# US 2010/0167284 A1 · Nucleic acid size detection method > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2010/0167284 A1 - Document ID: US20100167284A1 - Status: Published application - Kind: A1 - Application number: 12/444,361 - Filed: 2007-04-11 - Published: 2010-07-01 - Earliest claimed: 2006-10-05 - Assignees: Quest Diagnostics Investments Incorporated; U.S. Genomics, Inc. - Inventors: Donghui Huang; Charles M. Strom; Steven J. Potts; Jenny Ellen Rooke - Web page: https://patentgrep.com/patent/US20100167284A1 ## CPC - C12Q 1/6827: for detection of mutation or polymorphism - C12Q 1/6858: Allele-specific amplification - Y10T 436/17: Nitrogen containing - C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES - Y10T: TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION ## Abstract The present invention provides methods of determining the size of a particular nucleic acid segment of interest in a sample of nucleic acids through fragmentation of DNA, size fractionation, an optional second fragmentation, and identification using a marker sequence. In particular aspects, an expansion or reduction of tandem repeat sequences can be detected. In further aspects, carriers and individuals afflicted with fragile X syndrome or other diseases associated with tandem repeats can be distinguished from normal individuals. ## Claims ### Claim 1 A method for determining the size of a nucleic acid segment in a nucleic acid sample, comprising, separating fragments of a nucleic acid, said fragments prepared from a nucleic acid-containing sample, wherein said fragments include some which contain said segment and a marker sequence, wherein the marker sequence is not contained within the segment, and said separating into fractions according to size under conditions in which a fragment containing said segment will be located in said fractions according to the size of the segment, and identifying those fraction(s) containing the segment by detecting the marker sequence, wherein the size of the segment is determined by the fraction in which it is identified. ### Claim 2 (depends on 1) The method of claim 1 wherein the nucleic acid segment is difficult to amplify by polymerase chain reaction. ### Claim 3 (depends on 2) The method of claim 2 wherein said nucleic acid segment is rich in guanine and cytosine bases. ### Claim 4 (depends on 1) A method according to claim 1, wherein said fragments are prepared using one or more restriction endonucleases. ### Claim 5 (depends on 1) A method according to claim 1, wherein said fractions are chosen to separate fragments containing segments having a normal size from fragments containing segments having an abnormal size. ### Claim 6 (depends on 1) A method according to claim 1, wherein said separating is into a number of fractions selected from the group consisting of 2-16. ### Claim 7 (depends on 1) method according to claim 1, wherein said separating is by electrophoresis. ### Claim 8 (depends on 7) A method according to claim 7, wherein said electrophoresis is capillary electrophoresis. ### Claim 9 (depends on 1) A method according to claim 1, wherein said separating is by chromatography. ### Claim 10 (depends on 1) A method according to claim 1, further comprising after said separating step, cleaving all or a portion of the segment from said marker sequence in fragments which contain said segment and said marker sequence. ### Claim 11 (depends on 10) A method according to claim 10, wherein said cleaving is accomplished using a restriction endonuclease. ### Claim 12 (depends on 1) A method according to claim 1, wherein said detecting further comprises amplifying said marker sequence. ### Claim 13 (depends on 12) A method according to claim 12, wherein said amplifying is accomplished with polymerase chain reaction (PCR). ### Claim 14 (depends on 13) A method according to claim 13, wherein said PCR includes a labeled primer. ### Claim 15 (depends on 1) The method of claim 1, wherein said detecting is accomplished using the Taqman PCR detection system. ### Claim 16 (depends on 1) A method according to claim 1, wherein said marker sequence is detected by hybridizing to two probes that hybridize simultaneously to the marker sequence. ### Claim 17 (depends on 1) A method according to claim 1, wherein said segment comprises a tandem repeat region. ### Claim 18 (depends on 17) A method according to claim 17, wherein the size determined is the number of repeats in said tandem repeat region. ### Claim 19 (depends on 19) A method according to claim 19, wherein said fractions are chosen to separate fragments with a normal number of tandem repeats from fragments containing an abnormal number of tandem repeats. ### Claim 20 (depends on 17) A method according to claim 17, wherein said tandem repeats are trinucleotide tandem repeats. ### Claim 21 (depends on 17) A method according to claim 17, wherein said tandem repeat is associated with a gene selected from the group consisting of