DOI: 10.1126/science.3461561. Direct Cloning and Sequence Analysis of Enzymatically Amplified Genomic Sequences

Direct Cloning and Sequence Analysis of Enzymatically Amplified Genomic Sequences

10.1126/science.3461561

Crossref journal-article

American Association for the Advancement of Science (AAAS)

Science (221)

Abstract

A method is described for directly cloning enzymatically amplified segments of genomic DNA into an M13 vector for sequence analysis. A 110-base pair fragment of the human β-globin gene and a 242-base pair fragment of the human leukocyte antigen DQα locus were amplified by the polymerase chain reaction method, a procedure based on repeated cycles of denaturation, primer annealing, and extension by DNA polymerase I. Oligonucleotide primers with restriction endonuclease sites added to their 5′ ends were used to facilitate the cloning of the amplified DNA. The analysis of cloned products allowed the quantitative evaluation of the amplification method's specificity and fidelity. Given the low frequency of sequence errors observed, this approach promises to be a rapid method for obtaining reliable genomic sequences from nanogram amounts of DNA.

Bibliography

Scharf, S. J., Horn, G. T., & Erlich, H. A. (1986). Direct Cloning and Sequence Analysis of Enzymatically Amplified Genomic Sequences. Science, 233(4768), 1076â1078.

Authors 3

Stephen J. Scharf (first)
Glenn T. Horn (additional)
Henry A. Erlich (additional)

References 11 Referenced 514

AUFFRAY, C, ISOTYPIC AND ALLOTYPIC VARIATION OF HUMAN CLASS-II HISTOCOMPATIBILITY ANTIGEN ALPHA-CHAIN GENES, NATURE 308: 327 (1984). (10.1038/308327a0) / NATURE (1984)
BUGAWAN T.L. unpublished data.
10.1073/pnas.81.7.1991
ERLICH, H, MOLECULAR ANALYSIS OF HLA CLASS-I AND CLASS-II ANTIGEN LOSS MUTANTS REVEALS A HOMOZYGOUS DELETION OF THE DR, DQ, AND PART OF THE DP REGION - IMPLICATIONS FOR CLASS-II GENE ORDER, HUMAN IMMUNOLOGY 16: 205 (1986). (10.1016/0198-8859(86)90049-2) / HUMAN IMMUNOLOGY (1986)
Mullis, K. B., Methods in Enzymology 155: 335 (1987). (10.1016/0076-6879(87)55023-6) / Methods in Enzymology (1987)
GUO, L.H., EXONUCLEASE-III - USE FOR DNA-SEQUENCE ANALYSIS AND IN SPECIFIC DELETIONS OF NUCLEOTIDES, METHODS IN ENZYMOLOGY 100: 60 (1983). (10.1016/0076-6879(83)00046-4) / METHODS IN ENZYMOLOGY (1983)
Maniatis T. Molecular Cloning: A Laboratory Manual (1982).
MESSING, J, NEW M13 VECTORS FOR CLONING, METHODS IN ENZYMOLOGY 101: 20 (1983). (10.1016/0076-6879(83)01005-8) / METHODS IN ENZYMOLOGY (1983)
PONCZ, M, NUCLEOTIDE-SEQUENCE ANALYSIS OF THE DELTA-BETA-GLOBIN GENE REGION IN HUMANS, JOURNAL OF BIOLOGICAL CHEMISTRY 258: 11599 (1983). (10.1016/S0021-9258(17)44270-0) / JOURNAL OF BIOLOGICAL CHEMISTRY (1983)
SAIKI, R.K., A NOVEL METHOD FOR THE DETECTION OF POLYMORPHIC RESTRICTION SITES BY CLEAVAGE OF OLIGONUCLEOTIDE PROBES - APPLICATION TO SICKLE-CELL-ANEMIA, BIO-TECHNOLOGY 3: 1008 (1985). / BIO-TECHNOLOGY (1985)
10.1126/science.2999980

Dates

Type	When
Created	18 years, 10 months ago (Oct. 5, 2006, 4:28 p.m.)
Deposited	1 year, 7 months ago (Jan. 13, 2024, 10:47 p.m.)
Indexed	1 month, 4 weeks ago (June 30, 2025, 6:21 p.m.)
Issued	38 years, 11 months ago (Sept. 5, 1986)
Published	38 years, 11 months ago (Sept. 5, 1986)
Published Print	38 years, 11 months ago (Sept. 5, 1986)

