WO1999016908A2 - Technique d'affichage pour identifier les polymorphismes dans le site d'insertion line-1 - Google Patents

Technique d'affichage pour identifier les polymorphismes dans le site d'insertion line-1 Download PDF

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Publication number
WO1999016908A2
WO1999016908A2 PCT/US1998/020620 US9820620W WO9916908A2 WO 1999016908 A2 WO1999016908 A2 WO 1999016908A2 US 9820620 W US9820620 W US 9820620W WO 9916908 A2 WO9916908 A2 WO 9916908A2
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dna
individual
insertion site
primer
genomic dna
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PCT/US1998/020620
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WO1999016908A3 (fr
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Gary D. Swergold
Fang-Miin Sheen
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THE GOVERNMENT OF THE UNITED STATES OF AMERICA, as represented by THE SECRETARY OF THE DEPARTMENT OF HEALTH AND HUMAN SERVICES 6011 Executive Boulevard, Suite 325
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Priority to AU95960/98A priority Critical patent/AU9596098A/en
Publication of WO1999016908A2 publication Critical patent/WO1999016908A2/fr
Publication of WO1999016908A3 publication Critical patent/WO1999016908A3/fr

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    • 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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present invention pertains, in general, to methods of human identification by DNA analysis.
  • the present invention pertains to a novel PCR-based method called
  • transposable element or transposon is a DNA sequence able to insert itself at a new location in the genome without having any sequence relationship with the target locus.
  • Transposons are an important force for change in both prokaryotic and eukaryotic genomes. Eukaryotic transposable elements are divided into two general groups. One group of elements is comparable to bacterial transposons . The other group of elements is similar to retroviruses in their general organization and by their use of RNA intermediates for transposition. Because of their relationship to retroviruses, these elements are called retrotransposons or retroposons.
  • Retrotransposons differ from retroviruses in that the retrotransposons do not pass through an independent infectious form. Both transposon classes, the bacterial-like transposons and the retrotransposons, generate short direct repeats of target DNA when an insertion occurs (see reference 34 for a general discussion of retroviruses and retroposons) .
  • a large portion of the moderately repetitive DNA of mammalian genomes consist of retrotransposons.
  • the major types of retrotransposons consist of short interspersed sequences or elements (SINES) and long interspersed sequences or elements (LINES) .
  • SINES short interspersed sequences or elements
  • LINES long interspersed sequences or elements
  • the most abundant family of SINES found in mammals consists of the Alu repeats, which are present at about a million copies per genome. These SINES are collectively called the "Alu family" because they usually contain the recognition sequence AGCT for the restriction enzyme Alul. Methods have been developed for generating polymorphic markers based on the amplification of microsatellites at the 3 ' end of Alu sequences (36) .
  • LINE-1 or LI Mammalian genomes also contain 20,000 to 100,000 copies of LINES called LINE-1 or LI. All mammalian species have both older elements and younger, actively transposing LINES. For most species the identity of the actively mobile groups is not known.
  • a typical LI is approximately 6,000 to 7,000 kb long, terminates in an A-rich tract, and may have open reading frames.
  • Human LI has an internal RNA polymerase promoter and two open reading frames (31-32) . The first reading frame encodes a protein (p40) of ca . 40 kd with no known function (32) . Antibodies having binding affinity to the p40 protein have been produced and isolated (35) .
  • Human LINE-1 sequences (LlHs) make up about 5% of the human genome and most are defective, primarily due to truncation and internal rearrangements (33) . The average human haploid genome harbors an estimated
  • LlHs LINE-1 retrotransposons
