 | | | | GPS™-1 Genome Priming System |
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Prices are in US dollars and valid only for US orders.
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Description:
The GPS™-1 Genome Priming System provides a simple in vitro method for generating a population of DNA sequencing templates with randomly interspersed primer-binding sites. This system is useful for sequencing DNAs that require more than one sequencing reaction to determine the entire sequence. GPS-1 is a faster alternative to primer walking, random subcloning and nested deletion methods; it can also be used for mapping projects.
GPS-1 is a Tn7 transposon-based in vitro system which uses TnsABC* Transposase to insert a transposon (Transprimer) randomly into the DNA target (1,2,3) (Figure 1). There is minimal site preference for Transprimer insertion and, due to target immunity, only one insertion occurs per target DNA molecule (4). Therefore, the in vitro reaction produces a population of target DNA molecules each containing the Transprimer element at a different position.
The GPS-1 System includes two Transprimer transposons-one encodes kanamycin resistance and the other encodes chloramphenicol resistance. This allows choice of antibiotic for selection of product DNA molecules. Since the transposon donor plasmid is unable to replicate (ori-) in ordinary lab strains of E. coli (5,6), only products (target DNA molecules containing Transprimer insertions) can be recovered. Unique priming sites on both ends of the Transprimer element, together with supplied primers (PrimerN and PrimerS), allow DNA sequence to be obtained from both strands of the target DNA at the position of the insertion. If gaps in the sequence remain after this initial random phase of sequencing, unique restriction sites within the Transprimer element enable mapping of insertions to specifically target any missing sequence.
Figure 1: GPS-1 System Overview
Figure 2: Reaction Overview
Advantages:- Can be used with plasmids, cDNAs, cosmids and large targets such as BACs.
- Transprimer is inserted randomly into the target DNA. There are no "hot spots" for insertion (3).
- Choice of drug resistance - Kanr and Camr Transprimers included.
- The entire protocol takes less than 90 minutes (Figure 2).
- Two parallel sequencing reactions can be performed on one insertion template.
- Unique rare-cutting restriction enzyme sites (NotI, PmeI, I-CeuI, SwaI, SpeI) within the Transprimer facilitate insert mapping. This is particularly useful when sequencing through repetitive DNA.
- High efficiency: 103-106 insertion products are recovered per reaction depending on whether a chemical transformation method or electroporation is used.
- Faster than primer walking, random subcloning and nested deletion methods.
Kit Components:
Control Target Plasmid (LITMUS 28)
pGPS1.1 Transprimer Donor
pGPS2.1 Transprimer Donor
PrimerN (30-mer)
PrimerS (30-mer)
Start Solution (20X)
TnsABC* Transposase
GPS™ Buffer Pack (10X)
Storage Conditions
Storage Temperature:
-20°C
References
- 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.
- Metcalf, W.W., Jiang, W. and Wanner, B.L. (1994) Gene, 138, 1-7.
Reagents Sold Separately
pGPS1.1 Transprimer Donor
pGPS2.1 Transprimer Donor
PrimerN (30-mer)
PrimerS (30-mer)
TnsABC* Transposase
GPS™ Buffer Pack
Companion Products
1 kb DNA Ladder
GPS™-LS Linker Scanning System
GPS™-M Mutagenesis System
I-CeuI
I-SceI
NotI
PI-SceI
PmeI
SpeI
SwaI
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