match the 10 variation probes. The match of each variation probe with its normal or control probe forms a pair of probes. Any probe sub-set comprises at least two probes for the nucleotide of interest, a probe harboring a variation and the corresponding normal or control probe.
  • the number of distinct probes in a probe sub-set can range from one (in the case of a non-variant segment) to about 10,000, typically one uses about 2-200 probes per probe sub-set.
  • the probes are all distinct probes. In one embodiment, there is at least a duplicate or replicate of a probe. In another embodiment, one uses triplicates of a probe. In one embodiment, four or five replicates of each of the different probes making up a probe sub-set can be used. In other embodiments, more than five replicates of each different probe are used on a solid support, in some embodiment, up to 10, or up to 50 replicates are used. In the case of a non-variant segment, the probe sub-set is only composed of normal (i.e., wild type) or control probes.
  • an indel probe set refers to the collection of all the probe subsets selected for interrogating a nucleotide or nucleotide of interest where an indel variation can occur, and detecting all the kinds of variation affecting this nucleotide of interest. Any combination of indel probe sub-sets can be selected for a given probe set. Probe sub-sets in a given probe set can have different number of probes, and different combinations of length, strand hybridization and position of the variation to be detected in the probe. However, the process of designing the probe sub-set and probe set follows the novel selection method claimed herein and remains the same.
  • a set of indel probe sets refers to the collection of all the probe sets (thus, all the probes) selected for interrogating a genetic variant segment or a non-variant segment.
  • a genetic variant segment where indels are known to occur encompassing 0.7 kilobases (kb) long is selected for interrogation.
  • the investigator can select any number of probe sets covering these regions.
  • the full length of the segment of interest can be covered by designing indel probe sets for all the nt of the variant segment, i. e. 700 nt. For example, one can decide to have 700 different indel probe sets.
  • Each indel probe set can consist of any number of probes. In one
  • the number of distinct probes can be different for each probe set.
  • the number of distinct probes in a set of probe sets can range from one to about 300,000, typically one uses about 25,000 - 200,000 probes per set of probe sets for a nucleic acid region covering 1 kb.
  • the probes can be all distinct probes, and they complement and interrogate a single genetic variant segment or non-variant segment.
  • a set of indel probe sets constitutes a library of probes.
  • the genetic variant segment where indels of interest can be found is between about 50 base- pair (bp) to about 5000 bp long. In one embodiment, the genetic variant segment where indels of interest can be found is between about 100 bp to about 2000 bp long.
  • a set of two probe sets is shown.
  • the two probe sets affect respectively nt 16 and 17 of a variant segment which is 37 bp long.
  • the first probe set is for interrogating variations of the 16 th nt.
  • Two probe sub-sets making up the probe set for nt 16 are shown: one designed to detect a one-nt deletion (Dell) of the nt of interest (nt 16), and the other designed to detect a deletion of nt 16 and the nt immediately Y adjacent, i.e. nt 17 (Del2).
  • the Dell variation 1 probe length is 30 nt, and the position of the nt of interest is position 16 (of the 30 nt).
  • the Dell variation 2 probe also is 30 bp-long, but the position of the nt of interest is position 13 (of the 30 nt of the probe).
  • the Del2 variation 1 of the second probe sub-set of this first probe set is the deletion of nt 16 and 17, the indel variation (16 th bp) is placed at position 16 of a 30 nt long fragment.
  • the Del2 variation 2 is designed to detect the deletion of nt 16 and 17, in other words, a two-nt deletion at the 16 th nt position, but the indel variation is located in position 13 of a 30 nt long probe.
  • the second probe set is designed to detect variations affecting the 17 th bp of the same variant segment.
  • Two probe sub-sets make up this second probe set for the 17 th bp. As shown in Fig. 3, the same characteristics of probes have been designed, although this has not to be the case on other embodiments.
  • the nt of interest is in position 16 of a 30 nt-long probe.
  • the indel variation is in position 13 of a 30 bp-long probe.
  • the second probe sub-set (Del2), deletions of 2 nt are interrogated, the indel variation is placed at position 16 of the 30 nt-long probe in variation 1, and in position 13 of the 30 nt- long probe in variation 2.
  • probes are designed for the variant segment LDLR gene Exon 2, from position 68 -121, in intron 1, to nucleotide in position 190 +102 (reference sequence NM_000527.3, SEQ ID NO: 4579), a 345 nt- long sequence.
  • the possible probes variety can be whether to have sense and antisense strands probes, have three different sizes of probes (probes of 21, 23 and 25 nt), 5 different positions of the indel variation in the probe , such as central (0), central-2 nt (-2), central-4 nt (-4), central+2 nt (+2), central+4 nt (+4) positions or more etc, for the detection of the deletion of one, two and three nt and the detection of the insertion of one, two and three nt.
  • the number of probes to be designed can be 103,500, including both normal and variation probes.
  • the term "normal probe” or “control probe” refers to a probe that has no indel genetic variation, meaning that the probe has the wild type sequence with no deletions, insertions or duplications or has a known SNP respectively.
  • the "normal probe” or “control probe” interrogates the control nucleic acid (cNA).
  • the "normal probe” or “control probe” interrogates the non-variant segment.
  • variable probe refers to a probe that has an indel genetic variation with respect to the normal/wild type or control sequence, meaning that the probe has a deletion, insertion or duplication within the sequence.
  • the "variation probe” interrogates the test nucleic acid (tNA). In another embodiment, the "variation probe” interrogates the genetic variant segment.
  • probe feature refers to a localized and concentrated deposit of multiple copies of the same probe on a solid support surface (a defined “spot” on the glass surface or oligonucleotides on one bead).
  • spot on the glass surface or oligonucleotides on one bead.
  • a probe feature is a spot or dot printed with multiple copies of the same probe.
  • the multiple copies can range from tens to hundreds to thousands, e.g., about 10-10,000, or 100-10,000. All of the whole integers numbering from 10 to 10,000 are included.
  • a probe feature refers to a single bead coated with at least about 100 copies of the same oligonucleotides probe that complement and interrogate a single genetic variant segment or non-variant segment.
  • concentration of the oligonucleotide solution determines the approximate copies of oligonucleotides coating the bead.
  • the bead can have about 100-10,000 copies of the same probe.
  • the raw value or signal intensity of the hybridization reaction in the methods herein is obtained from a probe feature, meaning from a "dot" or a single probe-coated bead. In other words, measuring the signal intensity after hybridization of the test sample or the control sample gives a raw signal value.
  • replica feature or “replicate probe feature” refer to a replicate or multiples of a probe feature all having a single/same type of probe to genetic variant segment or non-variant segment (parallel dots or spots with same probe or oligonucleotide sequence on a solid surface or parallel numbers of beads coated with the same probe).
  • a flat solid support such as a glass- chip
  • all replicate features of one probe feature have one type of probe and the replicate features can be arranged, for example in a row but not close to each other on the glass-chip surface.
  • replica feature refers to number of probe -coated beads.
  • 100 probe-coated beads are 100 replicate features or replicate probe features.
  • On a solid flat surface for each probe, there are at least four replicate features, at least five, at least six, at least seven, at least eight, at least nine, and at least ten replicate features.
  • For a spherical solid surface there are at least 100 replicate features, typically between about 100-5000 probe- coated beads. All of the whole integers going from 100 to 5,000 are included.
  • the term "interrogation” refers to the examination, investigation or study of the nucleotide sequence information in a NA, i.e., the genotype.
  • the term "median" when used in the analysis of the data obtained from the probe feature replicas refers to general meaning when used in statistical analysis. Median is the 'middle value' in a list of values when arranged in increasing order. For example, for a list of the following numbers: 9, 3, 44, 17, 15 (odd amount of numbers), after lining up these numbers: 3, 9, 15, 17, 44 in increasing order (smallest to largest), the median is 15 which is the number in the middle of the ordered list. In the situation, wherein an even number of replicates are present, a median is found by finding the middle pair of numbers, and then find the value that would be half way between them. This is easily done by adding them together and dividing by two. In the present methods, the analysis of median is performed using computer-implemented software with the signal intensity values from the replicate features as an input and median as an output.
  • the term "mean" when used in the analysis of the data obtained from the probe feature replicas refers to general meaning when used in statistical analysis. Median is the average of a list of values, calculated by the formula:
  • the term "solid support”, on which the plurality of probes is deposited can be any solid support to which oligonucleotides can be attached. Practically any support, to which an oligonucleotide can be joined or immobilized, and which may be used in the production of DNA probe arrays and particle suspensions, can be used in the invention.
  • the said support can be of a non-porous material, for example, glass, silicone, plastic, or a porous material such as a membrane or filter (for example, nylon, nitrocellulose) or a gel.
  • the said support is a glass support, such as a glass slide.
  • the support is a particle in suspension, as described above, such as a microparticle.
  • Microparticles useful for the methods of the invention are commercially available for example from LUMINEX ® Inc., INVITROGEN TM (Carlsbad, Calif.), and Polysciences Inc. (Warrington, Pa.).
  • the solid support is a non-porous solid support.
  • the solid support is a porous solid support. Such supports are well known to one skilled in the art.
  • Embodiments of the invention provide (1) a library of probes which allows the detection of indels in a genetic variant segment; (2) a method of designing such a library of probes; (3) the use of the library of probes to detect the presence of indels in the test NA sample, the method comprises the immobilization of the probes on a solid support, the hybridization of test and optionally normal or control NA samples on the probes, the determination of the intensity for each NA-hybridized probe, and the analysis process and interpretation of the data generated by the hybridization; and (4) a solid support chip or spherical microbeads comprising a library of probes which allows the detection and
  • a specifically designed library of probes for detecting at least one indel variation in a genetic variant segment having a length of N number of base pairs (wherein N is typically a number between 25 and 5000, for example 50-2000), the library comprising a set of probe sets which comprises N number of probe sets (i.e. the same number of probe sets as there are nucleotides in the segment to be analyzed), wherein there is one probe set for each nucleotide position of the genetic variant segment.
  • Each probe set comprises at least one probe sub-set, wherein the at least one probe sub-set is for interrogating a single kind of indel; and further wherein the at least one probe sub-set comprises at least a pair of probes, a normal probe and a variant probe, both of which interrogate the same region on the genetic variant segment (i.e. are designed to bind to either to the normal or variant sequence in that specific location).
  • These probes form a pair of probes that have the same sequence length and are of the same type of nucleic acids (except for the difference in the normal and variant sequence to be detected). The length of the probes is between 15-50 nucleotides.
  • the normal probe comprises a sequence corresponding and binding to the normal / wild type or control sequence of genetic variant segment and the variant probe comprises an indel-containing variant sequence of genetic variant segment.
  • the indel in the variant probe is located at -4, -3, -2, -1, 0, +1, +2, +3 or +4 position in the variant probe or located up to 25 nt off the central nt for probes longer, e. g. up to 50nt, wherein the position 0 is the center nucleotide of the probe when the probe has an odd number of nucleotides; and wherein the position -1 and +1 are the two central nucleotides of the probe when the probe has an even number of nucleotides, e. g.
  • the library of probes can consist of the design of all the probes able to detect any one or all kinds of possible indels that one would desire or is likely to detect. If the indels are known indels that one looks for, e.g., as a disease screen or a drug resistance or tolerance screen, probes for only those specific indels can be included into the library. For example, if the genetic region of interest is 100 bp long in the genome, all possible indels for this region would encompass all indel types occurring at each of the 100 nt of this 100 bp-long sequence. For example, a deletion, an insertion and a duplication individually for the position 1 in this 100 bp-long sequence, and so forth for all the following 99 positions in this 100 bp-long sequence.
  • the library of probes comprises or consists essentially of or consists of a control and/or a normal probe.
  • the control and/or normal probes are provided on a solid support.
  • Control or normal/wild type probes for a known non-variant segment(s) on the X- chromosome exhibit gender dimorphism, meaning that the control, i.e. known nt at position x or the normal wild type nt at position x, is present depends on whether hybridization is performed on a male or female subject (one copy in males, two in females).
  • Such control probes and their respective X chromosome non-variant segments can be used as controls to verify that the control nt, e.g.
  • an X chromosome non-variant segment(s) can be selected from two well characterized genes: the PLP locus and F9 locus on the human X-chromosome. These non-variant segments can be used for the normalization.
  • the first gene is PLP (for Proteolipid Protein 1, located Xq22), a gene whose duplications and deletions are responsible of the Pelizaeus-Merzbacher disease (PMD). This disease is an X-linked recessive hypomyelinative leukodystrophy (HLD1) in which myelin is not formed properly in the central nervous system. PMD is characterized clinically by nystagmus, spastic quadriplegia, ataxia, and developmental delay. PLP1 is located at position chromosome X:
  • the second gene is the F9 (for coagulation factor IX, located Xq22) which is responsible of Hemophilia B. Deletions of this gene cause Hemophilia B.
  • F9 is located at position chromosome X: 138,440,061-138,473,783 (SEQ ID NO: 4582) with respect to human genome assembly 18 March 2006 (GENBANKTM accession number for its mRNA: NM_000133.3; SEQ ID NO: 4583).
  • the indel variation can be deletion of one, two, three, four, five, six, seven, eight, nine or ten adjacent nucleotides (nts).
  • the deletion can be up to 50 nt, e. g. 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 and 50 nt.
  • the indel variations also can be insertions of one, two, three, four, five, six, seven, eight, nine or ten adjacent nts.
  • the insertions or deletions can be located at any of the nt in the sequence.
  • the insertion can be up to 50 nt, e. g. 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 and 50 nt.
  • the insertion of one or more nucleotides can be a duplication of one or more nt, up to ten nt, of the reference/control or wild type sequence, for example, two, three, four, five, six, seven, eight, nine or ten nts.
  • the duplication can be up to 50 nt, e. g. 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 and 50 nt.
  • probes encompassed for each nt position in the genetic variant segment include: (1) probes with different length, e. g. probes that are anywhere from 15- 50 nt long, such as 21, 23, 25, 27, 30 and 32 nt long; (2) the probes that have the indel variation located at different interrogating position inside the variant probe, e. g.
  • the position 0 is the center nucleotide of the probe when the probe has an odd number of nts; and wherein the position - 1 and +1 are the two central nts of the probe when the probe has an even number of nucleotides (see Fig. 8); and (3) probes that complement or hybridize to the sense strand or the anti-sense strand of the genetic variant segment.
  • the strand to which the probe complements or hybridizes would be the stand interrogated by the probe of the variant segment.
  • the best probe for its detection might be a probe 25 bp long, interrogating the sense strand of the variant segment and the indel variation is located at the 0 position (i. e. nt number 13) of the 25 bp long probe (see Fig.
  • the best probe for its detection might be a probe 23 base-pair long, interrogating the anti-sense strand of the variant segment and with the indel variation is located at the -3 position (i. e. nt number 9) of the 23 bp-long probe (see Fig. 8).
  • the at least one probe sub-set comprises more than one pair of probes, e. g. anywhere from 5-100 pairs of probes.
  • Member probes constituting a pair of probes have the same length and interrogate the same region on the variant segment and they interrogate the same strand.
