The new GPS-1 Genome Priming System allows researchers to prepare DNA segments for sequencing faster than any other method available today. With the race to complete the Human Genome Project heating up, this new system will allow scientists to pick up the pace. Furthermore sequencing is the best method of learning a proteinís amino acid sequence, so this exciting new method will enhance molecular biologyís understanding of protein conformation by allowing them to study more sequences in less time. During this golden age of molecular biology, the GPS-1 Genome Priming System fits right in with the other amazing advances of the day.
How Does It Work?
The system uses Tn-7 transposons (Transprimer) (1) that randomly insert into target DNA with universal primers. Transposons are segments of DNA that can move to differents spots along a chromosome. Therefore, they can move anywhere along a genome. When this in vitro method is carried out with billions of molecules, it can be assured that the transposons will insert in thousands of different sites within the Genomic Library. Once the transposon has been added to a genomic library, the primers allow template strands of the different fragments to be produced. These strands can then be sequenced using any method available and overlaps can be compared to discover the DNA sequence. Because the transposon is inserted randomly, an entire genome can be sequenced at once, as opposed to cumbersome, time-consuming methods like primer walking. Also new primers do not need to be developed because the transposon comes with primers ready to be used.
Figure 1. (1)
Simply isolate 'Your Favorite DNA' to be sequenced and follow the reaction protocol provided by New England BioLabs Inc. (1) to create fragments ready for sequencing. The plasmids lack the origin of replication (ori-), so "only products (target DNA molecules containing Transprimer insertions) can be recovered." (1) The company provides two transposons to be inserted, one with kanamycin resistance and the other chloramphenicol resistance. Therefore, you have the two controls to ensure that only plasmids with transposon insertions are replicated.
Mixing TnsABC Transposase, an enzyme which inserts the transposons into the target DNA, the target DNA, and the appropriate buffer is all thatís necessary to create a Genomic Library using the provided plasmid vectors. Once the vectors are transformed into competent E. coli cells that are sensitive to the antibiotic, to which the chosen transposon has a resistance, simply plate the cells with "drug combinations appropriate to select for the target plasmid alone and for the target with a transprimer insertion" (1). A plate should have around "100 colonies" which is a sufficient number to complete the sequencing (1). Using the provided primers (Primer N and Primer S) both strands of DNA can be sequenced.
Figure 2. (1)
The Tn7 transposon carries a special immunity from Transposase. The enzyme will not insert the vector into target DNA if it already contains an insertion. Therfeore only one insertion of a tansposon will be made per target DNA molecule.
Because of the ability to choose a drug resistance, only colonies with insertions will be replicated.
Fast. Instead of the old methods of preparing DNA for sequencing like primer walking, where only short segments of DNA could be sequenced at a time. The GPS-1 Genome Priming System allows an entire Genome to be prepared for sequencing in under 90 minutes.
1) Easy. The protocol provided by BioLabs Inc. is easy to follow and requires no special equipment that could not be found in your basic molecular biology laboratory.
2) Controls. The convenient design of the plasmid a transposon allows the researcher to selectively choose drug resistance and guard against double insertions.
3) Efficient. 100-10000 "insertion products are recovered per
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