It is known that certain complex DNA segments, known as transposons, are able to insert into many sites in the genome of their host organisms. Transposons exist in prokaryotes, such as bacteria, as well as in eukaryotes.
Recently, a useful bacterial transposon referred to as .gamma..delta., or Tn1000, was discovered and characterized. .gamma..delta. is a discrete six kilobase (kb) segment of bacterial DNA which can insert at high frequency into numerous sites in the plasmids of gram negative bacteria. It encodes the enzyme (transposase) for its own transposition, the enzyme (resolvase) for resolution of the cointegrate product that is produced by intermolecular transposition, and another gene, the function of whose product is unknown. The DNA sequence of .gamma..delta. has been deposited in GenBank by R. Reed.
Further background information on .gamma..delta. and related transposons can be had by reference to the recent review articles by Guyer, Meth. Enzymol. 101:362-363 (1983); Berg and Berg, Uses of transposable elements and maps of known insertions. In: Neidhardt, et al., (eds.), Escherichia coil and Salmonella typhimurium: Cellular and Molecular Biology. Amer. Soc. for Microbiology, Washington, D.C. pp. 1071-1109 (1987); Berg, Berg and Groisman, Transposable elements and the genetic engineering of bacteria. pp. 879-925. In: Berg and Howe (eds.), Mobile DNA. Amer. Soc. for Microbiology, Washington, DC.(1989); Sherratt, Tn3 and related transposable elements: Site-specific recombination and transposition. pp163-184 In: Berg and Howe (eds.), Mobile DNA. Amer. Soc. for Microbiology, Washington, D.C. (1989), and Berg et al., Transposon-facilitated sequencing of DNAs cloned in plasmids. Meth. Enzymol. 218:279-306 (1993).
Two widely used methods for DNA sequence analysis are the base-specific chemical cleavage method (Maxam and Gilbert, PNAS 74:560, 1977) and the enzymatic chain termination method (Sanger et al., PNAS 74:5463, 1977). The enzymatic chain termination method has gained wide acceptance as the method of choice. Generally in this method the DNA segment of interest is cloned in an appropriate vector and a short oligonucleotide complementary to a sequence adjacent to the cloning site is used to prime DNA chain terminators. Typically, only a few hundred base pairs (bp) can be sequenced from the primer site. Usually the sequence of longer DNA stretches is assembled from numerous random shod DNA sequences with the aid of computers. Further background information on dideoxy sequencing can be had by reference to the recent book by Sambrook et al. ( Molecular Cloning, Cold Spring Harbor Press, 1989).
The strategies to bring more distant DNA regions near to the primer site include: i) subcloning small DNA fragments into a plasmid, as noted above; ii) isolating nested deletions derivatives in vivo in a transposon vector (Ahmed, J. Mol. Biol. 178:941-948,1984; Meth. Enzymol. 155:177-204, 1987); iii)isolating nested deletions derivatives in vitro in a plasmid vector with appropriate restriction sites (Henikoff, Gene 28:351-359, 1984); iv) making new oligonucleotide primers complementary to the end of each sequenced segment (primer waking) (Winnoto et al., Nature 324: 679, 1986); or v) using random insertions of transposons into the chromosome or a large cloned piece of DNA (Adachi, et al., Nucleic Acids Res. 15: 771, 1987; Liu et al., Nucleic Acids Res. 15:9461-9469, 1987; Chow and Berg, PNAS 85: 6468-6472; Nag et al., Gene 64: 135-145, 1988; Phadnis et al., PNAS 86: 5908-5912, 1989; Strausbaugh et al., PNAS 87:6213-621 7,1990).
Major problems associated with each of these methods are summarized here: i) Subcloning is inefficient and often does not yield complete coverage, even with many-fold redundant sequencing, and aligning short DNA sequence fragments is sometimes difficult, especially if the clone contains repeated sequences. ii) Previously described vectors for isolating nested deletions into cloned DNA in vivo depend on intramolecular transposition by the transposon Tn9. These vectors contain the plasmid replication origin exterior to the transposon, between the transposon and cloned fragment. Consequently, deletions that extend into the cloned fragment in one direction are not recoverable because they delete the plasmid replication origin. Therefore, a second clone with the fragment in the opposite orientation must be used to access the other strand. In addition, Tn9 transposes nonrandomly, making it difficult to achieve complete coverage. iii) Previously described vectors for isolating nested deletions in vitro depend on the ability of specific exonucleases to digest DNA cleaved in a specific way. This process is often difficult to control and cannot readily be used to isolate deletions that extend more than a few kb from the deletion site. iv) Primer walking is a generally reliable method, but it is slow and expensive. In addition, it may be difficult to design unique primers for regions containing repeated sequences. v) Random insertions of a transposon can be done by very simple steps using in vivo reactions in Escherichia Coil cells, but insertion sites are difficult to map, and there is no way to select for insertions in a specific region. This invention avoids or minimizes many of the above described limitations.
Short DNA sequences that serve as initiation sites for replication (replication origins) are found in every autonomously replicating unit (replicon). Bacterial plasmids are small (generally up to 150 kb) replicons that are not absolutely required for bacterial growth under most conditions. Naturally occurring plasmids contain a replication origin, and other genes or genetic information, often including transposons. Natural transposons found in Escherichia Coli and related bacteria do not contain replication origins. Plasmid replication origins have, however, been cloned between the ends of transposons on a plasmid for various purposes, including to study the process of transposition (Cohen et al., Cold Spring Harbor Symp. Quant. Biol. 43: 1269-1255,1978); to insert the transposon into chromosomal DNA in vivo and then to clone DNA adjacent to the transposon insertion site either in vitro (Yakobson and Guiney, J. Bacteriol. 160:451-453, 1984; Furuichi et al., J. Bacteriol. 164:270-275, 1985; Koncz et al., Mol. Gen. Genet. 207:99-105, 1987) or in vivo (Groisman et al., PNAS 81:1480-1483); and for undefined uses (Mizuuchi et al., U.S. Pat. No. 4.716.105, 1987).