Funders 0

None

BibTeX

@article{Scharf_1986, title={Direct Cloning and Sequence Analysis of Enzymatically Amplified Genomic Sequences}, volume={233}, ISSN={1095-9203}, url={http://dx.doi.org/10.1126/science.3461561}, DOI={10.1126/science.3461561}, number={4768}, journal={Science}, publisher={American Association for the Advancement of Science (AAAS)}, author={Scharf, Stephen J. and Horn, Glenn T. and Erlich, Henry A.}, year={1986}, month=sep, pages={1076–1078} }

JSON

{
  "indexed": {
    "date-parts": [
      [
        2025,
        6,
        30
      ]
    ],
    "date-time": "2025-06-30T22:21:35Z",
    "timestamp": 1751322095902
  },
  "reference-count": 11,
  "publisher": "American Association for the Advancement of Science (AAAS)",
  "issue": "4768",
  "content-domain": {
    "domain": [],
    "crossmark-restriction": false
  },
  "published-print": {
    "date-parts": [
      [
        1986,
        9,
        5
      ]
    ]
  },
  "abstract": "<jats:p>A method is described for directly cloning enzymatically amplified segments of genomic DNA into an M13 vector for sequence analysis. A 110-base pair fragment of the human \u03b2-globin gene and a 242-base pair fragment of the human leukocyte antigen DQ\u03b1 locus were amplified by the polymerase chain reaction method, a procedure based on repeated cycles of denaturation, primer annealing, and extension by DNA polymerase I. Oligonucleotide primers with restriction endonuclease sites added to their 5\u2032 ends were used to facilitate the cloning of the amplified DNA. The analysis of cloned products allowed the quantitative evaluation of the amplification method's specificity and fidelity. Given the low frequency of sequence errors observed, this approach promises to be a rapid method for obtaining reliable genomic sequences from nanogram amounts of DNA.</jats:p>",
  "DOI": "10.1126/science.3461561",
  "type": "journal-article",
  "created": {
    "date-parts": [
      [
        2006,
        10,
        5
      ]
    ],
    "date-time": "2006-10-05T20:28:52Z",
    "timestamp": 1160080132000
  },
  "page": "1076-1078",
  "source": "Crossref",
  "is-referenced-by-count": 514,
  "title": "Direct Cloning and Sequence Analysis of Enzymatically Amplified Genomic Sequences",
  "prefix": "10.1126",
  "volume": "233",
  "author": [
    {
      "given": "Stephen J.",
      "family": "Scharf",
      "sequence": "first",
      "affiliation": [
        {
          "name": "Cetus Corporation, Department of Human Genetics, Emeryville, CA 94608."
        }
      ]
    },
    {
      "given": "Glenn T.",
      "family": "Horn",
      "sequence": "additional",
      "affiliation": [
        {
          "name": "Cetus Corporation, Department of Human Genetics, Emeryville, CA 94608."
        }
      ]
    },
    {
      "given": "Henry A.",
      "family": "Erlich",
      "sequence": "additional",
      "affiliation": [
        {
          "name": "Cetus Corporation, Department of Human Genetics, Emeryville, CA 94608."
        }
      ]
    }
  ],
  "member": "221",
  "reference": [
    {
      "key": "e_1_2_1_1_1",
      "doi-asserted-by": "crossref",
      "first-page": "327",
      "DOI": "10.1038/308327a0",
      "volume": "308",
      "year": "1984",
      "unstructured": "AUFFRAY, C, ISOTYPIC AND ALLOTYPIC VARIATION OF HUMAN CLASS-II HISTOCOMPATIBILITY ANTIGEN ALPHA-CHAIN GENES, NATURE 308: 327 (1984).",
      "journal-title": "NATURE"
    },
    {
      "key": "e_1_2_1_2_1",
      "unstructured": "BUGAWAN T.L. unpublished data."
    },
    {
      "key": "e_1_2_1_3_1",
      "doi-asserted-by": "publisher",
      "DOI": "10.1073/pnas.81.7.1991"
    },
    {
      "key": "e_1_2_1_4_1",
      "doi-asserted-by": "crossref",
      "first-page": "205",