  • the pairs of probes of the at least one probe set have different length, at least three different length, e. g. anywhere from 15-50 nt long. For example, there are pairs of probes that are 15, 20, 25, 30, 35, 40, 45 and 50 nt long making up the at least one probe sub-set. For example, there are pairs of probes that are 21, 23 and 25 nt long making up the at least one probe sub-set.
  • the pairs of probes of the at least one probe set interrogate a different strand of the genetic variant segment, e.g. some pairs of probes interrogate the sense strand of the genetic variant segment and other pairs of probes interrogate the anti-sense strand of the genetic variant segment. These pairs of probes can have the same length or different length.
  • the probes are all designed to hybridize to either the sense or the anti-sense strand.
  • the pairs of probes of the at least one probe set have different positions of the indel variation located within the probe.
  • a library of probes are probes for detecting and analysing/characterizing several combinations of indel variations in a genetic variant segment. This facilitates simultaneous detection and analysis of several indels in the genetic variant segment.
  • a library of probes can have probes for detecting and analysis/characterization insertions of one, two, and three nts at all the nt positions and probes for detecting and
  • Another library of probes can have probes for insertions of four and five nts at all the nt positions and probes for deletions of four and five nts at all the nt positions in the genetic variant segment.
  • Libraries can have probes that detecting and analysing/characterization all possible
  • probes for detecting and analysing/characterization of several combinations of indel variations if more than one indel is present in a genetic variant segment.
  • the library of probes can be used to detecting and analysing/characterization of several combinations of indel variations in more than one genetic variant segment, e. g. several genetic variant segments.
  • an investigator may wish to analyze several genetic variant segments found in a test NA sample.
  • Such a library of probes will facilitate simultaneous detection and analysis of several indels in the test NA sample which may have only one or several genetic variant segments.
  • the library of probes comprises one set of probe sets and as many probe sets as the length or number of nt (or base pairs) in the variant segment under interrogation, wherein each probe set comprises a single probe sub-set, wherein each probe sub-set itself comprises a single pair of probes consisting of one control or normal/wild type probe (C) and one variation probe (V).
  • Fig. 4 A shows an example of this embodiment of the library of probes for a single indel detection whether it is a one nt deletion or a one nt- insertion.
  • the library is for interrogating a variant segment having a length of N bp-long (where N can be any number of nucleotides, such as, 25-5000 or any size as described already above in connection with some embodiments), and each probe sub-set itself comprises a single pair of probes.
  • N can be any number of nucleotides, such as, 25-5000 or any size as described already above in connection with some embodiments
  • each probe sub-set itself comprises a single pair of probes.
  • N bp-long segment there should be N number of probe sets (i.e. as many probe sets as there are nucleotides in the segment that on wishes to analyze) making up the one set of probe sets.
  • Replicas of each of the probes for each pair of probes can be placed on the solid support, e.g., replicas of the C and V probes (see Figure 4) of each pair of probes can be placed on the solid support.
  • a library of probes is the set of probe sets or the entire collection of probe sets.
  • the number of probe sets comprising in the library is as many as the length of nt (or base pairs) in the variant segment under interrogation.
  • Each probe set comprises one or more probe sub-sets wherein each probe sub-set comprises one or more pairs of probes.
  • a pair of probes consists of one control or normal/wild type probe and one variant probe.
  • Each pair of probes making up a probe sub-set differ from the other pairs of probes within the same probe sub-set in term of the length, the location of the indel variation inside the variant probe, and/or the interrogation strand: sense strand or the anti-sense strand.
  • a probe sub-set is a collection of pairs of probes that investigates a single type of indel variation at a specific X position in a genetic variant segment.
  • a probe set is then a collection of probe sub-sets that investigates indel variations at a specific X position in a genetic variant segment (see Fig. 3, probe sub-set nt 16 dell and nt 16 del2 make up the probe set for nt 16).
  • the collection of probe sub-sets i. e. a probe set
  • the collection of probe sub-sets investigates more than one type of indel variation at a specific X position in a genetic variant segment (e.g.
  • the probe set can be the collection of probe sub-sets that investigates all the desired types of indel variation at X position.
  • a probe sub-set comprises pairs of probes that investigate a deletion of one nt at the 16 th position, probe sub-set nt 16, Dell.
  • Another probe sub-set comprises pairs of probes that investigate an insertion of one nt at the 16 th position, probe sub-set nt 16, Insl.
  • Another probe sub-set comprises pairs of probes that investigate a duplication of one nt at the 16 th position, probe sub-set nt 16, Dupl.
  • Another probe sub-set comprises pairs of probes that investigate a duplication of two nt at the 16 th position, probe sub-set nt 16, Dup2.
  • the collection of probe sub-sets for the 16 th nt: Dell, Insl, Dupl and Dup2 make up a probe set for nt at the 16 th position of the genetic variant segment.
  • control or normal/wild type probe and the variant probe making a pair of probe have the same length, i. e. both the normal and variant probes have the same number of bases or nt (Fig. 1 and Fig. 3).
  • control or normal/wild type probe and the variant probe making a pair of probe hybridize to the same strand of the genetic variant segment, i. e. both the normal and variant probes hybridize to the sense strand of the genetic variant segment or both hybridize to the anti-sense strand of the genetic variant segment.
  • the normal/control probe and the variant probe interrogate about the same region in the genetic variant segment.
  • each of these probe sub-sets comprises at least a pair of probes consisting of one control or normal probe and one variant probe.
  • each probe subset comprises several pairs of probes wherein each pair consists of one control or normal probe and one variant probe.
  • the probes of the different pairs of probes can differ from each other by the length, the location of the indel variation inside the variant probe or the interrogation strand: sense strand or the anti-sense strand.
  • Fig. 3 shows two pairs of probes for the probe sub-set nt 16 dell, two pairs of probes for the probe sub-set nt 16 del2, two pairs of probes for the probe sub-set nt 17 dell and two pairs of probes for the probe sub-set nt 17 del2.
  • the two pairs of probes for the probe sub-set nt 16 dell investigates a single nt deletion at nt position 16. All four probes of these two pairs are 30 mers, meaning they each consist of 30 nt.
  • the probes from the two pairs differ by the location of the deletion on the probe; in nt 16 dell variation 1, the deletion is located at position 16 of the 30 mer, or position +1, and in nt 16 dell variation 2, the deletion is located at position 13 of the 30 mer, or position -3.
  • the two pairs of probes for the probe sub-set nt 16 del2 investigates a single nt deletion at nt position 16 and a deletion of one 3' contiguous of nt position 16. All four probes of these two pairs are 30 mers, meaning they each consist of 30 nt.
  • the probes from the two pairs differ by the location of the deletion on the probe; in nt 16 dell variation 1, the deletion is located at position 16 of the 30 mer, or position +1, and in nt 16 dell variation 2, the deletion is located at position 13 of the 30 mer, or position -3.
  • the library of probes comprises one set of probe sets and as many probe sets as the length of the variant segment under interrogation (e.g., if the length of the segment to be analyzed is 2000 base pairs, 2000 probe sets are designed according the novel system or rules set forth in this specification), wherein each probe set comprises a single probe sub-set, wherein the probe sub-set comprises at least one pair of probes, wherein each pair of probes consists of one normal/wild type probe and one variant probe, wherein the probes of each pair of probes making up the probe sub-set differ from each other in terms of length, indel variation location inside the probe and strand of interrogation of the variant segment.
  • Fig. 4B shows one exemplary system how to make a library of probes for detecting a single type of indel present in a variant segment having a length of N bp-long.
  • For each probe set there is a single probe sub-set consisting of ten pairs of probes.
  • one probe sub-set investigates the single nt deletion or insertion at position X in the genetic variant segment.
  • the second probe sub-set investigates the single nt deletion or insertion plus another nt deletion or insertion 3 'side of position X in the genetic variant segment.
  • indel for example, two types of indels; e.g., a deletion of only one nt in a genetic variant segment and an insertion of only one nt in a genetic variant segment.
  • the only one-nt deletion in a genetic variant segment and the only one-nt insertion in a genetic variant segment represent two distinct kinds or types of indels that can occur for each and every nt position in the variant segment.
  • the library of probes comprises one set of probe sets and as many probe sets as the length of the variant segment under interrogation, wherein each probe set comprises two probe sub-sets, wherein each probe sub-set comprises at least one pair of probes, wherein each pair of probes consists of one control or normal/wild- type probe and one variation probe, wherein the probes of each pair of probes making up the probe subset differ from each other in terms of length, indel variation location inside the probe and strand of interrogation of the variant segment. There is one probe sub-set for each distinct indel being investigated.
  • the first and a second probe sub-set for the probe set that correspond to the nt at the X position in the genetic variant segment; the first probe sub-set that investigates the single nt deletion at position X in the genetic variant segment and the second probe sub-set investigates the single nt insertion at position X in the genetic variant segment.
  • the application therefore provides various combinations of indels that can be investigated simultaneously with a library of probes, where the library of probes is designed according to the rules set forth in this specification and examples.
  • one decides to detect several kinds of indel variation in a genetic variant segment, for example, deletions of one, two, three, four and five nts.
  • a library of probes for detecting and analysis of a genetic variant segment with one, two, three, four and five nts deletions has one set of probes sets. There are as many probe sets as the length of the variant segment under interrogation, each probe set comprising of five probe sub-set, each probe sub-set for investigating each of the different type of indel, i. e. deletions of one, two, three, four and five nts; each probe sub-set comprising at least one pair of probes which consist of one normal probe and one variant probe.
  • the probes of each pair of probes within a probe sub-set differ from each other in terms of length, interrogating position inside the probe and strand of interrogation of the variant segment.
  • a library of probes for detecting and analysis of a genetic variant segment with one, two, and three nt deletions and one nt insertion will have one set of probes sets. There are again as many probe sets as the length of the variant segment under interrogation. Each probe set comprising four probe sub-sets, each probe sub-set for investigating each of the different type of indel, i. e.
  • each probe sub-set comprises at least one pair of probes which consist of one control or normal / wild type probe and one variant probe.
  • the probes of each pair of probes within a probe sub-set differ from each other in terms of length, interrogating position inside the probe and strand of interrogation of the variant segment.
  • Fig. 4C shows one embodiment of the library of probes for detecting four types of indel present in a variant segment having a length of N bp-long.
  • the library has four probe sub-sets, one probe sub-set for each of the different type of indel, i.e. deletions of one, two and three nt and insertion of one nt; each probe sub-set consisting of 32 pairs of probes.
  • N bp-long segment there are N numbers of probe sets making up the one set of probe sets. Replica of the C and V probes of a pair of probes can be placed on the solid support.
  • Methods of analysis for the detection of indels rely on, in general, comparisons of hybridization intensities among normal and variation probes, among tNA and cNA samples, among genetic variant and non-variant segments, and combinations thereof.
  • the method of detection of indels relies solely on the hybridization intensities among normal and variation probes of at least one test sample.
  • probes comprising the probe sub sets should have the same characteristics (length, length, position of the indel variation of interest - i.e. at position +1 from the middle of the probe or as described above for other alternative positions, and strand hybridized - i.e., sense or anti-sense) across all probe sets.
  • probe feature refers to a localized and concentrated deposit of multiple copies of the same probe on a solid support surface (for example, a defined "spot" on the glass surface or
  • the method of detecting and analyzing an least one indel in a genetic variant segment having a length of N nucleotide bases comprises:
  • the library of probes comprises one set of probe sets for interrogating a genetic variant segment
  • the set of probe sets comprise at least two probe sets, a first and a second probe set
  • each the at least two probe sets comprises at least one probe sub-set
  • the at least one probe sub-set comprise at least one pair of probes: a normal or control probe and a variation probe
  • the normal probe of the pair is complementary to the normal (wild type) or control sequence of the genetic variant segment
  • the variation probe is complementary to the normal sequence except for a position corresponding to the indel variation
  • the type of indel variation probes of the at least two probe sets are the same
  • the first probe set interrogates an indel located at position k in the genetic variant segment
  • the second probe set interrogates the indel located position k+1 in the genetic variant segment, and wherein the probes are placed on the solid support as probe features
  • step (f) applying an algorithm to the data from step (e), thereby determining the indel present in the genetic variant segment of the tNA sample, wherein algorithm comprises the steps of:
  • Ratio 1 IN (k) ⁇ )
  • k has values from 1 to N-l where 1 represent the first oligonucleotide of the variant segment and N is the last base;
  • the method comprises comparing all the ratios of each probe sub-set to confirm that they all indicate the same indel variation.
  • An example of this embodiment of method is shown in Fig. 5 and some exemplary intensity data generated by the hybridization of the test NA and the probe features and the calculation of the can be found in Tables 2-5.
  • the probe sub-sets within the probe sets for each contiguous nt in the variant segment has the same number of pairs of probes and the same type of pair of probes in term of length, interrogating position inside the probe and strand of interrogation of the variant segment.
  • the method compares results of a certain nucleotide with the results of the nucleotide to its right (k vs k+1).
  • the probes within each probe sub-set of a probe set must have matching probes in the probe sub-set of the adjacent probe set, matching in term of length, location of indel interrogation, and strand of interrogation between probe sub-sets in order to obtain the ratios for ratio comparison.
  • the matching up is necessary for consecutive nt positions in the segment investigated.
  • matches up means that the matched pair of probes are of the same length, interrogate the approximately same region in the variant segment, interrogate the same type of indel and the indel is located at the same position within the probe (see Fig. 1 and 3).
  • the method of detection and analysis of the indels relies on the hybridization of the tNA samples as well as one control NA sample.
  • the genetic variant segment and the non-variant segment form both the tNA and the cNA samples are used.
  • the method of detecting and analyzing an indel in a genetic variant segment having a length of N nucleotide bases comprises:
  • each of the probe set for the genetic variant segment comprises at least one probe sub-set, wherein the at least one probe sub-set comprises at least one pair of probes: a normal or control probe and a variation probe, wherein the normal probe of the pair is complementary to the normal (wild type) or control (i. e.
  • each of the probe set for the genetic non-variant comprises at least one probe sub-set, wherein the at least one probe sub-set comprises at least one probe: a normal (wild type) or control probe, and wherein the probes are placed on the solid support as probe features;
  • step (g) applying an algorithm to the data from step (f), thereby determining the indel variation present in the genetic variant segment of the tNA sample, wherein algorithm comprises the steps of:
  • step (i) computing a median or a mean of the intensity from step (f) for the normal or control probe features of all the probe sets of the set of probe sets interrogating the at least one non-variant segment wherein the median or mean is used as a normalization factor for intensities of step (f);