DRPLA, EPM1, FRAXA, FMR1, FRDA, huntingtin, JPH-3, AR, DM1, DM2, SCA1, SCA2, SCA3, SCA6, SCA7, SCA8, SCA10, SCA12, SCA17, PABPN1 and HOXD13. ### Claim 22 (depends on 21) A method according to claim 21 wherein said tandem repeat is associated with a gene selected from the group consisting of FMR1, FRDA, and DM1. ### Claim 23 (depends on 22) method according to claim 22, wherein said gene is FMR1. ### Claim 24 A method of detecting a mutation in a tandem repeat segment of a gene in a nucleic acid sample, wherein said mutation is characterized by a change in the number of repeats compared to the number of repeats in the wild type allele, said method comprising separating fragments of a nucleic acid, said fragments prepared from a nucleic acid-containing sample, wherein said fragments include some which contain said tandem repeat segment and a marker sequence, wherein the marker sequence is not contained within the segment, and said separating into fractions according to size under conditions in which a fragment containing the tandem repeat segment will be located in said fractions according to the number of repeats in the tandem repeat segment; identifying those fraction(s) containing the segment by detecting the marker sequence, wherein the number of repeats in the tandem repeat segment is determined by the fraction in which it is identified, and comparing the number of repeats in the tandem repeat segment from the nucleic acid sample to the number in the corresponding wild type allele, wherein a number of repeats differing from the number in the wild type allele is indicative of a mutation. ### Claim 25 (depends on 24) A method according to claim 24, further comprising, determining if a mutation is a premutation or a full mutation by comparing the number of repeats in the tandem repeat segment from the nucleic acid sample to the number in the corresponding full mutation allele, wherein a number of repeats greater than the wild type allele but less than the full mutation is indicative of a premutation allele, whereas a number of repeats greater than or equal to the full mutation is indicative of a full mutation allele. ### Claim 26 (depends on 24) A method according to claim 24, wherein said fragments are obtained using one or more restriction endonucleases. ### Claim 27 (depends on 26) A method according to claim 26, wherein said one or more restriction endonucleases are selected from the group consisting of AluI, SphI, BmtI, BlpI, MlyI, and BstNI. ### Claim 28 (depends on 27) A method according to claim 27, wherein said one or more restriction endonuclease is AluI. ### Claim 29 (depends on 27) A method according to claim 27, wherein said one or more restriction endonucleases are SphI and BmtI. ### Claim 30 (depends on 27) A method according to claim 27, wherein said one or more restriction endonucleases are BlpI and MylI. ### Claim 31 (depends on 24) A method according to claim 24, wherein said separating is by electrophoresis. ### Claim 32 (depends on 33) A method according to claim 33, wherein said electrophoresis is capillary electrophoresis. ### Claim 33 (depends on 24) A method according to claim 24, wherein said separating is by chromatography. ### Claim 34 (depends on 24) A method according to claim 24, wherein said fractions are chosen to separate fragments containing tandem repeat segments having a normal number of tandem repeats from fragments containing tandem repeat segments having an abnormal number of repeats. ### Claim 35 (depends on 24) A method according to claim 24, wherein said separating is into a number of fractions selected from the group consisting of 2-16. ### Claim 36 (depends on 24) A method according to claim 24, further comprising after said separating step, cleaving all or a portion of the tandem repeat segment from said marker sequence in fragments which contain said tandem repeat segment and said marker sequence. ### Claim 37 (depends on 36) A method according to claim 36, wherein said cleaving is accomplished using a restriction endonuclease. ### Claim 38 (depends on 37) A method according to claim 37, wherein said restriction endonuclease is selected from the group consisting of BsaWI, HphI, BbvI, BstNI, Hpy1881, SmlI, and BmtI. ### Claim 39 (depends on 24) A method according to claim 24, wherein said detecting further comprises amplifying said marker. ### Claim 40 (depends on 39) A method according to claim 39, wherein said amplifying is accomplished with polymerase chain reaction (PCR). ### Claim 41 (depends on 40) A method according to claim 40 wherein said amplifying employs a detectably labeled primer. ### Claim 42 (depends on 41) A method according to claim 41 said detecting is accomplished with electrophoresis. ### Claim 43 (depends on 24) The method of claim 24, wherein said detecting is accomplished using the Taqman PCR detection system. ### Claim 44 (depends on 24) A method according to claim 24, wherein said marker sequence is detected by hybridizing