Probe mapping to facilitate transposon-based DNA sequencing

Journal Article · Wed Aug 01 04:00:00 EDT 1990 · Proceedings of the National Academy of Sciences of the United States of America; (USA)

DOI:https://doi.org/10.1073/pnas.87.16.6213· OSTI ID:5726652

Strausbaugh, L D; Bourke, M T; Sommer, M T; Coon, M E; Berg, C M ^[1]

---

1. Univ. of Connecticut, Stoors, CT (USA)

A promising strategy for DNA sequencing exploits transposons to provide mobile sites for the binding of sequencing primers. For such a strategy to be maximally efficient, the location and orientation of the transposon must be readily determined and the insertion sites should be randomly distributed. The authors demonstrate an efficient probe-based method for the localization and orientation of transposon-borne primer sites, which is adaptable to large-scale sequencing strategies. This approach requires no prior restriction enzyme mapping or knowledge of the cloned sequence and eliminates the inefficiency inherent in totally random sequencing methods. To test the efficiency of probe mapping, 49 insertions of the transposon {gamma}{delta} (Tn1000) in a cloned fragment of Drosophila melanogaster DNA were mapped and oriented. In addition, oligonucleotide primers specific for unique subterminal {gamma}{delta} segments were used to prime dideoxynucleotide double-stranded sequencing. These data provided an opportunity to rigorously examine {gamma}{delta} insertion sites. The insertions were quire randomly distributed, even though the target DNA fragment had both A+T-rich and G+C-rich regions; in G+C-rich DNA, the insertions were found in A+T-rich valleys. These data demonstrate that {gamma}{delta} is an excellent choice for supplying mobile primer binding sites to cloned DNA and that transposon-based probe mapping permits the sequences of large cloned segments to be determined without any subcloning.

• OSTI ID: 5726652
• Journal Information: Proceedings of the National Academy of Sciences of the United States of America; (USA), Journal Name: Proceedings of the National Academy of Sciences of the United States of America; (USA) Vol. 87:16; ISSN PNASA; ISSN 0027-8424
• Country of Publication: United States
• Language: English

Related Subjects

550200* -- Biochemistry
59 BASIC BIOLOGICAL SCIENCES
AUTORADIOGRAPHY
BACTERIA
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
CLONING
DAYS LIVING RADIOISOTOPES
DNA
DNA HYBRIDIZATION
DNA SEQUENCING
DNA-CLONING
ESCHERICHIA COLI
EVEN-ODD NUCLEI
HYBRIDIZATION
ISOTOPES
LIGHT NUCLEI
MICROORGANISMS
MUTAGENESIS
NUCLEI
NUCLEIC ACIDS
ORGANIC COMPOUNDS
ORGANIC SULFUR COMPOUNDS
RADIOISOTOPES
STRUCTURAL CHEMICAL ANALYSIS
SULFUR 35
SULFUR ISOTOPES
THIOLS
TRANSPOSONS