  • step (ii) applying the normalization factor of step (i) to the intensities of step (f) to obtain a normalized intensity for all the probe features;
  • Ratio 1 IN ⁇ t ⁇ IN ⁇ (iv) . pairing the probe feature of variation probes hybridized with the genetic variant segment from the tNA sample with the probe feature of variation probes hybridized with the genetic variant segment from the cNA sample for all the probe sub-sets of all probe sets; pairing of the normal or control probes are performed too;
  • (v) computing a variation ratio (Ratio 2) between IV ⁇ over IV ⁇ where k can have values from 1 to N where 1 represent the first nucleotide base of the variant segment and n the last nucleotide base, where IV (k)t represents the value of intensity of the variation probe from the tNA sample and IV (k)C represents the intensity of the variation probe from the cNA sample:
  • Ratio 2 IV (k)t ⁇ IV (k)c
  • a Ratio (t/C) (k) is equal to about one, the indel variation of the nucleotide k position is normal (i.e. wild type); if the Ratio (t/C) (k) is more than two, preferentially more than 5, preferentially more than 10, this indicates a heterozygote indel variation at the nucleotide k position; if the ratio is more than 100, preferentially more than 200 fold, this indicates an homozygote indel variation at the nucleotide in k position.
  • the comparison is between each probe set of the tNA and probe set of the control tNA (for instance, probe set for nt 16 for tNA versus the probe set control for nt 16 for cNA, and probe set for nt 17 for tNA versus probe set for nt 17 for cNA).
  • all the probes in a sub-set for the tNA must have a corresponding probe in the cNA.
  • the number of pairs of probes for the probe sub-set need not be the same, e. g.
  • the control should have at least 7 probes, 3 matching the ones of set 16 and 4 matching the ones of set 17.
  • the probe features of normal probes hybridized with the genetic variant segment from the tNA sample are paired with the probe features of normal probes hybridized with the genetic variant segment from the cNA sample for all the probe sub-sets of all probe sets.
  • the method comprises comparing all the ratios of each probe sub-set to confirm that they all indicate the same indel variation.
  • An example of this embodiment of method is shown in Fig. 6.
  • control NA sample can be used for the method of detection and analysis of indels described.
  • Those skilled in the art can readily adopt the above described method to calculate the normalization factor of step (f) by using data from the various control NA samples, instead of data from just one cNA sample.
  • the normalization factor is computed by the median or the mean of all the normal probe features of the variant segment when several, preferentially more than 10, preferentially more than 50, preferentially more than 100 probe sub sets are used.
  • the method of detection of indels relies on the hybridization of the test samples as well as various control samples. In this embodiment, only the genetic variant segment is used.
  • the method of detecting and analyzing an indel in a genetic variant segment having a length of N nucleotide bases comprises:
  • the set of probe sets comprising at least one probe set
  • the at least one probe set for the genetic variant segment comprises at least one probe sub-set
  • the at least one probe sub-sets comprise at least one pair of probes: a normal probe and a variation probe, wherein the normal probe of the pair is complementary to the normal (wild type or control) sequence of the genetic variant segment, and the variation probe is complementary to the normal sequence except for a position corresponding to the indel variation, and wherein the probes are placed on the solid support as probe features;
  • step (g) applying an algorithm to the data from step (f), thereby determining the indel present in the genetic variant segment of the tNA sample, wherein algorithm comprises the steps of:
  • Ratio (k) intensity value for normal probe
  • step (ii). computing the mean and the standard deviation of the ratios obtained for all the control NA samples; and (iii). comparing the ratios obtained for each of the tNA sample with the mean ratio obtained for the cNA samples in step (ii), wherein if the ratio of the tNA sample is at least 5 standard deviations away from the mean ratio obtained with the cNA sample, the test NA has the indel variation at position k, either in an heterozygous or a homozygous state; wherein k has values from 1 to N-l where 1 represent the first oligonucleotide of the variant segment and N is the last base.
  • probes features of each pair of probes comprising the at least one probe sub-set are grouped together according to their length, position of the nucleotide of interest, and interrogation strand.
  • corresponding variation probe features are paired according to their length, position of the nucleotide of interest, and interrogation strand for the at least tNA sample and the cNA samples.
  • probe features are grouped together according to their probe sub-set.
  • the normal and their corresponding variation probe features are paired according to the probe feature groups.
  • the method comprises comparing all the ratios of each probe sub-set to confirm that they all indicate the same indel variation.
  • any of the method described can be used. It is contemplated that more than one described method is use for any given indel investigated. In some embodiments, one can use one of the four methods of indel detection. In some embodiments, two, three or all methods described are used for indel detection.
  • each probe of pairs of probes comprising the probe sub-sets are attached to a solid support to form probe features.
  • replicates of each probe features on the solid support are exemplified in Fig. 9.
  • the number of replicates for each probe is between 1-50, from 1 and up to 5, or from 1 up to 10.
  • the methods comprise measuring an intensity of the detectable label in non-probe positions of the solid support to obtain a background intensity value.
  • the methods comprise transforming the intensity of the detectable label obtained into a raw value for each probe or probe feature and the solid support background using a quantitation software.
  • One embodiment of the methods comprises amending the raw value for each of the probe feature or replicate probe feature by deducting the background raw value, thereby obtaining a net value for the each probe feature or replicate probe feature for both the at least one genetic variant segment and the at least one genetic non-variant segment.
  • One embodiment of the methods comprises selecting for subsequent analysis the probe features whose net values pass quality control thresholds or values signal to noise ratio of, typically, over three (SNR>3), in the probe feature positions wherein a signal is detected.
  • the method is computer implemented.
  • NA samples can be obtained from any appropriate biological sample which contains
  • the sample may be taken from a fluid or tissue, secretion, cell or cell line derived from the human body.
  • samples may be taken from blood, including serum, lymphocytes, lymphoblastoid cells, fibroblasts, platelets, mononuclear cells or other blood cells, from saliva, liver, kidney, pancreas or heart, urine or from any other tissue, fluid, cell or cell line derived from the human body.
  • a suitable sample may be a sample of cells from the buccal cavity.
  • the NA is obtained from a blood sample.
  • NA can be extracted and isolated from the biological sample using conventional techniques.
  • the nucleic acid to be extracted from the biological sample may be DNA, or RNA, typically total RNA.
  • RNA is extracted if the genetic variation to be studied is situated in the coding sequence of a gene.
  • the methods further comprise a step of obtaining cDNA from the RNA. This may be carried out using conventional methods, such as reverse transcription using suitable primers. Subsequent procedures are then carried out on the extracted DNA or the cDNA obtained from extracted RNA.
  • DNA as used herein, may include both DNA and cDNA.
  • any genetic variant segment can be analyzed using the computer- implemented algorithm as described. The genetic variations to be tested are located within known nucleic acid sequences and well characterized.
  • the NA samples which contain the genetic segment or segments of interest are subjected to an amplification reaction prior to analysis in order to obtain amplification products which contain the genetic variations to be identified.
  • the amplified nucleic acid regions are typically the variant and/or non-variant segment to be interrogated.
  • Any suitable technique or method can be used for amplification. In general, the technique allows the multiplex amplification of all the DNA sequences containing the genetic variations to be identified. In other words, where multiple genetic variations are to be analyzed, it is preferable to simultaneously amplify all of the corresponding target DNA regions in one reaction (comprising the variations). Carrying out the amplification in a single step (or as few steps as possible) simplifies the method. PCR amplification conditions are such that the final copy number after amplification reflects the initial copy number of the segments in the NA samples.
  • multiplex PCR can be carried out, using appropriate pairs of
  • oligonucleotide PCR primers which are capable of amplifying the target regions containing the genetic variations to be identified.
  • each genetic variant segment is amplified together with a genetic non- variant segment in the multiplex PCR reaction using the test or control NA sample as the DNA template.
  • the genetic variant and the genetic non-variant segments amplified together form an amplification group.
  • Any suitable pair of primers which allow specific amplification of a target DNA region may be used.
  • the primers allow amplification in the least possible number of PCR reactions.
  • genotyping e.g. DNA-array or particle suspension
  • the present method can comprise the use of one or more of these primers or one or more of the listed primer pairs. Examples presented in the present application provide additional exemplary primers.
  • several independent multiplex PCR amplification reactions are carried out for the test NA sample and the control sample.
  • at least four independent multiplex PCR amplification reactions are carried out for the test NA sample and the control sample.
  • about four independent multiplex PCR amplification reactions are carried out for the test NA sample and the control NA sample.
  • the PCR products from the independent amplifications for the test NA sample are pooled together. Likewise, those of the control NA samples are pooled together.
  • no or at least one genetic non-variant segment can be selected.
  • the genetic non-variant segment is encompassed within the test NA and control NA samples. For example, if neither the test nor the control exhibit Down syndrome, a test region from the Down syndrome region of chromosome 21 can be selected as a non- variant segment.
  • the NA in the test NA and control samples are detectably-labeled.
  • the aim is to be able to later detect hybridization between the genetic variant or non-variant segments and probe features fixed on a solid support.
  • Methods of labeling NA are well known to one skill in the art, e. g. US Patent No. 6,573,374 and US Patent No. 5,700,647 describe suitable labeling methods.
  • the attached label is detected by various methods known in the art, e.g. optically, wherein a photonic signal is converted to an electronic signal and registered by a computer, which outputs a signal in, for example, a numeric value.
  • a labeled nucleotide can be incorporated during the amplification reaction or labeled primers can be used for amplification.
  • the labeled nucleotide is a biotinylated nucleotide.
  • the labeled primer is a biotinylated primer.
  • Labeling can be direct using for example, fluorescent or radioactive markers or any other marker known by persons skilled in the art.
  • fluorophores include for example, Cy3 or Cy5.
  • enzymes may be used for sample labeling, for example alkaline phosphatase or peroxidase.
  • radioactive isotopes which can be used include for example 33 P, 125 I, or any other marker known by persons skilled in the art.
  • labeling of amplification products is carried out using a nucleotide which has been labeled directly or indirectly with one or more fluorophores.
  • labeling of amplification products is carried out using primers labeled directly or indirectly with one or more fluorophores.
  • Labeling may also be indirect, using, for example, chemical or enzymatic methods.
  • an amplification product may incorporate one member of a specific binding pair, for example avidin or streptavidin, conjugated with a fluorescent marker and the probe to which it will hybridize may be joined to the other member of the specific binding pair, for example biotin (indicator), allowing the probe/target binding signal to be measured by fluorimetry.
  • an amplification product may incorporate one member of a specific binding pair, for example, an anti-dioxigenin antibody combined with an enzyme (marker) and the probe to which it will hybridise may be joined to the other member of the specific binding pair, for example dioxigenin (indicator).
  • amplification product to probe the enzyme substrate is converted into a luminous or fluorescent product and the signal can be read by, for example, chemi-luminescence or fluorometry.
  • the NA or the amplification products can further undergo a fragmentation reaction, thereby obtaining some fragmentation products which comprise or contain the genetic variations to be identified or analyzed. Typically fragmentation increases the efficiency of the hybridization reaction. Fragmentation may be carried out by any suitable method known in the art, for example, by contacting the nucleic acid, e.g. the amplification products with a suitable enzyme such as a DNase.
  • the PCR products are fragmented to smaller sizes and then detectably labeled prior to hybridization with probes on a solid support. In one embodiment, the PCR products are fragmented to between about 12 -250nt in size. In one embodiment, the PCR products are fragmented to between about 25 -200nt in size. In one embodiment, the PCR products are fragmented to between about 25 -150nt in size. In one embodiment, the PCR products are fragmented to between about 25 -lOOnt in size. In one embodiment, the PCR products are fragmented to between about 25 -75nt in size. In one embodiment, the PCR products are fragmented to between about 25 -50nt in size.
  • labeling with a detectable label may be carried out pre-hybridization by labeling the fragmentation products.
  • Suitable labeling techniques are known in the art and may be direct or indirect as described herein.
  • Direct labeling may comprise the use of, for example, fluorophores, enzymes or radioactive isotopes.
  • the direct labeling comprises the use of biotin.
  • Indirect labeling may comprise the use of, for example, specific binding pairs that incorporate e.g. fluorophores, enzymes, etc.
  • the fragmentation products may undergo a direct or indirect labeling with one or various markers, for example biotin or one or various fluorophores, although other known markers can be used by those skilled in the art.
  • markers for example biotin or one or various fluorophores, although other known markers can be used by those skilled in the art.
  • hybridization intensity values for use in analysis methods, can be amended for each of the probe features by deducting the background raw value from the raw value, thereby obtaining a net value.
  • At least one oligonucleotide probe is designed and synthesize for each of the variant and non-variant segment to be interrogated.
  • at least two unique probes are designed and synthesize for each segment.
  • at least five, at least ten, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, and at least 100, including all the whole integers between 2-100,000 unique probes are designed and synthesize for each segment. All of the probes are unique, although they can have overlapping sequences.
  • the collection of unique probes designed and synthesized for each segment constitutes a set of probe set.
  • the set of probe sets for a segment that is interrogated comprises at least two unique probe sets for that segment.
  • a set of probe sets for a segment that is interrogated comprises at least five, at least ten, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least70, at least 75, at least 80, at least 85, at least 90, at least 95, and at least 100, including all the whole integers between 2-100, unique probe sets.
  • a first set of probe sets is provided for a genetic variant segment (form the test NA sample) to be interrogated.
  • a second set of probe sets is provided for a genetic non-variant segment (from the control NA sample) to be interrogated.
  • the library of probe is attached to a solid support as probe features in a specific arrangement wherein the location of each probe feature is known.
  • a probe feature is provided on a solid support; the probe feature being a localized and concentrated sample having multiple copies of the same probe is deposited and attached on a solid surface.
  • a probe feature is a minute spot or dot printed with multiple copies of the same probe (see Fig. 9). The multiple copies can range from hundreds to thousands, e.g. 100-10000. All of the whole integers between 100 to 10,000 are included is a single probe feature.
  • a probe feature refers to a single probe -coated bead. All the beads are coated with the multiple copies of same probe that complements and interrogates a single genetic variant segment or non-variant segment.
  • the range of numbers of probe-coated beads in "a probe feature" is between 100-1000, including all of the whole integers between 10-10000.
  • replicates of a probe feature are made on a solid support.
  • a solid support such as a glass-chip
  • all replicate features of one probe feature type have one type of probe and the replicates can be arranged in a row on the glass-chip surface. Multiple rows can be made and distributed in fix and known coordinates on the glass chip (see Fig. 9).
