Coincidence cloning of Alu PCR products

Journal Article · Thu Aug 01 04:00:00 EDT 1991 · Proceedings of the National Academy of Sciences of the United States of America; (United States)

DOI:https://doi.org/10.1073/pnas.88.15.6765· OSTI ID:5701513

Aslanidis, C; De Jong, P J ^[1]

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1. Lawrence Livermore National Lab., CA (United States)

Coincidence cloning allows the isolation of sequences held in common by two genomic DNA populations. Human DNA from two human-hamster hybrid cell lines was amplified by Alu-repeat primers (Alu PCR) and the products originating from the shared human chromosomal region were cloned. To achieve this, human sequences were amplified with very similar Alu primers from the two different human-hamster hybrid cell lines. The products were then digested with an appropriate restriction enzyme (either BanHI or Sal I), combined, denatured, and reannealed. The derived heteroduplex molecules (originating from the human regions common to both cell lines) had single BamHI and Sal I cohesive ends due to the primers used, so that they could be cloned in a double-digested plasmid vector. The authors used this method to enrich about 10-fold for Alu PCR products from the human chromosome 19q13.2 region, resulting in a region-specific clone collection. About 90% of the recombinants with BamHI-Sal I inserts are derived from the common region. This approach allows the boundaries for the regional probe isolation to be defined by combinations of hybrids rather than single hybrid cell lines, thus permitting greater flexibility in the selection of regions for probe isolation.

• DOE Contract Number: W-7405-ENG-48
• OSTI ID: 5701513
• Journal Information: Proceedings of the National Academy of Sciences of the United States of America; (United States), Journal Name: Proceedings of the National Academy of Sciences of the United States of America; (United States) Vol. 88:15; ISSN 0027-8424; ISSN PNASA
• Country of Publication: United States
• Language: English

Related Subjects

550400* -- Genetics
59 BASIC BIOLOGICAL SCIENCES
AUTORADIOGRAPHY
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
CHROMOSOMES
CLONING
COINCIDENCE METHODS
COUNTING TECHNIQUES
DAYS LIVING RADIOISOTOPES
DISEASES
DNA
DNA HYBRIDIZATION
DNA SEQUENCING
DNA-CLONING
GENE AMPLIFICATION
HEREDITARY DISEASES
HUMAN CHROMOSOME 19
HUMAN CHROMOSOMES
HYBRIDIZATION
ISOTOPES
LIGHT NUCLEI
NUCLEI
NUCLEIC ACIDS
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
PHOSPHORUS 32
PHOSPHORUS ISOTOPES
RADIOISOTOPES
STRUCTURAL CHEMICAL ANALYSIS