  • LINE-1 elements are sequences of DNA that are commonly known as jumping genes. They have the ability to create new copies of themselves and to insert the new copies into places in the DNA where they were not previously located. Individual persons differ in the location of many of the LINE-1 elements in their DNA. In addition, alleles differing in the length of LlHs poly-A tails exist. These differences contribute to the genetic differences between people. Genetic variation is an important determinant of the differences that make all people individuals (our phenotypes) . It contributes to our differential responses to environmental exposures, and drug sensitivities. It also allows individuals to be identified by their DNA.
  • LI display designed to identify loci that differ by the presence or absence of an insertion (i.e., dimorphic sites).
  • LI display we investigated the variability of LlHs insertion sites in the DNAs of 6 male individuals from diverse ethnic backgrounds. Of the previously reported 5 LlHs insertion site dimorphisms and 7 de novo transpositions, all but one belong to the Ta subset of elements (3-7) . In the present study, greater than 20% of the Ta sites tested and few of the non-TA sites were dimorphic. We isolated 6 dimorphic TA insertion sites and confirmed their status by Southern blotting, PCR with flanking primers, or both.
  • This invention comprises methods of human identification by analysis of DNA. More specifically, the present invention provides a novel display technique called LINE-1 Display which is designed to easily identify differences in the LlHs insertion sites between different individuals. LINE-1 Display is capable of detecting two types of polymorphisms: differences in insertion site locations, and differences in the lengths of LlHs A-rich tails. LINE-1 Display represents a powerful new tool for identifying both rare and common DNA polymorphisms.
  • This invention provides display techniques for identifying LINE-1 insertion site polymorphisms used for the forensic identification of individuals, body fluids, body parts, etc.
  • this invention provides methods for the identification of donated cells or organs as to the individual of donation. Even further, this invention provides methods used to identify the ethnicity of a person on the basis of DNA analysis. Prior art methods of using DNA markers are not easily capable of accomplishing the results which can be obtained using the present invention.
  • This invention provides display techniques for identifying LINE-1 insertion site polymorphisms used in genealogy. More specifically, this invention provides methods of using a sample of cells or blood or body fluids to identify an individual's ethnic background (s) . This invention further provides methods of determining the background of individuals who are interested in their past histories or for whom the information is lost.
  • the present invention also provides display techniques for identifying LINE-1 insertion site polymorphisms used for determining the relationships between genetic variation and disease susceptibilities.
  • the prior art teaches that researchers are increasingly investigating the contribution of genes to the propensity for acquiring specific diseases.
  • the methods of the present invention permit the identification of the direct contributions made by transposable element polymorphisms to disease susceptibilities and any associations between LINE insertions and disease susceptibility.
  • the present invention also provides display techniques for identifying LINE-1 insertion site polymorphisms used for identifying the relationships between genetic variation and drug responses.
  • the methods of the present invention permit the discovery of differential responses, both intended and adverse, to the administration of drugs by transposable element polymorphisms .
  • the present invention also provides display techniques for identifying LINE-1 insertion site polymorphisms used for the identification of stored body parts.
  • the methods of the present invention will allow the positive identification of body parts, including organs and cells, which have been stored for prolonged periods. Thus, the methods of the present invention will permit the positive identification of body parts subjected to long-term storage prior to their administration to recipient patients.
  • the present invention also provides display techniques for identifying LINE-1 insertion site polymorphisms used for cancer- related research, diagnosis, and treatment.
  • LINE-1 transposition may be involved in the development of certain types of cancer, especially breast and testicular.
  • the methods of the present invention allow this hypothesis to be investigated. If there is a relationship then our mapping method would be useful as a clinical test for margins of resection, for early diagnosis, determining the risk of disease, etc.
  • the monitoring of LINE-1 transposition would be a useful marker in developing breast cancer prophylaxis.