  • replicate features of one probe are many probe -coated beads, about 100 probe -coated beads. These beads all have probes of a single type.
  • the solid support has between 10- 50 replicate features for each unique probe. All whole integers between 10-50 are considered.
  • For each probe on a spherical solid support there are at least about 100 replicate features or probe -coated beads.
  • replicates of probe features of a first set of probe sets are provided for a genetic variant segment (form the test NA sample) to be interrogated.
  • replicates of probe features of a second set of probe sets are provided for a genetic non-variant segment (from the control NA sample) to be interrogated.
  • the replicates of probe features of the first and second set of probe sets are attached on same solid support.
  • two or more identical solid supports are used, each solid support having probe features.
  • One solid support is used to hybridize with the test NA sample and the other solid support is used to hybridize with the control NA sample (Fig. 6 and 7).
  • each solid support having all the replicates of a first and a second set of probe sets, wherein the first set of probe sets interrogates a genetic variant segment and the second set of probe sets interrogates a genetic non-variant segment.
  • One solid support is used to hybridize with the test NA sample and the other solid support is used to hybridize with the control NA sample (see Fig. 6 and 7).
  • each probe feature is provided in at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 9, at least 10 replicates and the probe features are attached to the flat surface at positions according to a known uniform spatial distribution, i.e., a support or surface with an ordered array of binding (e.g. hybridization) sites or probes.
  • a known uniform spatial distribution i.e., a support or surface with an ordered array of binding (e.g. hybridization) sites or probes.
  • anywhere form 10-50 replicate probe features are provided.
  • the arrangement of replicate features on the support is predetermined.
  • Each probe replicate is located at a known predetermined position on the solid support such that the identity (i.e. the sequence) of each probe can be determined from its position on the array.
  • the probes are uniformly distributed in a predetermined pattern.
  • the solid support is a flat surface.
  • a flat solid support is a glass-chip surface.
  • DNA-arrays in the form of DNA-chips to detect genetic variations, the present invention also contemplates the use of DNA particle or bead suspensions.
  • the solid support is a micron-size particle.
  • the beads are uniquely identifiable. Examples of particle identifiers on a particle are a bar code and a fluorescent dye.
  • the beads are bar-coded. These beads such as polymer or magnetic beads have unique spectroscopic signatures. Beads can be synthesized by dispersion polymerization of a family of styrene monomers and methacrylic acid to generate a spectroscopically encoded bead library. Raman spectroscopy is used to monitor complexing events on the barcoded beads. The genotyping assays from ILLUMINA ® , Inc.
  • the solid supports form particle suspensions. It has been found that these particle suspensions should comply with a number of requirements in order to be used in the present methods, for example in terms of the design of the probes, the number of probes provided for each genetic variation to be detected and the distribution of probes on the support. These are described in detail herein.
  • each probe is attached to at least 10 units of each particle species, wherein each particle species is distinguishable by a unique code from all other particle species.
  • each probe is attached to at least 1000 units of each particle species.
  • the labeled NA are contacted with a solid support having attached probes in a specified arrangement described herein as replicate features, allowing NA hybridization between the tNA and the cNA (collective hereby termed as target NA) with the probes in the replicate features and the formation of target-probe complexes.
  • target NA NA hybridization between the tNA and the cNA
  • specific hybridization complexes are formed between target NA and corresponding probes. Since the NAs are labeled, the target-probe complexes formed can therefore be detected.
  • the hybridization conditions allow specific hybridization between probes and corresponding target NA to form specific probe/target hybridization complexes while minimizing hybridization between probes carrying one or more mismatches to the DNA.
  • Such conditions may be determined empirically, for example by varying the time and/or temperature of hybridization and/or the number and stringency of the array washing steps that are performed following hybridization and are designed to eliminate all probe -DNA interactions that are non-specific.
  • the melting temperature of the probe/target complexes may occur at 75-85°C.
  • hybridizations can be for one hour, although higher and lower temperatures and longer or shorter hybridizations may also suffice. A skilled artisan can optimize these conditions using routine methods.
  • hybridization can be carried out using conventional methods and devices.
  • hybridization is carried out using an automated hybridization station.
  • the segments are placed in contact with the probes under conditions which allow hybridization to take place.
  • stable hybridization conditions allows the length and sequence of the probes to be optimized in order to maximize the discrimination between genetic variations A and B, e.g. between wild type and mutant sequences, as described herein.
  • a chip DNA array has from 300 to 40000 probe features, for example, from
  • the chip can have from 1000 to 20000 probes, such as 1000 to 15000 or 1000 to 10000, or 1000 to 5000.
  • a suitable chip may have from 2000 to 20000, 2000 to 10000 or 2000 to 5000 probe features.
  • a chip may have 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 12000, 14000, 16000, 18000 or 20000 probes.
  • Smaller chips 400 to 1000 probes such as 400, 500, 600, 700, 800, 900 or 950 probes are also envisaged.
  • the number of probes in a particle suspension will vary depending on the number of individually identifiable particles.
  • the chip DNA array of the invention comprises a support or surface with an ordered array of binding (e.g. hybridization) sites or probe features.
  • binding e.g. hybridization
  • probe features e.g. hybridization sites or probe features.
  • the arrangement of probes on the support is predetermined.
  • Each probe i.e each replicate feature
  • the probes are uniformly distributed in a predetermined pattern.
  • the probes deposited on the support are not grouped by genetic variation but have a random distribution. Typically they are also not grouped within the same genetic variation. If desired, this random distribution can be always the same. Therefore, typically the probes are deposited on the solid support (in an array) following a predetermined pattern so that they are uniformly distributed, for example, between the two areas that may constitute a DNA-chip, but not grouped according to the genetic variation to be characterized.
  • probe features are arranged on the support in subarrays.
  • Microarrays are in general prepared by selecting probes which comprise a given polynucleotide sequence, and then immobilizing such probes to a solid support or surface. Probes may be designed, tested and selected as described herein. In general, the probes can comprise DNA sequences. In some embodiments the probes may comprise RNA sequences, or copolymer sequences of DNA and RNA. The polynucleotide sequences of the probes may also comprise DNA and/or RNA analogues, or combinations thereof. For example, the polynucleotide sequences of the probes may be full or partial fragments of genomic DNA.
  • the polynucleotide sequences of the probes may also be synthesized nucleotide sequences, such as synthetic oligonucleotide sequences.
  • the probe sequences can be synthesized either enzymatically in vivo, enzymatically in vitro (e.g., by PCR), or non-enzymatically in vitro.
  • microarrays or chips can be made in a number of ways. However produced, microarrays typically share certain characteristics. The arrays are reproducible, allowing multiple copies of a given array to be produced and easily compared with each other. Preferably, microarrays are made from materials that are stable under binding (e.g., nucleic acid hybridization) conditions. The microarrays are preferably small, e.g., between 0.25 to 25 or 0.5 to 20 cm 2 , such 0.5 to 20 cm 2 or 0.5 to 15 cm 2 , for example, 1 to 15 cm 2 or 1 to 10 cm 2 , such as 2, 4, 6 or 9 cm 2 .
  • Replicate probe features can be attached to the solid support using conventional techniques for immobilization of oligonucleotides on the surface of the supports.
  • the techniques used depend, amongst other factors, on the nature of the support used - porous (membranes, micro-particles, etc.) or non-porous (glass, plastic, silicone, etc.)
  • the probes can be immobilized on the support either by using non-covalent immobilization techniques or by using immobilization techniques based on the covalent binding of the probes to the support by chemical processes.
  • non-porous supports e.g., glass, silicone, plastic
  • pre-treatment with reactive groups e.g., amino, aldehyde
  • a member of a specific binding pair e.g. avidin, streptavidin
  • the immobilization of the probes on the support can be carried out by conventional methods, for example, by means of techniques based on the synthesis in situ of probes on the support (e.g., photolithography, direct chemical synthesis, etc.) or by techniques based on, for example, robotic arms which deposit the corresponding pre-synthesized probe (e.g. printing without contact, printing by contact) (See U. S. Patent No. 7,281,419 for example).
  • the support is a glass slide and in this case, the probes, in the number of established replicates (for example, 6, 8 or 10) are printed on pre-treated glass slides, for example coated with aminosilanes, using equipment for automated production of DNA -chips by deposition of the oligonucleotides on the glass slides ("micro-arrayer"). Deposition is carried out under appropriate conditions, for example, by means of crosslinking with ultraviolet radiation and heating (80°C), maintaining the humidity and controlling the temperature during the process of deposition, typically at a relative humidity of between 40-50% and typically at a temperature of 20°C.
  • the replicate probe features are distributed uniformly amongst the areas or sectors (sub- arrays), which typically constitute a DNA-chip.
  • the number of replicas and their uniform distribution across the DNA-chip minimizes the variability arising from the printing process that can affect experimental results.
  • particle suspension technology allows for the detection of probes in a single vessel, with individual probes attached to a particle with a distinguishable characteristic.
  • the particles are encoded with one or more optically distinguishable dyes, a detectable label, or other identifying characteristic such as a bar code.
  • Other labeling methods include, but are not limited to a combination of fluorescent and non-fluorescent dyes, or avidin coating for binding of biotinylated ligands. Such methods of encoding particles are known in the art.
  • the intensity of detectable label at each probe position can be determined.
  • the intensity of the signal (the raw intensity value) is a measure of hybridization at each replicate feature.
  • the intensity of detectable label at each probe position can be determined using any suitable means.
  • the means chosen will depend upon the nature of the label.
  • an appropriate device for example, a scanner, collects the image of the hybridized and developed DNA-chip. An image is captured and quantified.
  • the hybridized and developed DNA-chip is placed in a scanner in order to quantify the intensity of labeling at the points where hybridization has taken place.
  • a fluorescence confocal scanner is used.
  • the DNA-chip is placed in the said apparatus and the signal emitted by the fluorpohore due to excitation by a laser is scanned in order to quantify the signal intensity at the points where hybridization has taken place.
  • Non-limiting examples of scanners which can be used according to the present invention include scanners marketed by the following companies: Axon, Agilent, Perkin Elmer, etc.
  • the signal from the particles is detected by the use of a flow cytometer.
  • detection of fluorescent labels may also be carried out using a microscope or camera that will read the image on the particles.
  • Flow cytometric software for detection and analysis of the signal is available for example from Luminex, Inc. (Austin, TX).
  • the measuring intensity of the detectable label for each probe is performed using scanning.
  • raw intensity values can be gathered for each probe replica and the background noise associated with each probe replica can also be assessed in order to produce "clean" values for signal intensity at each replicate feature position.
  • the inventors have found that this can be done by applying a specific algorithm to the intensity data.
  • the algorithm and computer software developed by the inventors allows analysis of the genetic variations with sufficient sensitivity and reproducibility as to allow use in a clinical setting.
  • amending the raw intensity value to obtain the clean intensity value for each probe replica comprises subtracting background noise from the raw value. Background noise is typically determined using appropriate controls such as area of chip with no NA or probe.
  • the algorithm as described herein is designed to be computer implemented, and thus in some embodiments, the methods described herein comprise the use of a computer system and a computer program.
  • Embodiments of the invention can be described through functional modules, which are defined by computer executable instructions recorded on computer readable media and which cause a computer to perform method steps when executed.
  • the modules are segregated by function for the sake of clarity. However, it should be understood that the modules/systems need not correspond to discreet blocks of code and the described functions can be carried out by the execution of various code portions stored on various media and executed at various times. Furthermore, it should be appreciated that the modules may perform other functions, thus the modules are not limited to having any particular functions or set of functions.
  • the computer readable storage media can be any available tangible media that can be accessed by a computer.
  • Computer readable storage media includes volatile and nonvolatile, removable and non-removable tangible media implemented in any method or technology for storage of information such as computer readable instructions, data structures, program modules or other data.
  • Computer readable storage media includes, but is not limited to, RAM (random access memory), ROM (read only memory), EPROM (eraseable programmable read only memory), EEPROM (electrically eraseable programmable read only memory), flash memory or other memory technology, CD-ROM (compact disc read only memory), DVDs (digital versatile disks) or other optical storage media, magnetic cassettes, magnetic tape, magnetic disk storage or other magnetic storage media, other types of volatile and nonvolatile memory, and any other tangible medium which can be used to store the desired information and which can accessed by a computer including and any suitable combination of the foregoing.
  • RAM random access memory
  • ROM read only memory
  • EPROM eraseable programmable read only memory
  • EEPROM electrically eraseable programmable read only memory
  • flash memory or other memory technology CD-ROM (compact disc read only memory), DVDs (digital versatile disks) or other optical storage media, magnetic cassettes, magnetic tape, magnetic disk storage or other magnetic storage media, other types of volatile and nonvolatile memory, and
  • Computer-readable data embodied on one or more computer-readable media may define instructions, for example, as part of one or more programs that, as a result of being executed by a computer, instruct the computer to perform one or more of the functions described herein, and/or various embodiments, variations and combinations thereof.
  • Such instructions may be written in any of a plurality of programming languages, for example, Java, J#, Visual Basic, C, C#, C++, Fortran, Pascal, Eiffel, Basic, COBOL assembly language, and the like, or any of a variety of combinations thereof.
  • the computer-readable media on which such instructions are embodied may reside on one or more of the components of either of a system, or a computer readable storage medium described herein, may be distributed across one or more of such components.
  • the computer-readable media can be transportable such that the instructions stored thereon can be loaded onto any computer resource to implement the aspects of the present invention discussed herein.
  • the instructions stored on the computer- readable medium, described above are not limited to instructions embodied as part of an application program running on a host computer. Rather, the instructions may be embodied as any type of computer code (e.g., software or microcode) that can be employed to program a computer to implement aspects of the present invention.
  • the computer executable instructions may be written in a suitable computer language or combination of several languages.
  • the functional modules of certain embodiments of the invention include at minimum a measuring module #40, a storage module #30, a comparison module #80, and an output module #110 (Fig. 10).
  • the functional modules can be executed on one, or multiple, computers, or by using one, or multiple, computer networks.
  • the measuring module has computer executable instructions to provide e.g., expression information in computer readable form.
  • the measuring module #40 can comprise any system for detecting a signal representing the detectable label from a target NA-probe complex (Fig. 10).