      "DOI": "10.1016/0198-8859(86)90049-2",
      "volume": "16",
      "year": "1986",
      "unstructured": "ERLICH, H, MOLECULAR ANALYSIS OF HLA CLASS-I AND CLASS-II ANTIGEN LOSS MUTANTS REVEALS A HOMOZYGOUS DELETION OF THE DR, DQ, AND PART OF THE DP REGION - IMPLICATIONS FOR CLASS-II GENE ORDER, HUMAN IMMUNOLOGY 16: 205 (1986).",
      "journal-title": "HUMAN IMMUNOLOGY"
    },
    {
      "key": "e_1_2_1_5_1",
      "doi-asserted-by": "crossref",
      "first-page": "335",
      "DOI": "10.1016/0076-6879(87)55023-6",
      "volume": "155",
      "year": "1987",
      "unstructured": "Mullis, K. B., Methods in Enzymology 155: 335 (1987).",
      "journal-title": "Methods in Enzymology"
    },
    {
      "key": "e_1_2_1_6_1",
      "doi-asserted-by": "crossref",
      "first-page": "60",
      "DOI": "10.1016/0076-6879(83)00046-4",
      "volume": "100",
      "year": "1983",
      "unstructured": "GUO, L.H., EXONUCLEASE-III - USE FOR DNA-SEQUENCE ANALYSIS AND IN SPECIFIC DELETIONS OF NUCLEOTIDES, METHODS IN ENZYMOLOGY 100: 60 (1983).",
      "journal-title": "METHODS IN ENZYMOLOGY"
    },
    {
      "key": "e_1_2_1_7_1",
      "unstructured": "Maniatis T. Molecular Cloning: A Laboratory Manual (1982)."
    },
    {
      "key": "e_1_2_1_8_1",
      "doi-asserted-by": "crossref",
      "first-page": "20",
      "DOI": "10.1016/0076-6879(83)01005-8",
      "volume": "101",
      "year": "1983",
      "unstructured": "MESSING, J, NEW M13 VECTORS FOR CLONING, METHODS IN ENZYMOLOGY 101: 20 (1983).",
      "journal-title": "METHODS IN ENZYMOLOGY"
    },
    {
      "key": "e_1_2_1_9_1",
      "doi-asserted-by": "crossref",
      "first-page": "11599",
      "DOI": "10.1016/S0021-9258(17)44270-0",
      "volume": "258",
      "year": "1983",
      "unstructured": "PONCZ, M, NUCLEOTIDE-SEQUENCE ANALYSIS OF THE DELTA-BETA-GLOBIN GENE REGION IN HUMANS, JOURNAL OF BIOLOGICAL CHEMISTRY 258: 11599 (1983).",
      "journal-title": "JOURNAL OF BIOLOGICAL CHEMISTRY"
    },
    {
      "key": "e_1_2_1_10_1",
      "first-page": "1008",
      "volume": "3",
      "year": "1985",
      "unstructured": "SAIKI, R.K., A NOVEL METHOD FOR THE DETECTION OF POLYMORPHIC RESTRICTION SITES BY CLEAVAGE OF OLIGONUCLEOTIDE PROBES - APPLICATION TO SICKLE-CELL-ANEMIA, BIO-TECHNOLOGY 3: 1008 (1985).",
      "journal-title": "BIO-TECHNOLOGY"
    },
    {
      "key": "e_1_2_1_11_1",
      "doi-asserted-by": "publisher",
      "DOI": "10.1126/science.2999980"
    }
  ],
  "container-title": "Science",
  "original-title": [],
  "language": "en",
  "link": [
    {
      "URL": "https://www.science.org/doi/pdf/10.1126/science.3461561",
      "content-type": "unspecified",
      "content-version": "vor",
      "intended-application": "similarity-checking"
    }
  ],
  "deposited": {
    "date-parts": [
      [
        2024,
        1,
        14
      ]
    ],
    "date-time": "2024-01-14T03:47:27Z",
    "timestamp": 1705204047000
  },
  "score": 1,
  "resource": {
    "primary": {
      "URL": "https://www.science.org/doi/10.1126/science.3461561"
    }
  },
  "subtitle": [],
  "short-title": [],
  "issued": {
    "date-parts": [
      [
        1986,
        9,
        5
      ]
    ]
  },
  "references-count": 11,
  "journal-issue": {
    "issue": "4768",
    "published-print": {
      "date-parts": [
        [
          1986,
          9,
          5
        ]
      ]
    }
  },
  "alternative-id": [
    "10.1126/science.3461561"
  ],
  "URL": "http://dx.doi.org/10.1126/science.3461561",
  "relation": {},
  "ISSN": [
    "0036-8075",
    "1095-9203"
  ],
  "subject": [],
  "container-title-short": "Science",
  "published": {
    "date-parts": [
      [
        1986,
        9,
        5
      ]
    ]
  }
}