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US-12618110-B2 - Nucleic acid analysis using emulsion PCR

US12618110B2US 12618110 B2US12618110 B2US 12618110B2US-12618110-B2

Abstract

The present invention provides methods for analyzing large nucleic acids including chromosomes and chromosomal fragments. In one aspect, the present invention provides a method of nucleic acid analysis comprising the steps of (a) obtaining a sample of nucleic acid comprising at least one chromosome or fragment greater than about 1,000 base pairs in length and containing a target region; (b) creating an emulsion in which each drop of the emulsion contains an average of between about 0-2, 0-1.75, 0-1.5, 0-1.0, 0-0.75, 0-0.5, or fewer chromosomes or fragments of step (a), (c) performing emulsion PCR, (d) quantifying the number of emulsion droplets containing amplified nucleic acid from the target region; (e) calculating the ratio of droplets containing amplified nucleic acid from the target region to total droplets; and (f) comparing the ratio of step (e) to a reference ratio representing a known genotype.

Inventors

  • Charles M. Strom

Assignees

  • QUEST DIAGNOSTICS INVESTMENTS LLC

Dates

Publication Date
20260505
Application Date
20210927

Claims (18)

  1. 1 . A method for determining the genotype of a subject suspected to carry a 2+0 genotype comprising: (a) creating an emulsion from an aqueous nucleic acid sample comprising a locus of interest on at least one stretched chromosome or a fragment thereof, wherein each emulsion droplet contains an average of between 0-2 nucleic acids from the aqueous nucleic acid sample; (b) amplifying the locus of interest within the emulsion; (c) quantifying the number of emulsion droplets that comprise an amplified locus of interest; and (d) calculating the ratio of droplets containing the amplified locus of interest to the total droplets and identifying the subject (i) as having a 2+0 genotype; (ii) as having a 1+1 genotype; or (iii) having a homozygous deletion.
  2. 2 . The method of claim 1 , wherein the subject has a 2+0 genotype when the number of emulsion droplets that comprise the amplified locus of interest is less than two times the number of droplets amplified from a sample known to have a 1+1 genotype.
  3. 3 . The method of claim 1 , wherein the subject has a 1+1 genotype when the number of emulsion droplets that comprise the amplified locus of interest is greater than two times of the number of droplets amplified from a sample known to have 1+1 genotype.
  4. 4 . The method of claim 1 , wherein the subject has a homozygous deletion of the locus of interest when there are no droplets comprising an amplified locus of interest.
  5. 5 . The method of claim 1 , wherein the aqueous nucleic acid sample is prepared by (i) retracting the meniscus of a solution of nucleic acid at a constant rate; (ii) anchoring the ends of the nucleic acid to a solid substrate; and (iii) recovering the nucleic acid from the solid substrate following steps (i) and (ii).
  6. 6 . The method of claim 1 , wherein the emulsion is created by mechanical agitation or microfluidic droplet generation.
  7. 7 . The method of claim 1 , wherein the emulsion droplets generated are each between about 15 and about 100 pL in volume.
  8. 8 . The method of claim 1 , wherein the fragment is greater than 10 kilobases (kb), greater than 100 kb, greater than 200 kb, greater than 300 kb, greater than 400 kb, greater than 500 kb, greater than 600 kb, greater than 700 kb, greater than 800 kb, greater than 900 kb, greater than 1,000 kb or more in length.
  9. 9 . The method of claim 8 , wherein the fragment is between about 200 and about 700 kb in length.
  10. 10 . The method of claim 1 , wherein the locus of interest is a SMN1 locus.
  11. 11 . The method of claim 1 , wherein the emulsion further comprises oligonucleotide primers for amplification of the locus of interest.
  12. 12 . The method of claim 1 , wherein the emulsion droplets are within an oil phase.
  13. 13 . The method of claim 12 , wherein the oil phase comprises a mineral oil, a silicone based oil, or a fluorinated oil.
  14. 14 . The method of claim 1 , wherein the emulsion further comprises a surfactant.
  15. 15 . The method of claim 14 , wherein the surfactant is a non-ionic surfactant.
  16. 16 . The method of claim 15 , wherein the surfactant is cyclopentasiloxane, dimethicone copolyol, polysiloxane, polyalkyl polyether copolymer, polyglycerol ester, poloxamer, PVP/hexadecane copolymer, sorbitan monooleate, or polyoxyethylenesorbitan monooleate.
  17. 17 . The method of claim 1 , wherein the at least one chromosome or a fragment thereof has been stretched lengthwise to about 150 percent of its molecular contour length.
  18. 18 . The method of claim 1 , wherein each emulsion droplet contains an average of between 0-0.75 nucleic acids from the aqueous nucleic acid sample.