  • Such systems can include DNA microarray readers, RNA expression array reader, flow cytometer or any other system which produces an electronic signal converted from the original label, such as a photonic signal or a radioactive signal.
  • the original signal intensity or frequency determines the electronic signal intensity or frequency.
  • the information determined in the determination system can be read by the storage module #30 (Fig. 10).
  • the "storage module” is intended to include any suitable computing or processing apparatus or other device configured or adapted for storing data or information. Examples of electronic apparatus suitable for use with the present invention include stand-alone computing apparatus, data telecommunications networks, including local area networks (LAN), wide area networks (WAN), Internet, Intranet, and Extranet, and local and distributed computer processing systems.
  • Storage modules also include, but are not limited to: magnetic storage media, such as floppy discs, hard disc storage media, magnetic tape, optical storage media such as CD-ROM, DVD, electronic storage media such as RAM, ROM, EPROM, EEPROM and the like, general hard disks and hybrids of these categories such as magnetic/optical storage media.
  • the storage module is adapted or configured for having recorded thereon genetic variation information. Such information may be provided in digital form that can be transmitted and read electronically, e.g., via the Internet, on diskette, via USB (universal serial bus) or via any other suitable mode of communication.
  • stored refers to a process for encoding information on the storage module.
  • Those skilled in the art can readily adopt any of the presently known methods for recording information on known media to generate manufactures comprising genetic variation information.
  • the reference data stored in the storage module to be read by the comparison module is e.g., genetic variation data from normal subjects.
  • the "comparison module” #80 can use a variety of available software programs and formats for the comparison operative to compare genetic variation data determined in the measuring module for the variant and non-variant segment.
  • the comparison module is configured to use pattern recognition techniques to compare information from one or more entries to one or more reference data patterns.
  • the comparison module may be configured using existing commercially-available or freely-available software for comparing patterns, and may be optimized for particular data comparisons that are conducted.
  • the comparison module provides computer readable information related to normalized ratios of intensities, median log 2 of intensities etc in the analysis and interpretation of the genetic variation in an individual.
  • the comparison module can include an operating system (e.g., UNIX) on which runs a relational database management system, a World Wide Web application, and a World Wide Web server.
  • World Wide Web application includes the executable code necessary for generation of database language statements (e.g., Structured Query Language (SQL) statements).
  • SQL Structured Query Language
  • the executables will include embedded SQL statements.
  • the World Wide Web application can include a configuration file which contains pointers and addresses to the various software entities that comprise the server as well as the various external and internal databases which must be accessed to service user requests.
  • the Configuration file also directs requests for server resources to the appropriate hardware— as may be necessary should the server be distributed over two or more separate computers.
  • the World Wide Web server supports a TCP/IP protocol.
  • Local networks such as this are sometimes referred to as "Intranets.”
  • An advantage of such Intranets is that they allow easy communication with public domain databases residing on the World Wide Web (e.g., the GENBANK or Swiss Pro World Wide Web site).
  • users can directly access data (via Hypertext links for example) residing on Internet databases using a HTML interface provided by Web browsers and Web servers.
  • the comparison module provides a computer readable comparison result that can be processed in computer readable form by predefined criteria, or criteria defined by a user, to provide a content-based in part on the comparison result that may be stored and output as requested by a user using an output module #110 (Fig. 10).
  • the content based on the comparison result can be an expression value compared to a reference showing the in a genetic variant segment of the test NA sample.
  • the content based on the comparison result is displayed on a computer monitor #120. In one embodiment of the invention, the content based on the comparison result is displayed through printable media #130, #140.
  • the display module can be any suitable device configured to receive from a computer and display computer readable information to a user.
  • Non-limiting examples include, for example, general-purpose computers such as those based on Intel PENTIUM-type processor, Motorola PowerPC, Sun UltraSPARC, Hewlett-Packard PA-RISC processors, any of a variety of processors available from Advanced Micro Devices (AMD) of Sunnyvale, California, or any other type of processor, visual display devices such as flat panel displays, cathode ray tubes and the like, as well as computer printers of various types.
  • general-purpose computers such as those based on Intel PENTIUM-type processor, Motorola PowerPC, Sun UltraSPARC, Hewlett-Packard PA-RISC processors, any of a variety of processors available from Advanced Micro Devices (AMD) of Sunnyvale, California, or any other type of processor, visual display devices such as flat panel displays, cathode ray tubes and the like, as well as computer printers of various types.
  • AMD Advanced Micro Devices
  • a World Wide Web browser is used for providing a user interface for display of the content based on the comparison result.
  • modules of the invention can be adapted to have a web browser interface.
  • a user may construct requests for retrieving data from the comparison module.
  • the user will typically point and click to user interface elements such as buttons, pull down menus, scroll bars and the like conventionally employed in graphical user interfaces.
  • the present invention therefore provides for systems (and computer readable media for causing computer systems) to perform methods for analyzing genetic variations in a tNA sample.
  • modules of the machine may assume numerous configurations. For example, function may be provided on a single machine or distributed over multiple machines.
  • a system for detecting analyzing an indel variation in NA sample comprising:
  • a measuring module measuring the raw intensity comprising a detectable signal from a replicate feature indicating the presence or level of a NA-probe complex on a solid support comprising the replicate feature;
  • a storage module configured to store data output from the measuring module; c. a comparison module adapted to compare the data stored on the storage module with reference and/or control data, and to provide a retrieved content, and d. an output module for displaying the retrieved content for the user, wherein the retrieved content the Ratio ⁇ ) or Ratio (k)/(k+1) for the kth nucleotide of the genetic variant segment indicates that the presence of an indel in the NA sample.
  • a computer readable storage medium comprising:
  • a comparison module that compares the data stored on the storing data module with a reference data and/or control data, and to provide a comparison content
  • an output module displaying the comparison content for the user, wherein the retrieved content the for the kth nucleotide of the genetic variant segment indicates that the presence of an indel in the NA sample.
  • control data comprises data from an individual with normal / wild type genotype at the genetic variant segment under interrogation.
  • genes or genetic variant segments are selected on the basis of the pathogenicity of indels they may contain.
  • the probes for detecting indels are oligonucleotide NA ranging from 15 to 50 nt and are designed for interrogating genes or genetic variant segments.
  • Indels to be detected can be deletions of one nt, deletions of two nt, deletions of three nt, duplications of one nt, insertions of one nt, insertions of more than one nt.
  • the deletion, insertions or duplications can be anywhere from one to ten nt at any of the nt positions in the genes or genetic variant segments.
  • the deletion insertions or duplications can be up to 50 nt, e. g. 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 and 50 nt.
  • the gene having genetic variant segments to be interrogated is the gene encoding LDLR (for Low Density Lipoprotein Receptor, located 19pl3.2). It is involved in the phenotype of Hypercholesterolemia, Autosomic Dominant (HAD mainly called Familial
  • Hypercholesterolemia hereafter named FH
  • FH Hypercholesterolemia
  • Genetic variant segment 1 Promoter and exon 1 of LDLR gene (SEQ ID NO: 4585).
  • Table 1 One embodiment of a library of probes designed according to the system described herein is found in Table 1, comprising SEQ ID NO: 1-4575. This library of probe is designed to detect all possible indels for the exon 2 of the human LDLR gene.
  • genes having genetic variant segments that can be interrogated are the human apolipoprotein B (including Ag(x) antigen) (APOB) gene (SEQ ID NO: 4584), the various exons in PCSK9 (Proprotein convertase subtilisin/kexin type 9) gene, in particular, exons 2, 4, 7 and 10, as provided in SEQ ID NOS: 4602-4605, respectively and the cystic fibrosis transmembrane conductance regulator (CFTR) gene that is responsible for the genetic disorder cystic fibrosis. This gene is located on chromosome 7: 116907153-117096054 (approx. 188kb) (SEQ ID NO: 4606).
  • APOB apolipoprotein B
  • PCSK9 Protein convertase subtilisin/kexin type 9
  • CFTR cystic fibrosis transmembrane conductance regulator
  • the "A" of the ATG of the signal peptide" or base position number 1 is the 469th nucleotide in the genomic sequence of human LDLR sequence (SEQ ID NO: 4579).
  • the base position in the LDLR genomic sequence that corresponds to the 1st base position in the LDLR mRNA is 469.
  • the second number within each position formula refers to the number of the bases that is to be added or subtracted from the base position in the genomic where that base position corresponds to the first number of the position formula which is that in the mRNA.
  • the sequences of a number of indel oligonucleotide probes are selected from these variant segments.
  • test NA sample The patient's NA, such as DNA, to be genotyped, called test NA sample, is amplified to produce various genetic variant segments as listed herein and can be complementary of the entire size of the probes. Together with the patient's DNA, one or more control NA sample is amplified under the same conditions of the test target NA.
  • the targets are fragmented and labeled and then hybridized onto the probes that are immobilized on solid supports.
  • Solid supports such as flat glass chips or beads are scanned to obtain intensities of each single probe.
  • Additional embodiments of the invention provides a DNA chip comprising a plurality of probe features deposited on a solid support, the chip being suitable for use in a method of the invention described herein; a computational method for obtaining a genotype from DNA-chip hybridization intensity data wherein the method comprises using ratios for each segment to be genotyped; a computer system comprising a processor and means for controlling the processor to carry out a computational method of the invention; and a computer program comprising computer program code which when run on a computer or computer network causes the computer or computer network to carry out a
  • the invention provides a library of probes for detecting at least one indel variation in a genetic variant segment having a length of N number of base pairs, the library comprising (a) a set of probe sets which comprises N number of probe sets, wherein there is one probe set for each nucleotide position of the genetic variant segment; wherein each probe set comprise of at least one probe sub-set, wherein the at least probe sub-set is for interrogating a single kind of indel; wherein the at least probe sub-set comprises at least a pair of probes, a normal or control probe and a variant probe, both of which interrogate a substantially similar region on the genetic variant segment, wherein the both probes forming the pair of probes have the same sequence length, interrogated the same strand of genetic variant segment and are of the same type of nucleic acids, and wherein the length of the probes are between 15-50 nucleotides; wherein the normal probe comprises the normal / wild type or control sequence of genetic variant segment and the variant
  • control probes are designed and selected for the library.
  • normal / wild type probes are designed and selected for the library.
  • the library comprises only control probes and not normal probes, in addition to the corresponding variant probes.
  • the library comprises only normal probes and not control probes, in addition to the corresponding variant probes.
  • oligonucleotide probes are designed for each position to be tested in the genetic variant segment. In one embodiment, for each position of the segment to be interrogated, 36 probes are designed to detect changes from the normal or control sequence.
  • all these probes are to be oriented in the 3' to 5' direction on the solid support. In another embodiment, all these probes are to be oriented in the 3' to 5' direction on the solid support.
  • indels of 2 or more nts are always considered to the "right" (i.e., 30 of the nt interrogated in the genetic variant segment.
  • the oligonucleotide probes can be about 15-50 nt long.
  • the probes typically have the base to be examined (the site of the indel genetic variation) at the center of the probe, i.e., in the middle, such that for example a probe of 25 nucleotides long has the location of the genetic variation as nucleic acid 13 from the 5'end.
  • the indel variation is located at the "0" position within the probe, "0" refers to the central nt in the oligonucleotide probe.
  • the indel variation can also be 2-4 nucleic acids 3' or 5' of the center of the probe, i. e. at the -4, -3, -2, -1, +1, +2, +3 or +4 position within the probe, wherein the probe oriented in the 3' to 5' direction on the solid support.
  • one oligonucleotide probe where the base of interest (i. e. indel variation) located at the central base is deleted with respect to the normal or wild type allele. This probe detects a single nt deletion. The indel variation is located at the "0" position within the probe. The same oligonucleotide probe is also designed but on the other strand of the fragment is to be analyzed.
  • base of interest i. e. indel variation
  • one oligonucleotide probe where the base of interest is in an offset position of 4 nucleotides with respect to the central base towards the 5' end and is deleted compared to the normal or wild type allele. This probe detects a single nt deletion. This probe has the indel variation located at the -4 position with respect to the central nt in the oligonucleotide probe. A similar oligonucleotide probe is also designed but this probe hybridizes to the other strand of the fragment to be analyzed.
  • one oligonucleotide probe where the base of interest is in an offset position of 4 nucleotides with respect to the central base towards the 3' end and is deleted from the normal or wild type allele. This probe detects a single nt deletion. This probe has the indel variation located at the +4 position with respect to the central nt in the oligonucleotide probe.
  • a similar oligonucleotide probe is also designed but this probe hybridizes to the other strand of the fragment to be analyzed.
  • one oligonucleotide probe where the base of interest is located at the central base and its adjacent base on the 5' side is deleted with respect to the normal or wild type allele. This probe detects two nt deletion. The indel variation is located at the "0" position within the probe.
  • a similar oligonucleotide probe is also designed but this probe hybridizes to the other strand of the fragment to be analyzed.
  • one oligonucleotide probe where the two bases of interest are in an offset position of 4 nucleotides towards the 5' with respect to the base in the central position and its adjacent base on the 5' side and are deleted respect to the normal or wild type allele.
  • This probe detects two nt deletions.
  • This probe has the indel variation located at the -4 position with respect to the central nt in the oligonucleotide probe.
  • a similar oligonucleotide probe is also designed but this probe hybridizes to the other strand of the fragment to be analyzed.
  • one oligonucleotide probe where the two bases of interest are in an offset position of 4 nucleotides towards the 3' respect to the base in the central position and its adjacent base on the 5' side and are deleted respect to the normal or wild type allele.
  • This probe detects two nt deletions.
  • This probe has the indel variation located at the +4 position with respect to the central nt in the oligonucleotide probe.
  • a similar oligonucleotide probe is also designed but this probe hybridizes to the other strand of the fragment to be analyzed.
  • one oligonucleotide probe where the bases of interest located the central base and its adjacent 2 base on the 5' side are deleted respect to the normal or wild type allele. This probe detects three nt deletions. The indel variation is located at the "0" position within the probe. A similar oligonucleotide probe is also designed but this probe hybridizes to the other strand of the fragment to be analyzed.
  • one oligonucleotide probe where the three bases of interest are in an offset position of 4 nucleotides towards the 5' respect to the base in the central position and its adjacent base on the 5' side and are deleted respect to the normal or wild type allele.
  • This probe detects three nt deletions.
  • This probe has the indel variation located at the -4 position with respect to the central nt in the oligonucleotide probe.