  • the present invention also provides display techniques for identifying LINE-1 insertion site polymorphisms used for the study, detection, diagnosis, and treatment of certain developmental abnormalities.
  • LINE-1 insertion may be involved in the development of certain developmental abnormalities.
  • LINE-1 insertion may contribute to the unexplained loss of fetuses and to the development of aneuploids by non-disjunction. If this is true the methods of the present invention would be useful clinically for explaining fetal loss and for developing methods to combat fetal loss (i.e. drug development) .
  • LlHs insertion sites are highly polymorphic in the human population.
  • LINE-1 Display represents a powerful new tool for identifying both rare and common DNA polymorphisms.
  • LlHs insertion site polymorphisms identified by the methods of the present invention will be valuable tools for the field of human molecular anthropology.
  • LlHs insertion site and tail length polymorphisms identified by the methods of the present invention will be useful for genetic mapping studies.
  • Non-human mammal identification by DNA analysis using LINE-1 Display has application in a number of areas, including the tracking of tainted or diseased meat supplies.
  • LINE-1 Display will also be useful for evolutionary studies of non-human mammals. Further objects and advantages of the present invention will be clear from the description that follows .
  • FIG. 1 LI display protocol. A truncated of full-length LIHs-Ta (rectangle) is depicted surrounded by flanking DNA (solid lines) . The relative locations of the ACA, 6015G (see Materials and Methods) , and Ace I sites are indicated. The dashed lines represent the products of 2 rounds of PCR amplifications. In each round, multiple separate reactions were performed. In the first round, each reaction contained a Ta- specific primer (ACA) and a single arbitrary 10 bp primer. In the second round, portions of each of the first round reactions were reamplified using a nested primer (NP) that hybridizes to a conserved region of the 3 ' UTR, and the same arbitrary primers that were used in the first round.
  • ACA Ta- specific primer
  • NP nested primer
  • the two bands in the Ca-2 and Dr samples of the LID 1 blot are located very near to one another; their positions are indicated by lines.
  • the absence of bands for the Ca-1 samples in the LID 2 and LID 4 blots was due to an insufficient loading of DNA.
  • LID 1-6 PCR amplification of LID 1-6 with 5' and 3' flanking primers.
  • the 5' flanking sequences of LID 1-4 and 6 were determined by amplifying the empty alleles from the Genome Walker kit (Clontech) using the LID-specific 3FPa primers.
  • the 5 ' flanking sequence of LID 5 was obtained from Genbank (accession AC002122) .
  • Genbank accession AC002122
  • 200 ng genomic DNA were amplified with the LID-specific primers 5FP and 3FPa.
  • the arrowheads indicate the location of the amplified products of the empty alleles.
  • the larger bands are the amplified products of the filled alleles.
  • Digital photographs of the ethidium bromide stained gels, 2D. , or the autoradiograms of the gel after blotting and hybridization with probe Hb, 2E. are shown.
  • FIG. 3 Distribution of LID 1-6 among the 6 subjects. Presence or absence of LIDs were determined by LI display and either Southern blotting, PCR with one or two flanking primers, or both. The presence of a LID in a given individual is indicated by a + . LIDs were present in the heterozygous state in all cases.
  • FIGS 4A and 4B Quantification of LlHs subsets in the human genome .
  • 4A Southern blot quantification. Genomic DNA from i) individual Ca-2, ii) mouse LMTK " cells, and iii) LMTK " cells to which plasmid pLl .2A (which contains a Ta subst LlHs) was added were digested with Sau 3A I and Ace I to release the LlHs 3'
  • UTRs (4) Samples ii) and iii) were mixed in varying ratios to represent 0, 700, 1050, 1400, and 2100 relative copies of LlHs per haploid genome . 1 ⁇ g samples of each were Southern blotted and hybridized to oligomer C, an LIHs-Ta specific probe (3) . The relative activity of the hybridized bands was measured on a phosphorimager (Molecular Dynamics) . Results indicate a relative copy number of 2250 for the Ca-2 band, and a linear relationship of copy number to signal in the standard lanes.
  • the subset of elements which we call Tb, includes element JH-28, a previously reported de novo LlHs insertion (30) .
  • the presence of a G residue at position 6015 was previously shown to be characteristic of subset Ta elements (9) .
