 | | | | GPS™-LS Linker Scanning System |
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Prices are in US dollars and valid only for US orders.
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Description:
The GPS™-LS Linker Scanning System provides a simple in vitro method for insertion of 15 bp "linkers" at random positions throughout your target DNA. The mutagenesis is accomplished by introduction of a transposon (Transprimer), the majority of which is then removed by restriction digest. Religation results in a 15 bp insertion, which includes a unique PmeI site (Figure 1).
Linker-scanning is a method for the genetic study of protein structure. Segments of protein sequence located at the surface of the three-dimensional structure or in connector regions will frequently tolerate insertions of a few amino acids and remain functional, while segments that are buried, or part of an active site, are generally intolerant of such insertions (10-13).
Linker-scanning mutagenesis produces a collection of mutant genes, each of which contain a small number of extra codons at a different position. These insertion mutants are then tested for functionality.
The GPS-LS System includes two Transprimer transposons-one encodes kanamycin resistance and the other encodes chloramphenicol resistance. Since these transposon donor plasmids are unable to replicate (ori-) in ordinary lab strains of E. coli (8,9), only products (target DNA molecules containing Transprimer insertions) can be recovered. PmeI digestion of the resulting Transprimer insertion mutants removes all but 10 bp of the Transprimer. Ligation then produces a molecule containing these 10 bp and the 5 bp duplication of target DNA created by the transposition reaction. Overall, this procedure creates a 15 bp insertion including a unique PmeI site. In 4/6 frames, this will lead to an insertion of five amino acids. In the remaining two frames, there is a TAA stop codon. For multidomain proteins, these stop codon insertions can be used to identify domains with partial function. In all cases the locations of the insertions of interest can be identified by sequencing of the original full-length Transprimer insertions using the supplied primers, PrimerN and PrimerS, or by restriction mapping of the 15 bp insertions using the PmeI site located at the insertion site.
In addition to its use to explore protein structure and function, GPS-LS can be used as a means to investigate areas important in DNA, such as promoter regions or binding motifs. GPS-LS will also allow secondary insertion of a Tn7-based transposon into a target that has already received one, after Pme I-mediated deletion of the original Transprimer insertion. Finally, the GPS-LS Transprimers can be used for sequence acquisition or mutagenesis in exactly the same way as Transprimer-1 and Transprimer-2 in the GPS-1 and GPS-M systems.
Figure 1: GPS-LS Linker-Scanning System Overview
Advantages:- Transprimer is inserted at very many locations in the target DNA. There are no "hot spots" for insertion (7).
- Flexible: The 8-base PmeI site at the linker insertion site is rarely found in target DNA.
- Faster and less expensive than other linker-scanning methods.
- No oligonucleotide synthesis required for mutagenesis.
- No special strains, no mating protocols, and no phage infection procedures are required.
- Large regions can be scanned with the products of one GPS-LS reaction.
- Transprimer insertions can be mapped using rare-cutter restriction sites within the Transprimer and can be located precisely using the supplied sequencing primers.
- Deletion of the body of the Transprimer leaves a 15 bp "linker" containing a unique (and rare) Pme I site that can act as a physical marker or as a site for further modifications.
- 2 of every 3 insertions are in-frame, allowing identification of regions of the target sequence tolerant of pentapeptide insertion.
- Choice of drug-resistance: Kanr and Camr Transprimers included.
- High efficiency: 103 - 106 Transprimer insertions recovered per reaction.
- Chemical transformation or electroporation can be used.
Kit Components:
BSA
Control Target Plasmid (LITMUS 28)
NEBuffer 4 (10X)
pGPS4 Transprimer Donor
pGPS5 Transprimer Donor
PmeI
PrimerN (30-mer)
PrimerS (30-mer)
Start Solution (20X)
TnsABC* Transposase
GPS™ Buffer Pack (10X)
Storage Conditions
Storage Temperature:
-20°C
References
- Barany, F. (1985) Proc. Natl. Acad. Sci. USA, 82, 4202-4206.
- Metcalf, W.W., Jiang, W. and Wanner, B.L. (1994) Gene, 138, 1-7.
- Ware, J. et al. (2002) Int. J. Parasitol., 32, 159-166.
- Chandler, M. et al. (1982) J. Mol. Biol., 154, 229-243.
- Gwinn, M.L. et al. (1997) J. Bacteriol., 179, 7315-7320.
- Goff, S.P. and Prasad, V.R. (1991) R. Sauer (Eds.), Methods Enzymol., 208, pp. 586-603. San Diego: Academic Press.
- Stone, J.C., Atkinson, T., Smith, M. and Pawson, T. (1984) Cell, 37, 549-558.
- Hallet, B., Sherratt, D.J. and Hayes, F. (1997) Nucl. Acids Res., 25, 1866-1867.
- Craig, N.L. (1996) Curr. Top. Microbiol. Immunol., 204, 27-48.
- Stellwagen, A.E. and Craig, N.L. (1997) Genetics, 145, 573-585.
- Biery, M.C., Stewart, F.J., Stellwagen, A.E., Raleigh, E.A. and Craig, N.L. (2000) Nucl. Acids Res., 28, 1067-1077.
- Stellwagen, A.E. and Craig, N.L. (1997) EMBO J., 16, 6823-6834.
- Kolter, R., Inuzuka, M. and Helinski, D.R. (1978) Cell, 15, 1199-1208.
Reagents Sold Separately
BSA
NEBuffer 4
pGPS4 Transprimer Donor
pGPS5 Transprimer Donor
PmeI
PrimerN (30-mer)
PrimerS (30-mer)
TnsABC* Transposase
GPS™ Buffer Pack
Companion Products
1 kb DNA Ladder
AscI
GPS™-1 Genome Priming System
GPS™-M Mutagenesis System
I-CeuI
I-SceI
NotI
pGPS1.1 Transprimer Donor
pGPS2.1 Transprimer Donor
pGPS3 Transprimer Donor
PI-SceI
SpeI
SwaI
Legal
Patents:
Johns Hopkins University: Licensed Under U.S. Patent No. 6,420,524
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