  • a similar oligonucleotide probe is also designed but this probe hybridizes to the other strand of the fragment to be analyzed.
  • one oligonucleotide probe where the three bases of interest are in an offset position of 4 nucleotides towards the 3' respect to the base in the central position and its adjacent base on the 5' side and are deleted respect to the normal or wild type allele.
  • This probe detects three nt deletions.
  • This probe has the indel variation located at the +4 position with respect to the central nt in the oligonucleotide probe.
  • a similar oligonucleotide probe is also designed but this probe hybridizes to the other strand of the fragment to be analyzed.
  • one oligonucleotide probe where the base of interest located at the central base is duplicated respect to the normal or wild type allele. This probe detects a single nt duplication. The indel variation is located at the "0" position within the probe. A similar oligonucleotide probe is also designed but this probe hybridizes to the other strand of the fragment to be analyzed.
  • one oligonucleotide probe where the base of interest is in an offset position of 4 nucleotides respects to the central base towards the 5' end and is duplicated respect from the normal or wild type allele. This probe detects a single nt duplication. This probe has the indel variation located at the -4 position with respect to the central nt in the oligonucleotide probe. A similar oligonucleotide probe is also designed but this probe hybridizes to the other strand of the fragment to be analyzed.
  • one oligonucleotide probe where the base of interest is in an offset position of 4 nucleotides respects to the central base towards the 3' end and is duplicated respect from the normal or wild type allele. This probe detects a single nt duplication. This probe has the indel variation located at the +4 position with respect to the central nt in the oligonucleotide probe. A similar oligonucleotide probe is also designed but this probe hybridizes to the other strand of the fragment to be analyzed.
  • one oligonucleotide probe where the bases of interest being the central base and its adjacent base on the 5' side are duplicated respect to the normal or wild type allele. This probe detects a two- nt duplication. The indel variation is located at the "0" position within the probe. A similar oligonucleotide probe is also designed but this probe hybridizes to the other strand of the fragment to be analyzed.
  • one oligonucleotide probe where the two bases of interest are in an offset position of 4 nucleotides towards the 5' respect to the base in the central position and its adjacent base on the 5' side and are duplicated respect to the normal or wild type allele.
  • This probe detects a two- nt duplication.
  • This probe has the indel variation located at the -4 position with respect to the central nt in the oligonucleotide probe.
  • a similar oligonucleotide probe is also designed but this probe hybridizes to the other strand of the fragment to be analyzed.
  • one oligonucleotide probe where the two bases of interest are in an offset position of 4 nucleotides towards the 3' respect to the base in the central position and its adjacent base on the 5' side and are duplicated respect to the normal or wild type allele.
  • This probe detects a two- nt duplication.
  • This probe has the indel variation located at the +4 position with respect to the central nt in the oligonucleotide probe.
  • a similar oligonucleotide probe is also designed but this probe hybridizes to the other strand of the fragment to be analyzed.
  • one oligonucleotide probe where the bases of interest being the central base and its adjacent 2 base on the 5' side are duplicated respect to the normal or wild type allele. This probe detects a three -nt duplication. The indel variation is located at the "0" position within the probe. A similar oligonucleotide probe is also designed but this probe hybridizes to the other strand of the fragment to be analyzed.
  • one oligonucleotide probe where the three bases of interest are in an offset position of 4 nucleotides towards the 5' respect to the base in the central position and its adjacent base on the 5' side and are duplicated respect to the normal or wild type allele.
  • This probe detects a three - nt duplication.
  • This probe has the indel variation located at the -4 position with respect to the central nt in the oligonucleotide probe.
  • a similar oligonucleotide probe is also designed but this probe hybridizes to the other strand of the fragment to be analyzed.
  • the library of probes comprises of oligonucleotide oligos of three different length, 21 nt, 23 nt, and 25 nt, all of which have the indel variation located in central (0) position and oligonucleotide probes interrogating the sense and the anti-sense strand are represented.
  • one oligonucleotide probe where the three bases of interest are in an offset position of 4 nucleotides towards the 3' respect to the base in the central position and its adjacent base on the 5' side and are duplicated respect to the normal or wild type allele.
  • This probe detects a three - nt duplication.
  • This probe has the indel variation located at the +4 position with respect to the central nt in the oligonucleotide probe.
  • a similar oligonucleotide probe is also designed but this probe hybridizes to the other strand of the fragment to be analyzed.
  • LDLRex2 (27 68 123 30) _101.
  • _dO_f 4 ACGAGTTCCAGTGCCAAGACGGGAA 5 CG AGTTCCAGTG CCAAG ACG G G A 6 GAGTTCCAGTGCCAAGACGGG
  • LDLRex2(27 68 123 30) _106_ _dO_f 34 TTCCAGTGCCAAGACGGGAAATGCA 35 TCCAGTG CCAAG ACGG G AAATG C 36 CCAGTG CCAAG ACGG G AAATG
  • LDLRex2(27 68 123 30) _109. _dO_f 52 CAGTG CCAAG ACGG G AAATG CATCT 53 AGTG CCAAG ACGG G AAATG CATC 54 GTGCCAAGACGGGAAATGCAT
  • _dO_f 58 AGTG CCAAG ACGG G AAATG CATCTC 59 GTGCCAAGACGGGAAATGCATCT 60 TG CCAAG ACGG G AAATG CATC
  • _dO_r 79 TAGGAGATGCATTTCCCGTCTTGGC 80 AGGAGATGCATTTCCCGTCTTGG 81 G GAG ATG CATTTCCCGTCTTG
  • LDLRex2 (27 68 123 30) _121. _d0_f 124 GGGAAATGCATCTCCTACAAGTGGG 125 G G AAATG CATCTCCTACAAGTGG 126 GAAATGCATCTCCTACAAGTG
  • LDLRex2 (27 68 123 30) _121. _d0_r 127 CCCACTTGTAGGAGATGCATTTCCC 128 CCACTTGTAG GAG ATG CATTTCC 129 CACTTGTAG GAG ATG CATTTC
  • LDLRex2 (27 68 123 30) _123. _d0_f 136 GAAATGCATCTCCTACAAGTGGGTC 137 AAATG CATCTCCTACAAGTGG GT 138 AATGCATCTCCTACAAGTGGG
  • LDLRex2 (27 68 123 30) _125. _d0_r 151 CAGACCCACTTGTAGGAGATGCATT 152 AGACCCACTTGTAGGAGATGCAT 153 G ACCCACTTGTAG GAG ATG CA
  • LDLRex2 (27 68 123 30) _126. _d0_r 157 G CAG ACCCACTTGTAG GAG ATG CAT 158 CAGACCCACTTGTAGGAGATGCA 159 AGACCCACTTGTAGGAGATGC
  • LDLRex2 (27 68 123 30) _127. _d0_f 160 TGCATCTCCTACAAGTGGGTCTGCG 161 GCATCTCCTACAAGTGGGTCTGC 162 CATCTCCTACAAGTGGGTCTG
  • LDLRex2 (27 68 123 30) _128. _d0_f 166 GCATCTCCTACAAGTGGGTCTGCGA 167 CATCTCCTACAAGTG GGTCTG CG 168 ATCTCCTACAAGTG GGTCTG C
  • LDLRex2 (27 68 123 30) _128. _d0_r 169 TCG CAG ACCCACTTGTAG GAG ATG C 170 CG CAG ACCCACTTGTAG GAG ATG 171 G CAG ACCCACTTGTAG GAG AT
  • LDLRex2 (27 68 123 30) _129. _d0_r 175 ATCGCAGACCCACTTGTAGGAGATG 176 TCG CAG ACCCACTTGTAG GAG AT 177 CG CAG ACCCACTTGTAG GAGA
  • LDLRex2 (27 68 123 30) _130.
  • _d0_r 181 CATCGCAGACCCACTTGTAGGAGAT 182 ATCGCAGACCCACTTGTAGGAGA 183 TCG CAG ACCCACTTGTAG GAG
  • LDLRex2 (27 68 123 30) _131_ _d0_r 187 CCATCGCAGACCCACTTGTAGGAGA 188 CATCGCAGACCCACTTGTAGGAG 189 ATCGCAGACCCACTTGTAGGA
  • LDLRex2(27 68 123 30) _132_ _d0_f 190 CTCCTACAAGTG G GTCTG CG ATG GC 191 TCCTACAAGTGG GTCTG CG ATG G 192 CCTACAAGTG GGTCTG CG ATG
  • LDLRex2 (27 68 123 30) _133_ _d0_r 199 TG CCATCG CAG ACCCACTTGTAG G A 200 GCCATCGCAGACCCACTTGTAGG 201 CCATCGCAGACCCACTTGTAG
  • LDLRex2(27 68 123 30) _134_ _d0_f 202 CCTACAAGTG GGTCTG CG ATGG CAG 203 CTACAAGTG G GTCTG CG ATG GCA 204 TACAAGTGG GTCTG CG ATG GC
  • LDLRex2 (27 68 123 30) _136_ _d0_r 217 CGCTGCCATCGCAGACCCACTTGTA 218 GCTGCCATCGCAGACCCACTTGT 219 CTG CCATCG CAG ACCCACTTG
  • LDLRex2(27 68 123 30) _138. _d0_f 226 CAAGTGGGTCTGCGATGGCAGCGCT 227 AAGTGGGTCTGCGATGGCAGCGC 228 AGTG GGTCTG CG ATGG CAG CG
  • LDLRex2 (27 68 123 30) _138. _d0_r 229 AGCGCTGCCATCGCAGACCCACTTG 230 G CG CTG CCATCG CAG ACCCACTT 231 CGCTGCCATCGCAGACCCACT
  • LDLRex2 (27 68 123 30) _139.
  • _d0_f 232 AAGTGGGTCTGCGATGGCAGCGCTG 233 AGTGGGTCTGCGATGGCAGCGCT 234 GTGGGTCTGCGATGGCAGCGC
  • LDLRex2 (27 68 123 30) _139. _d0_r 235 CAGCGCTGCCATCGCAGACCCACTT 236 AGCGCTGCCATCGCAGACCCACT 237 G CGCTG CCATCG CAG ACCCAC
  • LDLRex2 (27 68 123 30) _141. _d0_f 244 GTGGGTCTGCGATGGCAGCGCTGAG 245 TGGGTCTGCGATGGCAGCGCTGA 246 GGGTCTGCGATGGCAGCGCTG
  • LDLRex2 (27 68 123 30) _141. _d0_r 247 CTCAGCGCTGCCATCGCAGACCCAC 248 TCAGCGCTGCCATCGCAGACCCA 249 CAGCGCTGCCATCGCAGACCC
  • LDLRex2 (27 68 123 30) _142. _d0_r 253 ACTCAG CGCTG CCATCG CAG ACCCA 254 CTCAGCGCTGCCATCGCAGACCC 255 TCAGCGCTGCCATCGCAGACC
  • LDLRex2 (27 68 123 30) _143. _d0_f 256 GGGTCTGCGATGGCAGCGCTGAGTG 257 GGTCTGCGATGGCAGCGCTGAGT 258 GTCTGCGATGGCAGCGCTGAG
  • LDLRex2 (27 68 123 30) _144. _d0_r 265 GCACTCAGCGCTGCCATCGCAGACC 266 CACTCAG CGCTG CCATCG CAG AC 267 ACTCAGCGCTGCCATCGCAGA
  • LDLRex2 (27 68 123 30) _145. _d0_f 268 GTCTGCGATGGCAGCGCTGAGTGCC 269 TCTGCGATGGCAGCGCTGAGTGC 270 CTGCGATGGCAGCGCTGAGTG
  • LDLRex2 (27 68 123 30) _145. _d0_r 271 GGCACTCAGCGCTGCCATCGCAGAC 272 GCACTCAGCGCTGCCATCGCAGA 273 CACTCAG CGCTG CCATCG CAG
  • LDLRex2 (27 68 123 30) _147. _d0_f 280 CTGCGATGGCAGCGCTGAGTGCCAG 281 TGCGATGGCAGCGCTGAGTGCCA 282 GCGATGGCAGCGCTGAGTGCC
  • LDLRex2 (27 68 123 30) _148_ _d0_f 286 TGCGATGGCAGCGCTGAGTGCCAGG 287 GCGATGGCAGCGCTGAGTGCCAG 288 CGATGGCAGCGCTGAGTGCCA
  • LDLRex2 (27 68 123 30) _149_ _d0_f 292 GCGATGGCAGCGCTGAGTGCCAGGA 293 CGATGGCAGCGCTGAGTGCCAGG 294 GATGGCAGCGCTGAGTGCCAG
  • LDLRex2 (27 68 123 30) _150.
  • _d0_f 298 CGATGGCAGCGCTGAGTGCCAGGAT 299 GATGGCAGCGCTGAGTGCCAGGA 300 ATGGCAGCGCTGAGTGCCAGG
  • LDLRex2 (27 68 123 30) _151. _d0_r 307 CATCCTGGCACTCAGCGCTGCCATC 308 ATCCTGGCACTCAGCGCTGCCAT 309 TCCTG GCACTCAG CGCTG CCA
  • LDLRex2 (27 68 123 30) _152.
  • _d0_r 313 CCATCCTGGCACTCAGCGCTGCCAT 314 CATCCTGGCACTCAGCGCTGCCA 315 ATCCTGGCACTCAGCGCTGCC
  • LDLRex2 (27 68 123 30) _154. _d0_f 322 GGCAGCGCTGAGTGCCAGGATGGCT 323 GCAGCGCTGAGTGCCAGGATGGC 324 CAGCGCTGAGTGCCAGGATGG
  • LDLRex2 (27 68 123 30) _154. _d0_r 325 AGCCATCCTGGCACTCAGCGCTGCC 326 G CCATCCTG GCACTCAG CGCTG C 327 CCATCCTGGCACTCAGCGCTG
  • LDLRex2 (27 68 123 30) _155. _d0_r 331 GAGCCATCCTGGCACTCAGCGCTGC 332 AGCCATCCTGGCACTCAGCGCTG 333 G CCATCCTG GCACTCAG CGCT
  • LDLRex2(27 68 123 30) _156. _d0_f 334 CAGCGCTGAGTGCCAGGATGGCTCT 335 AGCGCTGAGTGCCAGGATGGCTC 336 GCGCTGAGTGCCAGGATGGCT
  • LDLRex2 (27 68 123 30) _156. _d0_r 337 AGAGCCATCCTGGCACTCAGCGCTG 338 GAGCCATCCTGGCACTCAGCGCT 339 AGCCATCCTGGCACTCAGCGC
  • LDLRex2 (27 68 123 30) _157. _d0_r 343 CAGAGCCATCCTGGCACTCAGCGCT 344 AGAGCCATCCTGGCACTCAGCGC 345 GAGCCATCCTGGCACTCAGCG
  • LDLRex2 (27 68 123 30) _158.
  • _d0_f 346 GCGCTGAGTGCCAGGATGGCTCTGA 347 CGCTG AGTG CCAG G ATG GCTCTG 348 G CTG AGTGCCAG G ATG GCTCT
  • LDLRex2 (27 68 123 30) _159. _d0_r 355 ATCAGAGCCATCCTGGCACTCAGCG 356 TCAGAGCCATCCTGGCACTCAGC 357 CAGAGCCATCCTGGCACTCAG
  • _d0_f 358 GCTGAGTGCCAGGATGGCTCTGATG 359 CTGAGTGCCAGGATGGCTCTGAT 360 TGAGTGCCAGGATGGCTCTGA
  • LDLRex2 (27 68 123 30) _161. _d0_f 364 CTGAGTGCCAGGATGGCTCTGATGA 365 TGAGTGCCAGGATGGCTCTGATG 366 GAGTGCCAGGATGGCTCTGAT
  • LDLRex2 (27 68 123 30) _161. _d0_r 367 TCATCAGAGCCATCCTGGCACTCAG 368 CATCAGAGCCATCCTGGCACTCA 369 ATCAGAGCCATCCTGGCACTC
  • _d0_f 370 TGAGTGCCAGGATGGCTCTGATGAG 371 GAGTGCCAGGATGGCTCTGATGA 372 AGTGCCAGGATGGCTCTGATG
  • LDLRex2 (27 68 123 30) _162. _d0_r 373 CTCATCAGAGCCATCCTGGCACTCA 374 TCATCAGAGCCATCCTGGCACTC 375 CATCAGAGCCATCCTGGCACT
  • LDLRex2 (27 68 123 30) _165. _d0_f 388 GTGCCAGGATGGCTCTGATGAGTCC 389 TGCCAGGATGGCTCTGATGAGTC 390 GCCAGGATGGCTCTGATGAGT
  • LDLRex2 (27 68 123 30) _166.
  • _d0_f 394 TGCCAGGATGGCTCTGATGAGTCCC 395 GCCAGGATGGCTCTGATGAGTCC 396 CCAGGATGGCTCTGATGAGTC
  • LDLRex2 (27 68 123 30) _168.
  • _d0_f 406 CCAGGATGGCTCTGATGAGTCCCAG 407
  • CAGGATGGCTCTGATGAGTCCCA 408 AGGATGGCTCTGATGAGTCCC
  • LDLRex2 (27 68 123 30) _169.
  • _d0_f 412 CAGGATGGCTCTGATGAGTCCCAGG 413 AGGATGGCTCTGATGAGTCCCAG 414 GGATGGCTCTGATGAGTCCCA
  • _d0_f 418 AGGATGGCTCTGATGAGTCCCAGGA 419 GGATGGCTCTGATGAGTCCCAGG 420 GATGGCTCTGATGAGTCCCAG
  • LDLRex2 (27 68 123 30) _171.
  • _d0_f 424 GGATGGCTCTGATGAGTCCCAGGAG 425 GATGGCTCTGATGAGTCCCAGGA 426 ATGGCTCTGATGAGTCCCAGG
  • LDLRex2 (27 68 123 30) _171. _d0_r 427 CTCCTGGGACTCATCAGAGCCATCC 428 TCCTGGGACTCATCAGAGCCATC 429 CCTGGGACTCATCAGAGCCAT
  • LDLRex2 (27 68 123 30) _173.
  • _d0_f 436 ATGGCTCTGATGAGTCCCAGGAGAC 437 TGGCTCTGATGAGTCCCAGGAGA 438 GGCTCTGATGAGTCCCAGGAG
  • LDLRex2 (27 68 123 30) _173.
  • _d0_r 439 GTCTCCTGGGACTCATCAGAGCCAT 440 TCTCCTGGGACTCATCAGAGCCA 441 CTCCTGGGACTCATCAGAGCC
  • LDLRex2 (27 68 123 30) _174.
  • _d0_f 442 TGGCTCTGATGAGTCCCAGGAGACG 443 GGCTCTGATGAGTCCCAGGAGAC 444 G CTCTG ATG AGTCCCAG GAGA
  • LDLRex2 (27 68 123 30) _174. _d0_r 445 CGTCTCCTGGGACTCATCAGAGCCA 446 GTCTCCTGGGACTCATCAGAGCC 447 TCTCCTGGGACTCATCAGAGC
  • LDLRex2 (27 68 123 30) _175.
  • _d0_f 448 GGCTCTGATGAGTCCCAGGAGACGT 449 G CTCTG ATG AGTCCCAG G AG ACG 450 CTCTGATGAGTCCCAGGAGAC
  • LDLRex2 (27 68 123 30) _175. _d0_r 451 ACGTCTCCTGGGACTCATCAGAGCC 452 CGTCTCCTGGGACTCATCAGAGC 453 GTCTCCTGGGACTCATCAGAG
  • LDLRex2 (27 68 123 30) _176.
  • _d0_f 454 G CTCTG ATG AGTCCCAG GAG ACGTG 455 CTCTGATGAGTCCCAGGAGACGT 456 TCTGATGAGTCCCAGGAGACG
  • LDLRex2 (27 68 123 30) _176. _d0_r 457 CACGTCTCCTGGGACTCATCAGAGC 458 ACGTCTCCTGGGACTCATCAGAG 459 CGTCTCCTGGGACTCATCAGA
  • LDLRex2 (27 68 123 30) _177. _d0_f 460 CTCTGATGAGTCCCAGGAGACGTGC 461 TCTGATGAGTCCCAGGAGACGTG 462 CTGATGAGTCCCAGGAGACGT
  • _d0_r 463 GCACGTCTCCTGGGACTCATCAGAG 464 CACGTCTCCTGGGACTCATCAGA 465 ACGTCTCCTGGGACTCATCAG
  • LDLRex2 (27 68 123 30) _179_d0_f 472 CTGATGAGTCCCAGGAGACGTGCTG 473 TGATGAGTCCCAGGAGACGTGCT 474 GATGAGTCCCAGGAGACGTGC
  • LDLRex2(27 68 123 30) _179_d0_r 475 CAGCACGTCTCCTGGGACTCATCAG 476 AGCACGTCTCCTGGGACTCATCA 477 G CACGTCTCCTGG GACTCATC
  • LDLRex2(27 68 123 30) _183_d0_r 499 CTCACAG CACGTCTCCTGG GACTCA 500 TCACAG CACGTCTCCTGG GACTC 501 CACAG CACGTCTCCTGG G ACT
  • LDLRex2(27 68 123 30) _185_d0_f 508 AGTCCCAGGAGACGTGCTGTGAGTC 509 GTCCCAG GAG ACGTG CTGTG AGT 510 TCCCAGGAGACGTGCTGTGAG
  • LDLRex2(27 68 123 30) _189_d0_r 535 AGGGGACTCACAGCACGTCTCCTGG 536 GGGGACTCACAGCACGTCTCCTG 537 GGGACTCACAGCACGTCTCCT
  • LDLRex2(27 68 123 30) _190 + 2_d0_r 583 CAAAGGGGACTCACAGCACGTCTCC 584 AAAGGGGACTCACAGCACGTCTC 585 AAGGGGACTCACAGCACGTCT
  • LDLRex2(27 68 123 30) _190_d0_f 628 CAGGAGACGTGCTGTGAGTCCCCTT 629 AGGAGACGTGCTGTGAGTCCCCT 630 GGAGACGTGCTGTGAGTCCCC
  • LDLRex2(27;68;123;30) _68 - 13 _dO_ r 661 CTGAGAGAGGAAAAGGAGAAAGG 662 TGAGAGAGGAAAAGGAGAAAG 663 GAGAGAGAGGAAAAGGAGAAA
  • LDLRex2 (27 68 123 30) _73. _d0_r 757 TTTCGCATCTGTCGCCCACTGAGAG 758 TTCGCATCTGTCGCCCACTGAGA 759 TCG CATCTGTCG CCCACTG AG
  • _d0_f 766 CTCAGTGGGCGACAGATGCGAAAGA 767 TCAGTGGGCGACAGATGCGAAAG 768 CAGTGGGCGACAGATGCGAAA
  • LDLRex2 (27 68 123 30) _75. _d0_r 769 TCTTTCGCATCTGTCGCCCACTGAG 770 CTTTCG CATCTGTCG CCCACTG A 771 TTTCGCATCTGTCGCCCACTG
  • LDLRex2 (27 68 123 30 _79. _d0_f 790 GTGGGCGACAGATGCGAAAGAAACG 791 TGGGCGACAGATGCGAAAGAAAC 792 GGGCGACAGATGCGAAAGAAA
  • LDLRex2 (27 68 123 30 _79. _d0_r 793 CGTTTCTTTCGCATCTGTCGCCCAC 794 GTTTCTTTCGCATCTGTCGCCCA 795 TTTCTTTCG CATCTGTCG CCC
  • LDLRex2 (27 68 123 30 _80. _d0_f 796 TGGGCGACAGATGCGAAAGAAACGA 797 GGGCGACAGATGCGAAAGAAACG 798 GGCGACAGATGCGAAAGAAAC
  • LDLRex2 (27 68 123 30 _80. _d0_r 799 TCGTTTCTTTCGCATCTGTCGCCCA 800 CGTTTCTTTCGCATCTGTCGCCC 801 GTTTCTTTCGCATCTGTCGCC
  • LDLRex2 (27 68 123 30 _81. _d0_f 802 GGGCGACAGATGCGAAAGAAACGAG 803 GGCGACAGATGCGAAAGAAACGA 804 GCGACAGATGCGAAAGAAACG
  • LDLRex2 (27 68 123 30 _81. _d0_r 805 CTCGTTTCTTTCGCATCTGTCGCCC 806 TCGTTTCTTTCGCATCTGTCGCC 807 CGTTTCTTTCGCATCTGTCGC
  • LDLRex2 (27 68 123 30 _82. _d0_f 808 GGCGACAGATGCGAAAGAAACGAGT 809 GCGACAGATGCGAAAGAAACGAG 810 CGACAGATGCGAAAGAAACGA
  • LDLRex2 (27 68 123 30 _82. _d0_r 811 ACTCGTTTCTTTCG CATCTGTCG CC 812 CTCGTTTCTTTCGCATCTGTCGC 813 TCGTTTCTTTCGCATCTGTCG
  • LDLRex2 (27 68 123 30 _83. _d0_f 814 GCGACAGATGCGAAAGAAACGAGTT 815 CGACAGATGCGAAAGAAACGAGT 816 GACAGATGCGAAAGAAACGAG
  • LDLRex2 (27 68 123 30 _83. _d0_r 817 AACTCGTTTCTTTCGCATCTGTCGC 818 ACTCGTTTCTTTCG CATCTGTCG 819 CTCGTTTCTTTCGCATCTGTC
  • LDLRex2 (27 68 123 30 _84. _d0_f 820 CGACAGATGCGAAAGAAACGAGTTC 821 GACAGATGCGAAAGAAACGAGTT 822 ACAGATGCGAAAGAAACGAGT
  • LDLRex2 (27 68 123 30 _84. _d0_r 823 GAACTCGTTTCTTTCGCATCTGTCG 824 AACTCGTTTCTTTCGCATCTGTC 825 ACTCGTTTCTTTCG CATCTGT
  • LDLRex2 (27 68 123 30 _85. _d0_f 826 GACAGATGCGAAAGAAACGAGTTCC 827 ACAGATGCGAAAGAAACGAGTTC 828 CAG ATG CG AAAG AAACG AGTT
  • LDLRex2 (27 68 123 30 _85. _d0_r 829 GGAACTCGTTTCTTTCGCATCTGTC 830 GAACTCGTTTCTTTCGCATCTGT 831 AACTCGTTTCTTTCGCATCTG
  • LDLRex2 (27 68 123 30 _86. _d0_f 832 ACAGATGCGAAAGAAACGAGTTCCA 833 CAGATGCGAAAGAAACGAGTTCC 834 AGATGCGAAAGAAACGAGTTC
  • LDLRex2 (27 68 123 30) _89. _d0_f 850 GATGCGAAAGAAACGAGTTCCAGTG 851 ATGCGAAAGAAACGAGTTCCAGT 852 TGCGAAAGAAACGAGTTCCAG
  • LDLRex2 (27 68 123 30) _91. _d0_r 865 GGCACTGGAACTCGTTTCTTTCGCA 866 G CACTG G AACTCGTTTCTTTCGC 867 CACTGGAACTCGTTTCTTTCG
  • LDLRex2 (27 68 123 30) _93. _d0_f 874 CGAAAGAAACGAGTTCCAGTGCCAA 875 GAAAGAAACGAGTTCCAGTGCCA 876 AAAGAAACGAGTTCCAGTGCC
  • LDLRex2 (27 68 123 30) _94. _d0_f 880 GAAAGAAACGAGTTCCAGTGCCAAG 881 AAAG AAACG AGTTCCAGTG CCAA 882 AAGAAACGAGTTCCAGTGCCA
  • LDLRex2 (27 68 123 30) _95. _d0_f 886 AAAG AAACG AGTTCCAGTG CCAAG A 887 AAGAAACGAGTTCCAGTGCCAAG 888 AGAAACGAGTTCCAGTGCCAA
  • LDLRex2 (27 68 123 30) _96. _d0_f 892 AAGAAACGAGTTCCAGTGCCAAGAC 893 AGAAACGAGTTCCAGTGCCAAGA 894 GAAACGAGTTCCAGTGCCAAG
  • LDLRex2 (27 68 123 30) _97. _d0_f 898 AGAAACGAGTTCCAGTGCCAAGACG 899 GAAACGAGTTCCAGTGCCAAGAC 900 AAACGAGTTCCAGTGCCAAGA
  • LDLRex2 (27 68 123 30) _97. _d0_r 901 CGTCTTGGCACTGGAACTCGTTTCT 902 GTCTTGGCACTGGAACTCGTTTC 903 TCTTGG CACTG G AACTCGTTT
  • LDLRex2 (27 68 123 30) _98. _d0_f 904 GAAACGAGTTCCAGTGCCAAGACGG 905 AAACGAGTTCCAGTGCCAAGACG 906 AACGAGTTCCAGTGCCAAGAC
  • LDLRex2 (27 68 123 30) _98. _d0_r 907 CCGTCTTGG CACTG G AACTCGTTTC 908 CGTCTTGGCACTGGAACTCGTTT 909 GTCTTGGCACTGGAACTCGTT
  • LDLRex2 (27 68 123 30 _100. .dl_r 916 TTCCCGTCTTGGCCTGGAACTCGTT 917 TCCCGTCTTGGCCTGGAACTCGT 918 CCCGTCTTG GCCTG GAACTCG
  • LDLRex2 (27 68 123 30 _101_ .dlj 919 ACGAGTTCCAGTCCAAGACGGGAAA 920 CGAGTTCCAGTCCAAGACGGGAA 921 GAGTTCCAGTCCAAGACGGGA
  • LDLRex2 (27 68 123 30 _101_ .dl_r 922 TTTCCCGTCTTGGACTGGAACTCGT 923 TTCCCGTCTTGGACTGGAACTCG 924 TCCCGTCTTG GACTG GAACTC