  • the present invention is directed to display techniques for identifying LINE-1 insertion site polymorphisms.
  • variable bands represented dimorphic LIHs-Ta insertions sites which we called LINE Insertion Dimorphisms (LID) .
  • LID LINE Insertion Dimorphisms
  • Each of the 10 clones was unique and consisted of the terminal 80 bp of a LlHs 3' UTR (64 bp of amplified sequence plus 16 bp from primer NP) , an A-rich region, and a region of 3' flanking sequence (not shown) . Only 3 nucleotide differences from the LRE-1 sequence were detected among the terminal 64 bp of LlHs 3' UTR sequence determined from each of the clones (2 were present in poly-A addition signals) . All 10 clones contained a 6015G suggesting that they were members of subset Ta (see below) . Next we amplified the genomic DNA of each individual with primers ACA and 3FPa.
  • Each of the 3FPa primers was specific for the non-LlHs 3' flanking DNA of one of the cloned variable bands ( Figure 1) and each of the amplifications was done with a single 3FPa primer.
  • Figure 1 the presence or absence of unique bands of the predicted size matched the pattern seen m LI display ( Figures 2A and 2B) .
  • Figures 2A and 2B the pattern seen m LI display
  • amplification of genomic DNA with primers ACA and 3FPa resulted in bands of identical length in all 6 individuals (not shown) .
  • These 4 clones may represent monomorphic LlHs insertions .
  • Amplification of empty alleles is expected to result in oands that are shorter than amplification of the occupied alleles by a size that depends on the length of the LlHs insertion (10) . In each case, both the empty alleles
  • LIDs have great potential as markers for a variety of genomic studies.
  • the insertion of a transposable element into a particular chromosomal locus likely represents a unique historic event (10) .
  • alleles bearing LlHs insertions are identical by descent, not just by state. This is not the case for most other types of genetic markers (11) . It is also reasonable to infer that the ancestral allele is the one without the insertion (12) .
  • a possible role for LIHs-associated polymorphisms in the mapping of centromeres had been suggested
  • Alu dimorphisms and LIDs may represent complementary sets of genomic markers.
  • LlHs elements are not distributed randomly in the human genome (20-23) .
  • LI display may help establish if the selection occurs at the time of transposition or thereafter. LI display will also be useful for investigating other questions of LlHs biology including the rate of somatic and germ-line transposition, and a possible role for LlHs m the development of cancer (24-25).
  • Materials and Methods Genomic DNA Melanesian (Me) , pygmy (Py) , Druze (Dr) , and Caucasian-1 (Ca-1) DNA were isolated from tissue culture cell lines GM10540, GM10492, GM11522, and GM05386 (Coriell Cell Repository) , respectively.
  • Oligonucleotides Arbitrary oligonucleotide decamers were purchased from Operon (27) . The other oligonucleotides used in this study were prepared by the Center for Biologies Evaluation and Research core facility, or purchased from Life Technologies. The sequence of the LI display oligonucleotides were:
  • LI display PCR The first round of LI display PCR reactions were carried out with 25 ng genomic DNA, 0.5 ⁇ M primer ACA, and 0.3 ⁇ M decamer primer in 20 mM Tris pH 8.4 , 1.5 mM MgCl 2 , 50 mM CaCl 2 , 0.2 mM deoxynucleotides, and 2.5 U Taq polymerase for 40 cycles of 94°C for 30 sec, 36°C for 30 sec, and 72°C for 30 sec.
  • the second round of reactions were carried out using the same conditions except that the primer NP was substituted for primer ACA, and the template consisted of 2.5 ⁇ l of the products of the first round reactions.
  • LI display DNA fragments were isolated from agarose gels with the QIAquick gel extraction kit (Qiagen) and cloned by the TA method (Invitrogen) .
  • DNA sequences were determined with either the SequiTherm EXCEL kit (Epicentre Technologies) using 3: P-labeled primers, or with the Thermo Sequanase kit (Amersham) using JJ P- labeled dideoxynucleotides . Sequence analyses and database searching were preformed with the MacVector program version 6.0 (Oxford Molecular Group) .
  • PCR products were separated electrophoretically in a 3% 3:1 Nusieve agarose gel (FMC) and alkaline blotted onto a Nytran Plus membrane (Schleicher and Schuell) .
  • Hybridizations were performed with 32 P-end-labeled oligonucleotides (10 q cpm/ ⁇ g) in 5x SSPE/0.3% SDS/10 ⁇ g/ml salmon sperm DNA at 42°C overnight.
  • the membranes were washed in 2x SSPE at 25°C for 15 minutes, 2x SSPE/0.1% SDS at 25° for 45 minutes, and twice in 0.5x SSPE/0.1% SDS at 42°C for 15 minutes.
  • Chromosoma 91:28-38.