Description

CROSS REFERENCE TO RELATED APPLICATIONS This application is a Continuation of U.S. application Ser. No. 15/455,491, filed Mar. 10, 2017, which is a Continuation of U.S. application Ser. No. 14/369,426, which is the U.S. National Stage of PCT/US2012/071758, filed Dec. 27, 2017, which claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application Ser. No. 61/582,200, filed Dec. 30, 2011, which is incorporated by reference in its entirety. FIELD OF THE INVENTION The present invention relates to methods of nucleic acid analysis, in particular methods using emulsion PCR in analysis of large nucleic acid fragments or whole chromosomes. BACKGROUND OF THE INVENTION The following discussion of the background of the invention is merely provided to aid the reader in understanding the invention and is not admitted to describe or constitute prior art to the present invention. Many modern advances in cellular and molecular biology are rooted in the advent of large-scale amplification of nucleic acids and analytical methods dependent thereon. A number of methods are known in the art for performing such amplification of template nucleic acid molecules to generate populations of substantially identical copies. One technique that is particularly amenable to high throughput applications is emulsion polymerase chain reaction (“emulsion PCR” or “emPCR”). Emulsion PCR is performed by isolation of individual DNA molecules along with primer-coated beads in aqueous droplets within an oil phase. A PCR step coats each bead with clonal copies of the DNA molecule which are then immobilized for later sequencing. Emulsion PCR is used in a number of commercial methods, such as those of 454 Life Sciences, and SOLiD sequencing, (developed by Agencourt, now Applied Biosystems). Current emulsion PCR techniques involve the use of small fragments of DNA, which renders it unsuitable for analysis of certain genotypes such as those depending on an allelic linkage, and other applications for which assessment of large nucleic acids are required. Therefore, a need remains for emulsion PCR-based analytical methods that may applied to the evaluation of large nucleic acids including, for example, for monosomal analysis. SUMMARY OF THE INVENTION The present invention is based on the discovery that certain nucleic acid preparation techniques, such as those used in molecular combing, will facilitate the use of large nucleic acid fragments in emulsion PCR and subsequent analysis. In one aspect, the present invention provides a method of nucleic acid analysis comprising the steps of (a) obtaining a sample of nucleic acid comprising at least one chromosome or fragment greater than about 1000 base pairs in length and containing a target region; (b) creating an emulsion in which each drop of the emulsion contains an average of between about 0-2, 0-1.75, 0-1.5, 0-1.0, 0-0.75, 0-0.5, or fewer chromosomes or fragments of step (a), (c) performing emulsion PCR, (d) quantifying the number of emulsion droplets containing amplified nucleic acid from the target region; (e) calculating the ratio of droplets containing amplified nucleic acid from the target region to total droplets; and (f) comparing the ratio of step (e) to a reference ratio representing a known genotype. In another aspect, the present invention provides a method for determining the genotype of a subject (e.g., human) suspected to carry a 2+0 genotype comprising the steps of (a) obtaining a DNA sample comprising the locus of interest from the subject, (b) creating an emulsion in which each drop of the emulsion contains an average of between about 0-2, 0-1.75, 0-1.5, 0-1.0, 0-0.75, 0-0.5, or fewer chromosomes or fragments of step (a), (c) performing emulsion PCR, (d) quantifying the number of emulsion droplets containing amplified nucleic acid from the locus; (e) comparing the number of emulsion droplets from step (d) with the number of emulsion droplets containing amplified nucleic acid from the locus of a control 1+1 genotype sample, wherein a 1+1 genotypic sample will show successful amplification in about two times the emulsion droplets as the control and a negative sample with no successful amplification will indicate that the subject has a deletion of both alleles. In some embodiments, the locus of interest is the SMN1 gene on human chromosome 5. A “2+0 genotype” of a diploid cell, as used herein, refers to a duplication of a genetic sequence on a chromosome with a deletion or other types of disruption of the sequence at the counterpart chromosome. The wild-type of a 2+0 genotype can be referred to as a “1+1 genotype” in which each chromosome of a pair contains a copy of the sequence. A 2+0 genotype can result from improper genetic recombination or translocation between the chromosomes, without limitation. Since a 2+0 genotype and the corresponding 1+1 genotype have the same number of copies of the genetic sequence in a cell, they cannot be distinguished by dosage analysis (e.g., quanti