  • LDLRex2 (27 68 123 30 _102. .dlj 925 CGAGTTCCAGTGCAAGACGGGAAAT 926 GAGTTCCAGTGCAAGACGGGAAA 927 AGTTCCAGTGCAAGACGGGAA
  • LDLRex2 (27 68 123 30 _102. .dl_r 928 ATTTCCCGTCTTG CACTG GAACTCG 929 TTTCCCGTCTTGCACTGGAACTC 930 TTCCCGTCTTGCACTGGAACT
  • LDLRex2 (27 68 123 30 _103. .dlj 931 GAGTTCCAGTGCAAGACGGGAAATG 932 AGTTCCAGTG CAAG ACGG G AAAT 933 GTTCCAGTGCAAGACGGGAAA
  • LDLRex2 (27 68 123 30 _103. .dl_r 934 CATTTCCCGTCTTG CACTG GAACTC 935 ATTTCCCGTCTTG CACTG G AACT 936 TTTCCCGTCTTGCACTGGAAC
  • LDLRex2 (27 68 123 30 _104 .dlj 937 AGTTCCAGTGCCAGACGGGAAATGC 938 GTTCCAGTGCCAGACGGGAAATG 939 TTCCAGTGCCAGACGGGAAAT
  • LDLRex2 (27 68 123 30 _104 .dl_r 940 G CATTTCCCGTCTG GCACTG G AACT 941 CATTTCCCGTCTGGCACTGGAAC 942 ATTTCCCGTCTGGCACTGGAA
  • LDLRex2 (27 68 123 30 _105. .dlj 943 GTTCCAGTGCCAGACGGGAAATGCA 944 TTCCAGTGCCAGACGGGAAATGC 945 TCCAGTGCCAGACGGGAAATG
  • LDLRex2 (27 68 123 30 _105. .dl_r 946 TG CATTTCCCGTCTGG CACTG G AAC 947 GCATTTCCCGTCTGGCACTGGAA 948 CATTTCCCGTCTGG CACTG G A
  • LDLRex2 (27 68 123 30 _106. .dlj 949 TTCCAGTGCCAAACGGGAAATGCAT 950 TCCAGTGCCAAACG G G AAATG CA 951 CCAGTGCCAAACGGGAAATGC
  • LDLRex2 (27 68 123 30 _106. .dl_r 952 ATGCATTTCCCGTTTGGCACTGGAA 953 TG CATTTCCCGTTTGG CACTG G A 954 G CATTTCCCGTTTG G CACTG G
  • LDLRex2 (27 68 123 30 _107. .dlj 955 TCCAGTGCCAAGCGGGAAATGCATC 956 CCAGTGCCAAGCGGGAAATGCAT 957 CAGTGCCAAGCGGGAAATGCA
  • LDLRex2 (27 68 123 30 _107. .dl_r 958 GATG CATTTCCCGCTTG G CACTG G A 959 ATGCATTTCCCGCTTGGCACTGG 960 TGCATTTCCCGCTTGGCACTG
  • LDLRex2 (27 68 123 30 _108. .dlj 961 CCAGTG CCAAG AGG G AAATG CATCT 962 CAGTG CCAAG AGG G AAATG CATC 963 AGTG CCAAG AGG G AAATG CAT
  • LDLRex2 (27 68 123 30 _108. .dl_r 964 AGATGCATTTCCCTCTTGGCACTGG 965 GATGCATTTCCCTCTTGGCACTG 966 ATGCATTTCCCTCTTG G CACT
  • LDLRex2 (27 68 123 30 _109. .dlj 967 CAGTGCCAAGACGGAAATGCATCTC 968 AGTGCCAAGACGGAAATGCATCT 969 GTGCCAAGACGGAAATGCATC
  • LDLRex2 (27 68 123 30 _109. .dl_r 970 G AG ATG C ATTTCCGTCTTG G CACTG 971 AGATGCATTTCCGTCTTGGCACT 972 GATG CATTTCCGTCTTGG CAC
  • LDLRex2 (27 68 123 30 _110. .dlj 973 AGTGCCAAGACGGAAATGCATCTCC 974 GTGCCAAGACGGAAATGCATCTC 975 TGCCAAGACGGAAATGCATCT
  • LDLRex2 (27 68 123 30 _110. .dl_r 976 GGAGATGCATTTCCGTCTTGGCACT 977 GAGATGCATTTCCGTCTTGGCAC 978 AGATGCATTTCCGTCTTGGCA
  • LDLRex2 (27 68 123 30 _111_ .dlj 979 GTGCCAAGACGGAAATGCATCTCCT 980 TGCCAAGACGGAAATGCATCTCC 981 GCCAAGACGGAAATGCATCTC
  • LDLRex2 (27 68 123 30 _111_ .dl_r 982 AG GAG ATG CATTTCCGTCTTGG CAC 983 GGAGATGCATTTCCGTCTTGGCA 984 GAG ATG CATTTCCGTCTTG G C
  • LDLRex2 (27 68 123 30) _113. .dlj 991 GCCAAGACGGGAATGCATCTCCTAC 992 CCAAGACGGGAATGCATCTCCTA 993 CAAGACGGGAATGCATCTCCT
  • LDLRex2 (27 68 123 30) _114. .dlj 997 CCAAGACGGGAATGCATCTCCTACA 998 CAAGACGGGAATGCATCTCCTAC 999 AAGACGGGAATGCATCTCCTA
  • LDLRex2 (27 68 123 30) _115. .dlj 1003 CAAGACGGGAAAGCATCTCCTACAA 1004 AAG ACGG G AAAG CATCTCCTACA 1005 AGACGGGAAAGCATCTCCTAC
  • LDLRex2 (27 68 123 30 _116_ .dlj 1009 AAGACGGGAAATCATCTCCTACAAG 1010 AGACGGGAAATCATCTCCTACAA 1011 GACGGGAAATCATCTCCTACA
  • LDLRex2 (27 68 123 30 _116_ .dl_r 1012 CTTGTAGGAGATGATTTCCCGTCTT 1013 TTGTAGGAGATGATTTCCCGTCT 1014 TGTAG G AG ATG ATTTCCCGTC
  • LDLRex2 (27 68 123 30 _117. .dlj 1015 AGACGGGAAATGATCTCCTACAAGT 1016 GACGGGAAATGATCTCCTACAAG 1017 ACGGGAAATGATCTCCTACAA
  • LDLRex2 (27 68 123 30 _117. .dl_r 1018 ACTTGTAGGAGATCATTTCCCGTCT 1019 CTTGTAG G AG ATCATTTCCCGTC 1020 TTGTAG G AG ATCATTTCCCGT
  • LDLRex2 (27 68 123 30 _118. .dlj 1021 G ACGG G AAATG CTCTCCTACAAGTG 1022 ACGGGAAATGCTCTCCTACAAGT 1023 CG G G AAATG CTCTCCT AC A AG
  • LDLRex2 (27 68 123 30 _118. .dl_r 1024 CACTTGTAGGAGAGCATTTCCCGTC 1025 ACTTGTAGGAGAGCATTTCCCGT 1026 CTTGTAGGAGAGCATTTCCCG
  • LDLRex2 (27 68 123 30 _119. .dlj 1027 ACGGGAAATGCACTCCTACAAGTGG 1028 CGGGAAATGCACTCCTACAAGTG 1029 GGGAAATGCACTCCTACAAGT
  • LDLRex2 (27 68 123 30 _119. .dl_r 1030 CCACTTGTAGGAGTGCATTTCCCGT 1031 CACTTGTAGGAGTGCATTTCCCG 1032 ACTTGTAGGAGTGCATTTCCC
  • LDLRex2 (27 68 123 30 _120. .dlj 1033 CGGGAAATGCATTCCTACAAGTGGG 1034 GGGAAATGCATTCCTACAAGTGG 1035 G G AAATG CATTCCTACAAGTG
  • LDLRex2 (27 68 123 30 _120. .dl_r 1036 CCCACTTGTAGGAATGCATTTCCCG 1037 CCACTTGTAGGAATGCATTTCCC 1038 CACTTGTAGGAATGCATTTCC
  • LDLRex2 (27 68 123 30 _121_ .dlj 1039 GGGAAATGCATCCCTACAAGTGGGT 1040 G G AAATG CATCCCTACAAGTGG G 1041 GAAATGCATCCCTACAAGTGG
  • LDLRex2 (27 68 123 30 _121_ .dl_r 1042 ACCCACTTGTAGG G ATG CATTTCCC 1043 CCCACTTGTAGGGATGCATTTCC 1044 CCACTTGTAGGGATGCATTTC
  • LDLRex2 (27 68 123 30 _122. .dlj 1045 G G AAATG CATCTCTACAAGTGG GTC 1046 GAAATGCATCTCTACAAGTGGGT 1047 AAATG CATCTCTACAAGTGG G
  • LDLRex2 (27 68 123 30 _122. .dl_r 1048 GACCCACTTGTAGAGATGCATTTCC 1049 ACCCACTTGTAGAGATGCATTTC 1050 CCCACTTGTAGAGATGCATTT
  • LDLRex2 (27 68 123 30 _123. .dlj 1051 GAAATGCATCTCTACAAGTGGGTCT 1052 AAATG CATCTCTACAAGTGG GTC 1053 AATG CATCTCTACAAGTG GGT
  • LDLRex2 (27 68 123 30 _123. .dl_r 1054 AG ACCCACTTGTAG AG ATG CATTTC 1055 GACCCACTTGTAGAGATGCATTT 1056 ACCCACTTGTAGAGATGCATT
  • LDLRex2 (27 68 123 30 _124. .dlj 1057 AAATG CATCTCCACAAGTG GGTCTG 1058 AATG CATCTCCACAAGTGG GTCT 1059 ATGCATCTCCACAAGTGGGTC
  • LDLRex2 (27 68 123 30 _124. .dl_r 1060 CAGACCCACTTGTGGAGATGCATTT 1061 AGACCCACTTGTGGAGATGCATT 1062 G ACCCACTTGTG GAG ATG CAT
  • LDLRex2 (27 68 123 30 _125_ .dl_r 1066 GCAGACCCACTTGAGGAGATGCATT 1067 CAGACCCACTTGAGGAGATGCAT 1068 AGACCCACTTGAGGAGATGCA
  • LDLRex2 (27 68 123 30 _126_ .dlj 1069 ATGCATCTCCTAAAGTGGGTCTGCG 1070 TGCATCTCCTAAAGTGGGTCTGC 1071 GCATCTCCTAAAGTGGGTCTG
  • LDLRex2 (27 68 123 30 _126_ .dl_r 1072 CGCAG ACCCACTTTAG GAG ATG CAT 1073 GCAG ACCCACTTTAG GAG ATG CA 1074 CAGACCCACTTTAGGAGATGC
  • LDLRex2 (27 68 123 30) _130. .dl_r 1096 CCATCGCAGACCCCTTGTAGGAGAT 1097 CATCGCAGACCCCTTGTAGGAGA 1098 ATCGCAGACCCCTTGTAGGAG
  • LDLRex2 (27 68 123 30 _131_ .dl_r 1102 GCCATCGCAGACCACTTGTAGGAGA 1103 CCATCG CAG ACCACTTGTAG GAG 1104 CATCG CAG ACCACTTGTAG G A
  • LDLRex2 (27 68 123 30 _132_ .dlj 1105 CTCCTACAAGTG GTCTG CG ATG GCA 1106 TCCTACAAGTG GTCTG CG ATG GC 1107 CCTACAAGTGGTCTGCGATGG
  • LDLRex2 (27 68 123 30 _132_ .dl_r 1108 TGCCATCGCAGACCACTTGTAGGAG 1109 GCCATCGCAGACCACTTGTAGGA 1110 CCATCG CAG ACCACTTGTAG G
  • LDLRex2 (27 68 123 30 _133. .dlj 1111 TCCTACAAGTG GTCTG CG ATG GCAG 1112 CCTACAAGTGGTCTGCGATGGCA 1113 CTACAAGTGGTCTGCGATGGC
  • LDLRex2 (27 68 123 30 _133. .dl_r 1114 CTGCCATCGCAGACCACTTGTAGGA 1115 TGCCATCGCAGACCACTTGTAGG 1116 G CCATCG CAG ACCACTTGTAG
  • LDLRex2 (27 68 123 30 _134 .dlj 1117 CCTACAAGTGGGCTGCGATGGCAGC 1118 CTACAAGTGGGCTGCGATGGCAG 1119 TACAAGTGGGCTGCGATGGCA
  • LDLRex2 (27 68 123 30 _134 .dl_r 1120 G CTG CCATCG CAG CCCACTTGTAG G 1121 CTGCCATCGCAGCCCACTTGTAG 1122 TGCCATCGCAGCCCACTTGTA
  • LDLRex2 (27 68 123 30 _135. .dlj 1123 CTACAAGTGGGTTGCGATGGCAGCG 1124 TACAAGTGGGTTGCGATGGCAGC 1125 ACAAGTGGGTTGCGATGGCAG
  • LDLRex2 (27 68 123 30 _135. .dl_r 1126 CGCTGCCATCGCAACCCACTTGTAG 1127 GCTGCCATCGCAACCCACTTGTA 1128 CTG CCATCG CAACCCACTTGT
  • LDLRex2 (27 68 123 30 _136. .dlj 1129 TACAAGTGGGTCGCGATGGCAGCGC 1130 ACAAGTGGGTCGCGATGGCAGCG 1131 CAAGTGGGTCGCGATGGCAGC
  • LDLRex2 (27 68 123 30 _136. .dl_r 1132 GCGCTG CCATCG CG ACCCACTTGTA 1133 CGCTGCCATCGCGACCCACTTGT 1134 GCTGCCATCGCGACCCACTTG
  • LDLRex2 (27 68 123 30 _137_ .dlj 1135 ACAAGTGGGTCTCGATGGCAGCGCT 1136 CAAGTGGGTCTCGATGGCAGCGC 1137 AAGTGGGTCTCGATGGCAGCG
  • LDLRex2 (27 68 123 30 _137_ .dl_r 1138 AGCGCTGCCATCGAGACCCACTTGT 1139 GCGCTGCCATCGAGACCCACTTG 1140 CGCTGCCATCGAGACCCACTT
  • LDLRex2 (27 68 123 30 _138. .dlj 1141 CAAGTGGGTCTGGATGGCAGCGCTG 1142 AAGTGGGTCTGGATGGCAGCGCT 1143 AGTGGGTCTGGATGGCAGCGC
  • LDLRex2 (27 68 123 30 _138. .dl_r 1144 CAGCGCTGCCATCCAGACCCACTTG 1145 AGCGCTGCCATCCAGACCCACTT 1146 GCGCTG CCATCCAG ACCCACT
  • LDLRex2 (27 68 123 30 _139. .dlj 1147 AAGTGGGTCTGCATGGCAGCGCTGA 1148 AGTGGGTCTGCATGGCAGCGCTG 1149 GTGGGTCTGCATGGCAGCGCT
  • LDLRex2 (27 68 123 30 _139. .dl_r 1150 TCAGCGCTGCCATGCAGACCCACTT 1151 CAGCGCTGCCATGCAGACCCACT 1152 AGCGCTGCCATGCAGACCCAC
  • LDLRex2 (27 68 123 30 _140. .dlj 1153 AGTGGGTCTGCGTGGCAGCGCTGAG 1154 GTGGGTCTGCGTGGCAGCGCTGA 1155 TGGGTCTGCGTGGCAGCGCTG
  • LDLRex2 (27 68 123 30 _140. .dl_r 1156 CTCAGCGCTGCCACGCAGACCCACT 1157 TCAGCGCTGCCACGCAGACCCAC 1158 CAG CGCTG CCACG CAG ACCCA
  • LDLRex2 (27 68 123 30 _141_ .dlj 1159 GTGGGTCTGCGAGGCAGCGCTGAGT 1160 TGGGTCTGCGAGGCAGCGCTGAG 1161 GGGTCTGCGAGGCAGCGCTGA
  • LDLRex2 (27 68 123 30 _141_ .dl_r 1162 ACTCAG CG CTG CCTCGCAG ACCCAC 1163 CTCAGCGCTGCCTCGCAGACCCA 1164 TCAGCGCTGCCTCGCAGACCC
  • LDLRex2 (27 68 123 30 _142. .dlj 1165 TGGGTCTGCGATGCAGCGCTGAGTG 1166 GGGTCTGCGATGCAGCGCTGAGT 1167 GGTCTGCGATGCAGCGCTGAG
  • LDLRex2 (27 68 123 30 _142. .dl_r 1168 CACTCAGCGCTGCATCGCAGACCCA 1169 ACTCAG CG CTG CATCG CAG ACCC 1170 CTCAGCGCTGCATCGCAGACC
  • LDLRex2 (27 68 123 30) _143. .dlj 1171 GGGTCTGCGATGCAGCGCTGAGTGC 1172 GGTCTGCGATGCAGCGCTGAGTG 1173 GTCTGCGATGCAGCGCTGAGT
  • LDLRex2 (27 68 123 30) _143. .dl_r 1174 GCACTCAGCGCTGCATCGCAGACCC 1175 CACTCAGCGCTGCATCGCAGACC 1176 ACTCAGCGCTGCATCGCAGAC
  • LDLRex2 (27 68 123 30 _147_ .dlj 1195 CTGCGATGGCAGGCTGAGTGCCAGG 1196 TGCGATGGCAGGCTGAGTGCCAG 1197 GCGATGGCAGGCTGAGTGCCA
  • LDLRex2 (27 68 123 30 _147_ .dl_r 1198 CCTGG CACTCAGCCTG CCATCG CAG 1199 CTGG CACTCAGCCTG CCATCG CA 1200 TGGCACTCAGCCTGCCATCGC
  • LDLRex2 (27 68 123 30 _148_ .dlj 1201 TGCG ATG G CAG CCTG AGTG CCAG G A 1202 GCGATGGCAGCCTGAGTGCCAGG 1203 CGATGGCAGCCTGAGTGCCAG
  • LDLRex2 (27 68 123 30 _148_ .dl_r 1204 TCCTGGCACTCAGGCTGCCATCGCA 1205 CCTGGCACTCAGGCTGCCATCGC 1206 CTGGCACTCAGGCTGCCATCG
  • LDLRex2 (27 68 123 30 _149_ .dlj 1207 GCGATGGCAGCGTGAGTGCCAGGAT 1208 CGATGGCAGCGTGAGTGCCAGGA 1209 GATGGCAGCGTGAGTGCCAGG
  • LDLRex2 (27 68 123 30 _149_ .dl_r 1210 ATCCTGGCACTCACGCTGCCATCGC 1211 TCCTGGCACTCACGCTGCCATCG 1212 CCTG GCACTCACG CTG CCATC
  • LDLRex2 (27 68 123 30 _150_ .dlj 1213 CGATGGCAGCGCGAGTGCCAGGATG 1214 GATGGCAGCGCGAGTGCCAGGAT 1215 ATGGCAGCGCGAGTGCCAGGA
  • LDLRex2 (27 68 123 30 _150_ .dl_r 1216 CATCCTGGCACTCGCGCTGCCATCG 1217 ATCCTGGCACTCGCGCTGCCATC 1218 TCCTGG CACTCG CG CTG CCAT
  • LDLRex2 (27 68 123 30 _151_ .dlj 1219 GATGGCAGCGCTAGTGCCAGGATGG 1220 ATGGCAGCGCTAGTGCCAGGATG 1221 TGG CAG CG CTAGTG CCAG GAT
  • LDLRex2 (27 68 123 30 _151_ .dl_r 1222 CCATCCTGG CACTAG CG CTG CCATC 1223 CATCCTG GCACTAG CGCTG CCAT 1224 ATCCTGGCACTAGCGCTGCCA
  • LDLRex2 (27 68 123 30 _152_ .dlj 1225 ATGGCAGCGCTGGTGCCAGGATGGC 1226 TG G CAG CG CTG GTG CCAG GATGG 1227 GGCAGCGCTGGTGCCAGGATG
  • LDLRex2 (27 68 123 30 _152_ .dl_r 1228 G CCATCCTG GCACCAG CGCTG CCAT 1229 CCATCCTGGCACCAGCGCTGCCA 1230 CATCCTGGCACCAGCGCTGCC
  • LDLRex2 (27 68 123 30 _153. .dlj 1231 TGGCAGCGCTGATGCCAGGATGGCT 1232 GGCAGCGCTGATGCCAGGATGGC 1233 GCAGCGCTGATGCCAGGATGG
  • LDLRex2 (27 68 123 30 _153. .dl_r 1234 AGCCATCCTGG CATCAG CG CTG CCA 1235 G CCATCCTG GCATCAG CGCTG CC 1236 CCATCCTGGCATCAGCGCTGC
  • LDLRex2 (27 68 123 30 _154. .dlj 1237 GGCAGCGCTGAGGCCAGGATGGCTC 1238 GCAGCGCTGAGGCCAGGATGGCT 1239 CAGCGCTGAGGCCAGGATGGC
  • LDLRex2 (27 68 123 30 _154. .dl_r 1240 GAGCCATCCTGGCCTCAGCGCTGCC 1241 AGCCATCCTGGCCTCAGCGCTGC 1242 GCCATCCTGGCCTCAGCTG
  • LDLRex2 (27 68 123 30 _155. .dlj 1243 GCAGCGCTGAGTCCAGGATGGCTCT 1244 CAGCGCTGAGTCCAGGATGGCTC 1245 AGCGCTGAGTCCAGGATGGCT
  • LDLRex2 (27 68 123 30 _155. .dl_r 1246 AGAGCCATCCTGGACTCAGCGCTGC 1247 GAG CCATCCTG GACTCAG CGCTG 1248 AGCCATCCTGGACTCAGCGCT
  • LDLRex2(27 68 123 30 _156. .dlj 1249 CAG CGCTG AGTG CAG G ATG GCTCTG 1250 AGCG CTG AGTG CAG G ATG GCTCT 1251 GCGCTGAGTGCAGGATGGCTC
  • LDLRex2 (27 68 123 30 _156. .dl_r 1252 CAGAGCCATCCTGCACTCAGCGCTG 1253 AGAGCCATCCTGCACTCAGCGCT 1254 GAG CCATCCTG CACTCAGCG C
  • LDLRex2 (27 68 123 30 _157_ .dlj 1255 AGCGCTGAGTGCAGGATGGCTCTGA 1256 GCGCTGAGTGCAGGATGGCTCTG 1257 CGCTGAGTGCAGGATGGCTCT
  • LDLRex2 (27 68 123 30 _157_ .dl_r 1258 TCAGAGCCATCCTGCACTCAGCGCT 1259 CAGAGCCATCCTGCACTCAGCGC 1260 AGAGCCATCCTGCACTCAGCG
  • LDLRex2 (27 68 123 30 _158. .dlj 1261 GCGCTGAGTGCCGGATGGCTCTGAT 1262 CGCTGAGTGCCGGATGGCTCTGA 1263 GCTGAGTGCCGGATGGCTCTG
  • LDLRex2 (27 68 123 30 _158. .dl_r 1264 ATCAGAGCCATCCGGCACTCAGCGC 1265 TCAGAGCCATCCGGCACTCAGCG 1266 CAGAGCCATCCGGCACTCAGC
  • LDLRex2(27 68 123 30) _159. .dl_r 1270 CATCAG AG CCATCTG GCACTCAG CG 1271 ATCAG AG CCATCTG GCACTCAG C 1272 TCAGAGCCATCTGGCACTCAG
  • LDLRex2 (27 68 123 30) _161. .dlj 1279 CTGAGTGCCAGGTGGCTCTGATGAG 1280 TGAGTGCCAGGTGGCTCTGATGA 1281 G AGTG CCAG GTGG CTCTG ATG
  • LDLRex2 (27 68 123 30) _161. .dl_r 1282 CTCATCAGAGCCACCTGGCACTCAG 1283 TCATCAG AG CCACCTGG CACTCA 1284 CATCAGAGCCACCTGGCACTC
  • LDLRex2 (27 68 123 30 _164 .dlj 1297 AGTGCCAGGATGCTCTGATGAGTCC 1298 GTG CCAG GATGCTCTG ATG AGTC 1299 TGCCAGGATGCTCTGATGAGT
  • LDLRex2 (27 68 123 30 _164 .dl_r 1300 GGACTCATCAGAGCATCCTGGCACT 1301 GACTCATCAGAGCATCCTGGCAC 1302 ACTCATCAGAGCATCCTGGCA
  • LDLRex2 (27 68 123 30 _165_ .dlj 1303 GTGCCAGGATGGTCTGATGAGTCCC 1304 TGCCAGGATGGTCTGATGAGTCC 1305 G CCAG G ATG GTCTG ATG AGTC
  • LDLRex2 (27 68 123 30 _165_ .dl_r 1306 G GG ACTCATCAG ACCATCCTG G CAC 1307 GGACTCATCAGACCATCCTGGCA 1308 GACTCATCAGACCATCCTGGC
  • LDLRex2 (27 68 123 30 _166_ .dlj 1309 TGCCAGGATGGCCTGATGAGTCCCA 1310 GCCAGGATGGCCTGATGAGTCCC 1311 CCAGGATGGCCTGATGAGTCC
  • LDLRex2 (27 68 123 30 _167. .dlj 1315 GCCAGGATGGCTTGATGAGTCCCAG 1316 CCAGGATGGCTTGATGAGTCCCA 1317 CAGGATGGCTTGATGAGTCCC
  • LDLRex2 (27 68 123 30 _167. .dl_r 1318 CTGGGACTCATCAAGCCATCCTGGC 1319 TG G G ACTCATCAAG CCATCCTGG 1320 GGGACTCATCAAGCCATCCTG
  • LDLRex2 (27 68 123 30 _168_ .dlj 1321 CCAGGATGGCTCGATGAGTCCCAGG 1322 CAGGATGGCTCGATGAGTCCCAG 1323 AGGATGGCTCGATGAGTCCCA
  • LDLRex2 (27 68 123 30 _168_ .dl_r 1324 CCTGGGACTCATCGAGCCATCCTGG 1325 CTGGGACTCATCGAGCCATCCTG 1326 TGGGACTCATCGAGCCATCCT
  • LDLRex2 (27 68 123 30 _169_ .dlj 1327 CAGGATGGCTCTATGAGTCCCAGGA 1328 AGGATGGCTCTATGAGTCCCAGG 1329 G G ATGG CTCTATG AGTCCCAG
  • LDLRex2 (27 68 123 30 _169_ .dl_r 1330 TCCTGGGACTCATAGAGCCATCCTG 1331 CCTGGGACTCATAGAGCCATCCT 1332 CTGGGACTCATAGAGCCATCC
  • LDLRex2 (27 68 123 30 _170_ .dlj 1333 AGGATGGCTCTGTGAGTCCCAGGAG 1334 GGATGGCTCTGTGAGTCCCAGGA 1335 GATGGCTCTGTGAGTCCCAGG
  • LDLRex2 (27 68 123 30 _170_ .dl_r 1336 CTCCTGGGACTCACAGAGCCATCCT 1337 TCCTGGGACTCACAGAGCCATCC 1338 CCTGGGACTCACAGAGCCATC
  • LDLRex2 (27 68 123 30 _171. .dlj 1339 GGATGGCTCTGAGAGTCCCAGGAGA 1340 GATGGCTCTGAGAGTCCCAGGAG 1341 ATGGCTCTGAGAGTCCCAGGA
  • LDLRex2 (27 68 123 30 _171. .dl_r 1342 TCTCCTGGGACTCTCAGAGCCATCC 1343 CTCCTGGGACTCTCAGAGCCATC 1344 TCCTGGGACTCTCAGAGCCAT
  • LDLRex2 (27 68 123 30 _172. .dlj 1345 GATGGCTCTGATAGTCCCAGGAGAC 1346 ATGGCTCTGATAGTCCCAGGAGA 1347 TGGCTCTGATAGTCCCAGGAG
  • LDLRex2 (27 68 123 30 _172. .dl_r 1348 GTCTCCTGGGACTATCAGAGCCATC 1349 TCTCCTGGGACTATCAGAGCCAT 1350 CTCCTGGGACTATCAGAGCCA
  • LDLRex2 (27 68 123 30 _173_ .dlj 1351 ATGGCTCTGATGGTCCCAGGAGACG 1352 TGGCTCTGATGGTCCCAGGAGAC 1353 GGCTCTGATGGTCCCAGGAGA
  • LDLRex2 (27 68 123 30 _173. .dl_r 1354 CGTCTCCTGGGACCATCAGAGCCAT 1355 GTCTCCTGGGACCATCAGAGCCA 1356 TCTCCTGGGACCATCAGAGCC
  • LDLRex2(27 68 123 30) _174. .dl_r 1360 ACGTCTCCTGGGATCATCAGAGCCA 1361 CGTCTCCTGGGATCATCAGAGCC 1362 GTCTCCTG G G ATCATCAG AG C
  • LDLRex2 (27 68 123 30 _178_dl_ _r 1384 CAGCACGTCTCCTGGACTCATCAGA 1385 AGCACGTCTCCTGGACTCATCAG 1386 GCACGTCTCCTGGACTCATCA
  • LDLRex2 (27 68 123 30 _179_dl_ .f 1387 CTGATGAGTCCCGGAGACGTGCTGT 1388 TGATGAGTCCCGGAGACGTGCTG 1389 GATGAGTCCCGGAGACGTGCT
  • LDLRex2 (27 68 123 30 _179_dl_ _r 1390 ACAGCACGTCTCCGGGACTCATCAG 1391 CAG CACGTCTCCGG G ACTCATCA 1392 AG CACGTCTCCGG GACTCATC
  • LDLRex2 (27 68 123 30 _180_dl_ _r 1396 CACAGCACGTCTCTGGGACTCATCA 1397 ACAGCACGTCTCTGGGACTCATC 1398 CAGCACGTCTCTGGGACTCAT
  • LDLRex2 (27 68 123 30 _181_dl_ _r 1402 TCACAGCACGTCTCTGGGACTCATC 1403 CACAGCACGTCTCTGGGACTCAT 1404 ACAGCACGTCTCTGGGACTCA
  • LDLRex2(27 68 123 30 _182_dl_ _r 1408 CTCACAG CACGTCCCTGG G ACTCAT 1409 TCACAGCACGTCCCTGGGACTCA 1410 CACAG CACGTCCCTGG G ACTC
  • LDLRex2 (27 68 123 30 _183_dl_ _r 1414 ACTCACAGCACGTTCCTGGGACTCA 1415 CTCACAGCACGTTCCTGGGACTC 1416 TCACAGCACGTTCCTGGGACT
  • LDLRex2 (27 68 123 30 _184_dl_ f 1417 GAGTCCCAGGAGCGTGCTGTGAGTC 1418 AGTCCCAGGAGCGTGCTGTGAGT 1419 GTCCCAGGAGCGTGCTGTGAG
  • LDLRex2 (27 68 123 30 _184_dl_ _r 1420 G ACTCACAG CACG CTCCTGG G ACTC 1421 ACTCACAGCACGCTCCTGGGACT 1422 CTCACAG CACG CTCCTGG G AC
  • LDLRex2 (27 68 123 30 _185_dl_ f 1423 AGTCCCAGGAGAGTGCTGTGAGTCC 1424 GTCCCAGGAGAGTGCTGTGAGTC 1425 TCCCAGGAGAGTGCTGTGAGT
  • LDLRex2 (27 68 123 30 _185_dl_ r 1426 GGACTCACAGCACTCTCCTGGGACT 1427 GACTCACAGCACTCTCCTGGGAC 1428 ACTCACAGCACTCTCCTGGGA
  • LDLRex2 (27 68 123 30 _186_dl_ f 1429 GTCCCAGGAGACTGCTGTGAGTCCC 1430 TCCCAGGAGACTGCTGTGAGTCC 1431 CCCAG G AG ACTG CTGTG AGTC
  • LDLRex2 (27 68 123 30 _186_dl_ r 1432 GGGACTCACAGCAGTCTCCTGGGAC 1433 G G ACTCACAGCAGTCTCCTG G G A 1434 GACTCACAGCAGTCTCCTGGG
  • LDLRex2 (27 68 123 30 _187_dl_ r 1438 GGGGACTCACAGCCGTCTCCTGGGA 1439 GGGACTCACAGCCGTCTCCTGGG 1440 GGACTCACAGCCGTCTCCTGG
  • LDLRex2 (27 68 123 30 _188_dl_ f 1441 CCCAGGAGACGTCTGTGAGTCCCCT 1442 CCAGGAGACGTCTGTGAGTCCCC 1443 CAGGAGACGTCTGTGAGTCCC
  • LDLRex2 (27 68 123 30 _188_dl_ r 1444 AGGGGACTCACAGACGTCTCCTGGG 1445 GGGGACTCACAGACGTCTCCTGG 1446 GGGACTCACAGACGTCTCCTG
  • LDLRex2 (27 68 123 30 _189_dl_ f 1447 CCAG G AG ACGTGTGTG AGTCCCCTT 1448 CAGGAGACGTGTGTGAGTCCCCT 1449 AGGAGACGTGTGTGAGTCCCC
  • LDLRex2(27 68 123 30) _189_dl_ r 1450 AAGGGGACTCACACACGTCTCCTGG 1451 AG GG G ACTCACACACGTCTCCTG 1452 GGGGACTCACACACGTCTCCT
  • LDLRex2(27 68 123 30) _190 + 1_ dl_r 1456 CAAAG GG G ACTCAAG CACGTCTCCT 1457 AAAG G GG ACTCAAG CACGTCTCC 1458 AAGGGGACTCAAGCACGTCTC
  • LDLRex2 (27 68 123 30 _190 + 12_dl_r 1474 CATATCATGCCCAAGGGGACTCACA 1475 ATATCATGCCCAAGGGGACTCAC 1476 TATCATGCCCAAGGGGACTCA
  • LDLRex2 (27 68 123 30 _190 + 13_dl_f 1477 GTGAGTCCCCTTGGGCATGATATGC 1478 TGAGTCCCCTTGGGCATGATATG 1479 G AGTCCCCTTG GG CATG ATAT
  • LDLRex2 (27 68 123 30 _190 + 13_dl_r 1480 GCATATCATGCCCAAGGGGACTCAC 1481 CATATCATGCCCAAGGGGACTCA 1482 ATATCATGCCCAAGGGGACTC
  • LDLRex2 (27 68 123 30 _190 + 14_dl_f 1483 TGAGTCCCCTTTGGCATGATATGCA 1484 GAGTCCCCTTTGGCATGATATGC 1485 AGTCCCCTTTGGCATGATATG
  • LDLRex2 (27 68 123 30 _190 + 14_dl_r 1486 TGCATATCATG CCAAAG GG G ACTCA 1487 G CAT ATCATG CCAAAG GG GACTC 1488 CATATCATGCCAAAGGGGACT
  • LDLRex2 (27 68 123 30 _190 + 15_dl_f 1489 GAGTCCCCTTTGGCATGATATGCAT 1490 AGTCCCCTTTGGCATGATATGCA 1491 GTCCCCTTTGGCATGATATGC
  • LDLRex2 (27 68 123 30 _190 + 15_dl_r 1492 ATGCATATCATGCCAAAGGGGACTC 1493 TGCATATCATG CCAAAG GG G ACT 1494 G CAT ATCATG CCAAAG GG G AC
  • LDLRex2 (27 68 123 30 _190 + 2_dl_f 1495 GGAGACGTGCTGGAGTCCCCTTTGG 1496 GAGACGTGCTGGAGTCCCCTTTG 1497 AGACGTGCTGGAGTCCCCTTT
  • LDLRex2 (27 68 123 30 _190 + 2_dl_r 1498 CCAAAGGGGACTCCAGCACGTCTCC 1499 CAAAG GG G ACTCCAG CACGTCTC 1500 AAAG GG G ACTCCAG CACGTCT
  • LDLRex2 (27 68 123 30 _190 + 3_dl_f 1501 GAGACGTGCTGTAGTCCCCTTTGGG 1502 AGACGTGCTGTAGTCCCCTTTGG 1503 GACGTGCTGTAGTCCCCTTTG
  • LDLRex2 (27 68 123 30 _190 + 3_dl_r 1504 CCCAAAGGGGACTACAGCACGTCTC 1505 CCAAAGGGGACTACAGCACGTCT 1506 CAAAGGGGACTACAGCACGTC