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Abstract

La présente invention a trait à une nouvelle technique d'affichage appelée LINE-1 Display, élaborée pour détecter facilement les différences dans les sites d'insertions L1H entre différents individus. La technique LINE-1 Display est capable de détecter deux types de polymorphismes: les différences dans les lieux des sites d'insertion, et les différences dans les longueurs des queues polyA L1H. Les polymorphismes des sites d'insertion sont particulièrement utiles pour l'anthropologie moléculaire humaine, alors que les polymorphismes des longueurs sont particulièrement utiles dans l'étude des cartographies génétiques.
PCT/US1998/020620 1997-09-29 1998-09-29 Technique d'affichage pour identifier les polymorphismes dans le site d'insertion line-1 WO1999016908A2 (fr)

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AU95960/98A AU9596098A (en) 1997-09-29 1998-09-29 Display technique for identifying line-1 insertion site polymorphisms

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US6035397P 1997-09-29 1997-09-29
US60/060,353 1997-09-29

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015131110A1 (fr) * 2014-02-27 2015-09-03 Igenomx International Genomics Corporation Procédés pour l'analyse d'éléments mobiles somatiques, et leurs utilisations
US10604802B2 (en) 2014-02-04 2020-03-31 Jumpcode Genomics, Inc. Genome fractioning
US10968536B2 (en) 2015-02-25 2021-04-06 Jumpcode Genomics, Inc. Methods and compositions for sequencing
US11339427B2 (en) 2016-02-12 2022-05-24 Jumpcode Genomics, Inc. Method for target specific RNA transcription of DNA sequences

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
HOLMES S.E. ET AL.,: "A new retrotransposable human L1 elemet from the LRE2 locus on chromosome 1q produces a chimaeric insertion" NATURE GENETICS, vol. 7, - 1994 pages 143-148, XP002097819 cited in the application *
LAURENT A.M. ET AL., : "A rapid and simple method to isolate and characterize highly polymorphic markers from centromeric regions of the human chromosomes" NUCLEIC ACIDS RESEARCH, vol. 22, no. 2, - 1994 pages 194-199, XP002097820 *
SASSAMAN D. ET AL.,: "Many human L1 elemets are capable of retrotransposition" NATURE GENETICS, vol. 16, - 1997 pages 37-43, XP002097821 cited in the application *
SWERGOLD G.D. ET AL.,: "LINE-1 display (LID) mapping, a novel method for identifying insertion sites, reveals frequent insertion site polymorphisms in the human population" AM .J. HUMAN GENETICS, vol. 61, no. 4, - 1997 page sup.1230 XP002097818 *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10604802B2 (en) 2014-02-04 2020-03-31 Jumpcode Genomics, Inc. Genome fractioning
US11708606B2 (en) 2014-02-04 2023-07-25 Jumpcode Genomics, Inc. Genome fractioning
US11761039B2 (en) 2014-02-04 2023-09-19 Jumpcode Genomics, Inc. Genome fractioning
WO2015131110A1 (fr) * 2014-02-27 2015-09-03 Igenomx International Genomics Corporation Procédés pour l'analyse d'éléments mobiles somatiques, et leurs utilisations
AU2015222802B2 (en) * 2014-02-27 2021-07-01 Jumpcode Genomics, Inc. Methods for analysis of somatic mobile elements, and uses thereof
CN114250301A (zh) * 2014-02-27 2022-03-29 嘉普科德基因组学公司 用于分析体细胞可动因子的方法及其用途
US10968536B2 (en) 2015-02-25 2021-04-06 Jumpcode Genomics, Inc. Methods and compositions for sequencing
US11339427B2 (en) 2016-02-12 2022-05-24 Jumpcode Genomics, Inc. Method for target specific RNA transcription of DNA sequences

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