to two probes that hybridize simultaneously to the marker sequence. ### Claim 45 (depends on 24) A method according to claim 24, wherein said tandem repeats are trinucleotide tandem repeats. ### Claim at position 46 -64. (canceled) ### Claim 65 A method for screening male and female individuals for carrier status of mutations in the tandem repeat region of the FMR1 gene, said method comprising assaying nucleic acids from an individual to determine gender; and assaying said nucleic acid to determine the length of the tandem repeat region of the FMR1 gene wherein said determining comprises amplifying tandem repeat region, detecting an amplification product, and determining the number of tandem repeats in the amplification product, wherein in male individuals: the presence of an amplification product having less than 55 tandem repeats indicates the individual is not a carrier, the presence of an amplification product having 55 or more tandem repeats indicates the individual is a carrier, or in the absence of an amplification product, the carrier status is undetermined; and in female individuals: the presence of an amplification product having more than 55 tandem repeats indicates the individual is a carrier; or the presence of a single amplification product having less than 55 tandem repeats, the carrier status is undetermined. ### Claim 66 (depends on 65) A method according to claim 65, further comprising, analyzing undetermined individuals to determine carrier status, wherein said analyzing comprises, separating fragments of a nucleic acid, said fragments prepared from a nucleic acid containing sample of the individual, wherein said fragments include some which contain a tandem repeat segment of the FMR1 gene and a marker sequence, wherein the marker sequence is not contained within the tandem repeat segment, said separating into fractions according to size under conditions in which a fragment containing the tandem repeat segment having a normal number of repeats will be located in a first fraction; and a fragment containing a tandem repeat segment having a premutation will be located in a second fraction; and a fragment having a tandem repeat region having a full mutation will be located in a third fraction, identifying those fraction(s) containing the segment by detecting the marker sequence, wherein the number of repeats in the tandem repeat segment is determined by the fraction in which it is identified, wherein in male individuals: a positive result in the first fraction indicates the individual is not a carrier, a positive result in the second fraction indicates the individual is a premutation carrier, a positive result in the third fraction indicates the individual is affected; and in female individuals: a positive result in only the first fraction indicates the individual is homozygous for the a normal allele; a positive result in the second fraction indicates the individual is a premutation carrier; and a positive result in the third fraction indicates the individual is a full mutation carrier. ### Claim 67 (depends on 65) A method according to claim 65, wherein said assaying nucleic acids to determine gender comprises nucleic acid amplification. ### Claim 68 (depends on 67) A method according to claim 67, wherein said nucleic acid amplification comprises amplifying a region of the amelogenin gene which produces different sizes of amplification products from the amelogenin gene on the X chromosome and the amelogenin gene on the Y chromosome, determining the size of the amplification product or products, wherein the presence of one product of a single size indicates the gender is female and the presence of two products of different sizes indicates the gender is male. ### Claim 69 (depends on 67) A method according to claim 67, wherein said amplifying of a region of the amelogenin gene is performed in multiplex with the amplifying of the tandem repeat region of the FMR1 gene. ### Claim 70 (depends on 69) A method according to claim 69, wherein said multiplex amplification further comprises amplifying one or more control sequences. ### Claim 71 (depends on 66) A method according to claim 66, further comprising after said separating step cleaving all or a portion tandem repeat segment from said marker sequence fragments in which contain said tandem repeat segment and said marker sequence. ### Claim at position 54 -75. (canceled) ### Claim 76 (depends on 24) The method according to claim 24, wherein the gene is the FMR1 gene. ### Claim 77 (depends on 76) The method according to claim 76, wherein the identification of a tandem repeat segment in the first fraction indicates the individual has an FMR1 allele with a normal number of tandem repeats; the identification of a tandem repeat segment in the second fraction indicates the individual has a premutation FMR1 allele; and the identification of a tandem repeat segment in the third fraction indicates the individual has a full mutation FMR1 allele.