Abstract

L'invention concerne de nouveaux procédés de fabrication et de conception de banques de sondes d'acide nucléique pour la détection et la caractérisation exactes, fiables et spécifiques de petites insertions ou délétions de nucléotide (indels) ou de SNP dans un échantillon d'acide nucléique cible quelconque. L'invention concerne en outre une banque de sondes fabriquée par les procédés et les substrats en phase solide revêtus avec de telles banques de sondes. Les banques de sondes de l'invention permettent la détection d'indels dans un segment d'acide nucléique cible également appelé segment de variant génétique. L'invention concerne en outre des procédés d'utilisation de la banque de sondes pour détecter la présence d'indels dans l'échantillon d'acide nucléique d'essai.
PCT/IB2010/002354 2009-09-04 2010-09-03 Détection à rendement élevé de petites délétions et insertions génomiques WO2011027219A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US23987209P 2009-09-04 2009-09-04
US61/239,872 2009-09-04

Publications (1)

Publication Number Publication Date
WO2011027219A1 true WO2011027219A1 (fr) 2011-03-10

Family

ID=43431768

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/IB2010/002354 WO2011027219A1 (fr) 2009-09-04 2010-09-03 Détection à rendement élevé de petites délétions et insertions génomiques

Country Status (1)

Country Link
WO (1) WO2011027219A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014181107A1 (fr) * 2013-05-09 2014-11-13 Medical Research Council Procédé de dosage
EP2768983A4 (fr) * 2011-10-17 2015-06-03 Good Start Genetics Inc Méthodes d'identification de mutations associées à des maladies

Citations (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995011995A1 (fr) 1993-10-26 1995-05-04 Affymax Technologies N.V. Reseaux de sondes d'acide nucleique sur des microplaquettes biologiques
US5527675A (en) 1993-08-20 1996-06-18 Millipore Corporation Method for degradation and sequencing of polymers which sequentially eliminate terminal residues
US5539082A (en) 1993-04-26 1996-07-23 Nielsen; Peter E. Peptide nucleic acids
US5623049A (en) 1993-09-13 1997-04-22 Bayer Aktiengesellschaft Nucleic acid-binding oligomers possessing N-branching for therapy and diagnostics
US5700647A (en) 1991-06-21 1997-12-23 Canon Kabushiki Kaisha Carbocation containing cyanine-type dye
US5714331A (en) 1991-05-24 1998-02-03 Buchardt, Deceased; Ole Peptide nucleic acids having enhanced binding affinity, sequence specificity and solubility
US5786571A (en) 1997-05-09 1998-07-28 Lexmark International, Inc. Wrapped temperature sensing assembly
US6265166B1 (en) 1998-04-29 2001-07-24 Trustees Of Boston University Methods and compositions pertaining to PD-loops
US6461816B1 (en) * 1999-07-09 2002-10-08 Agilent Technologies, Inc. Methods for controlling cross-hybridization in analysis of nucleic acid sequences
DE10242206A1 (de) * 2001-09-12 2003-04-03 Max Delbrueck Centrum Methode zum Nachweis von Mutationen, Insertionen, Deletionen und Polymorphismen auf der DNA und ihre Verwendung
US6573374B1 (en) 1993-08-06 2003-06-03 Boehringer Mannheim Gmbh Nucleotides labelled with an infrared dye and their use in nucleic acid detection
US20030165823A1 (en) * 1993-10-26 2003-09-04 Cronin Maureen T. Arrays of nucleic acid probes on biological chips
EP1479782A1 (fr) * 2003-05-19 2004-11-24 Agilent Technologies, Inc. Procédé et système pour l'analyse d'un épissage variable de ARNm par hybridation à un réseau
US6949343B2 (en) 1997-10-27 2005-09-27 Boston Probes, Inc. Methods, kits and compositions pertaining to PNA Molecular Beacons
WO2006075039A2 (fr) * 2005-01-13 2006-07-20 Lacer, S.A. Methode de diagnostic in vitro de l'hypercholesterolemie familiale par detection de reordonnancements geniques
US7281419B2 (en) 2005-09-21 2007-10-16 The Board Of Trustees Of The University Of Illinois Multifunctional probe array system

Patent Citations (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5714331A (en) 1991-05-24 1998-02-03 Buchardt, Deceased; Ole Peptide nucleic acids having enhanced binding affinity, sequence specificity and solubility
US5773571A (en) 1991-05-24 1998-06-30 Nielsen; Peter E. Peptide nucleic acids
US5736336A (en) 1991-05-24 1998-04-07 Buchardt, Deceased; Ole Peptide nucleic acids having enhanced binding affinity, sequence specificity and solubility
US5700647A (en) 1991-06-21 1997-12-23 Canon Kabushiki Kaisha Carbocation containing cyanine-type dye
US5539082A (en) 1993-04-26 1996-07-23 Nielsen; Peter E. Peptide nucleic acids
US6573374B1 (en) 1993-08-06 2003-06-03 Boehringer Mannheim Gmbh Nucleotides labelled with an infrared dye and their use in nucleic acid detection
US5527675A (en) 1993-08-20 1996-06-18 Millipore Corporation Method for degradation and sequencing of polymers which sequentially eliminate terminal residues
US5623049A (en) 1993-09-13 1997-04-22 Bayer Aktiengesellschaft Nucleic acid-binding oligomers possessing N-branching for therapy and diagnostics
WO1995011995A1 (fr) 1993-10-26 1995-05-04 Affymax Technologies N.V. Reseaux de sondes d'acide nucleique sur des microplaquettes biologiques
US20030165823A1 (en) * 1993-10-26 2003-09-04 Cronin Maureen T. Arrays of nucleic acid probes on biological chips
US5786571A (en) 1997-05-09 1998-07-28 Lexmark International, Inc. Wrapped temperature sensing assembly
US6949343B2 (en) 1997-10-27 2005-09-27 Boston Probes, Inc. Methods, kits and compositions pertaining to PNA Molecular Beacons
US6265166B1 (en) 1998-04-29 2001-07-24 Trustees Of Boston University Methods and compositions pertaining to PD-loops
US6596486B2 (en) 1998-04-29 2003-07-22 Trustees Of Boston University Methods and compositions pertaining to PD-Loops
US6461816B1 (en) * 1999-07-09 2002-10-08 Agilent Technologies, Inc. Methods for controlling cross-hybridization in analysis of nucleic acid sequences
DE10242206A1 (de) * 2001-09-12 2003-04-03 Max Delbrueck Centrum Methode zum Nachweis von Mutationen, Insertionen, Deletionen und Polymorphismen auf der DNA und ihre Verwendung
EP1479782A1 (fr) * 2003-05-19 2004-11-24 Agilent Technologies, Inc. Procédé et système pour l'analyse d'un épissage variable de ARNm par hybridation à un réseau
WO2006075039A2 (fr) * 2005-01-13 2006-07-20 Lacer, S.A. Methode de diagnostic in vitro de l'hypercholesterolemie familiale par detection de reordonnancements geniques
US7281419B2 (en) 2005-09-21 2007-10-16 The Board Of Trustees Of The University Of Illinois Multifunctional probe array system

Non-Patent Citations (11)

* Cited by examiner, † Cited by third party
Title
"Analytical Tools for DNA, Genes and Genomes : Nuts & Bolts", 2005, DNA PRESS
"Computational Methods in Molecular Biology", 1998, ELSEVIER
"DNA Microarrays, Part B: Databases and Statistics", vol. 411, 2006, ACADEMIC PRESS
"Microarrays Methods and Applications", 2003, DNA PRESS
BRUCE CHRISTOPHER K ET AL: "Design and Validation of a Metabolic Disorder Resequencing Microarray (BRUM1)", HUMAN MUTATION, vol. 31, no. 7, July 2010 (2010-07-01), pages 858 - 865, XP002617796, ISSN: 1059-7794 *
DUNNEN; ANTONARAKIS, HUMAN MUTATION, vol. 15, 2000, pages 7 - 12
MAHARJAN RAM P ET AL: "Genome-wide analysis of single nucleotide polymorphisms in Bordetella pertussis using comparative genomic sequencing", RESEARCH IN MICROBIOLOGY, vol. 159, no. 9-10, November 2008 (2008-11-01), pages 602 - 608, XP002617797, ISSN: 0923-2508 *
OUELETTE; BZEVANIS: "Bioinformatics: A Practical Guide for Analysis of Gene and Proteins", 2001, WILEY & SONS, INC.
RASHIDI; BUEHLER: "Bioinformatics Basics: Application in Biological Science and Medicine", 2000, CRC PRESS
SALATHIA N ET AL: "Indel arrays: An affordable alternative for genotyping", PLANT JOURNAL, BLACKWELL SCIENTIFIC PUBLICATIONS, OXFORD, GB, vol. 51, no. 4, 1 August 2007 (2007-08-01), pages 727 - 737, XP002563916, ISSN: 0960-7412, [retrieved on 20070720], DOI: DOI:10.1111/J.1365-313X.2007.03194.X *
SETUBAL; MEIDANIS ET AL.: "Introduction to Computational Biology Methods", 1997, PWS PUBLISHING COMPANY

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2768983A4 (fr) * 2011-10-17 2015-06-03 Good Start Genetics Inc Méthodes d'identification de mutations associées à des maladies
US9228233B2 (en) 2011-10-17 2016-01-05 Good Start Genetics, Inc. Analysis methods
US9822409B2 (en) 2011-10-17 2017-11-21 Good Start Genetics, Inc. Analysis methods
US10370710B2 (en) 2011-10-17 2019-08-06 Good Start Genetics, Inc. Analysis methods
WO2014181107A1 (fr) * 2013-05-09 2014-11-13 Medical Research Council Procédé de dosage

Similar Documents

Publication Publication Date Title
US20180230513A1 (en) Complexity Management of Genomic DNA
San Segundo-Val et al. Introduction to the gene expression analysis
US11739371B2 (en) Arrays for single molecule detection and use thereof
US7300788B2 (en) Method for genotyping polymorphisms in humans
US20070065816A1 (en) Methods for genotyping
US20060199183A1 (en) Probe biochips and methods for use thereof
US20030082576A1 (en) High throughput polymorphism screening
Burgess Gene expression studies using microarrays
US6673536B1 (en) Methods of ranking oligonucleotides for specificity using wash dissociation histories
US20120215459A1 (en) High throughput detection of genomic copy number variations
WO2001066804A2 (fr) Methodes permettant d'optimiser la capacite d'hybridation de sondes polynucleotidiques, et permettant de localiser et de detecter des variations de sequence
Butcher et al. The nature of nurture: A genomewide association scan for family chaos
WO2007033210A9 (fr) Séquences étrangères
WO2011027219A1 (fr) Détection à rendement élevé de petites délétions et insertions génomiques
US20030082584A1 (en) Enzymatic ligation-based identification of transcript expression
US20120141986A1 (en) Multivalent substrate elements for detection of nucleic acid sequences
US20070231803A1 (en) Multiplex pcr mixtures and kits containing the same
EP1412526A2 (fr) Detection amelioree et distinction de l'expression genique differentielle par la ligature et l'amplification de sondes
US20070134678A1 (en) Comparative genome hybridization of organelle genomes
Zvara et al. Microarray technology
US8221978B2 (en) Normalization probes for comparative genome hybridization arrays
JPWO2005090565A1 (ja) Ｄｎａアレイと一塩基多型の検出方法
WO2007076577A1 (fr) Procédés pour puces à adn
US20090117552A1 (en) Method for Detection and Quantification of Target Nucleic Acids in a Sample
Zou et al. DNA microarrays: applications, future trends, and the need for standardization

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 10779844

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 10779844

Country of ref document: EP

Kind code of ref document: A1