DESCRIPTION OF THE INVENTION |
The present invention relates to a metho of interest when a region of the nucleot may be utilised in sequencing protocols, and may provide advantages over current |
d of amplifying a nucleotide sequence ide sequence is known. The method particularly in genome sequencing, sequencing techniques. |
The present invention may be used for pr using primers selected from a relatively approach can be used with many different for automation. |
imer walking on an amplifiable template small presynthesized library. This sequencing chemistries and is suitable |
In order that the present invention may forms will be described in the following anying drawings. |
be more clearly understood, preferred examples with reference to the accomp- |
Although the observation appears to have shown that successful PCR can be perform the template over the final 8 to 11 bp ( nucleotide matches the template for 8 or act as a PCR primer. |
been little appreciated, it has been ed with primers that only anneal to 23). In other words, provided an oligo- more bases at its 3' end, it can |
A complete octamer primer library which (of which at least eight bases were know a complete nonamer primer library would reduction of such a library set would st The present invention allows the number a library which may be used to amplify a sequence is known) to be greatly reduced |
could be used to amplify any template n) would contain 65.536 primers, while contain 262.144 primers. Even a 90% ill leave this library cumbersome. of primers in a complete library i.e. ny template (of which at least some by careful primer design. |
In accordance with this invention, using the specificity of, for example, a non-d be obtained from a much smaller degenera below. A schematic diagram of an example ted in FIG. 1 and describes the amplific of interest of which 9 bases (divided in known region") are known. |
a two-step amplification procedure, egenerate nonamer primer library can te nonamer primer library described of the two-step procedure is illustra- ation of a template nucleotide sequence to a "first known region" and a "second |
The invention requires the addition of a As shown in the example, each primer in partially degenerate (by introducing all 7, 8, and 9 (from the 3' end)), the requ Thus, the "nonamer" primer library can b each primer including 3 degenerate posit Although this reduces the number of prim transforms the nonamer primer library in members, and hence greatly reduces the a from this library may be prone to mispri on average, every 4 Kb of template, as c nonamer primer. |
5' tag sequence to the nonamer primer. the nonamer primer library is made four bases at, for example, positions ired library can be reduced 64-fold. e constructed from only 4096 primers, ions 7 to 9 bases from its 3' end. ers required, it also effectively to a hexamer primer library of 4096 mplification specificity. Primers ming as annealing sites would exist, ompared to every 262 Kb for a true |
A 5' tagged degenerate nonamer primer is it will anneal to 6 bases (the "second k 9 known bases of template. As the degene mixture of 64 related primers, amplifica will potentially result in a mixture of product may be a minor component. |
selected from the library, such that nown region") at the 3' end of the rate nonamer primer is actually a tion using this degenerate primer different products, of which the desired |
The desired product can then be selectiv use of a second primer. This second prim library which may include (as shown for which each primer contains the same 5' t first library (or a sequence which will of the 5' tag sequence of the primers in 64 possible combinations of 3 nucleotide is selected from this 64-member library of its combination of 3 nucleotides (at 5' to the 6 bases of template utilised t known region") (FIG. 2). Since non-desir plate mismatches in the 3 nucleotides (a will be selectively amplified from the p |
ely amplified from this mix by the er is selected from a second primer example in FIG. 2) 64-members, in ag sequence as the primers in the hybridise to the complementary strand the first library), and one of the s at its 3' end. The second primer on the basis of the complementarity the 3' end) to the 3 bases residing o select the first primer (the "first ed products will contain 3' primer-tem- s shown in FIG. 2), the desired product roduct mix. |
A reverse primer is, of course, required nced is from a cloned library, for examp primer can be used. The resultant PCR pr 5' tag sequence: the "known" 9 bp sequen Thus, as indicated above, the specificit primer library can be obtained using a c ate (hexamer) first primer library and a The product of the two PCRs can, of cour chemistry. Primer walking can be perform primer library by selecting the appropri a 3' match with the template at the desi The sequence of the region flanking the can then be obtained using either (a) th primer, as a primer. This process can be ation to select a new primer from the 5' entire sequence of the nucleotide sequen |
. However, if the template to be seque- le, a common vector-specific reverse oduct will contain, 5' to 3', the ce: and the flanking unknown sequence. y of, for example, a true nonamer ombination of a vastly smaller degener- second, 64-member primer library. se, be sequenced by any known sequence ed using a 5' tagged octamer or nonamer ate primer (ie. the primer which has red location) and performing a PCR. "known" sequence described above, e 5' tag alone, or (b) the second repeated by using the sequence inform- tagged primer library, until the ce of interest is determined. |
A number of adaptations/modifications of contemplated. For example, first, the pr two separate amplification reactions, or all three primers in one reaction but re (degenerate) primer 10 to 100-fold. In t is maintained as the early exhaustion of that amplification in the later stages i 5' tagged primer. |
the procedure outlined above are ocedure can be performed as either as a one-step procedure by including ducing the concentration of the first his way, amplification specificity the first 5' tagged primer ensures s predominantly due to the second |
Further, it is possible to perform the a and second primers i.e. without a conven be possible to use this approach as a me reactions by selecting two sets of prime primers specifically for each template ( minimum number of bases were known at ea |
mplification with two sets of first tional reverse primer. It may therefore, ans to perform general amplification rs, thus avoiding the need to synthesise provided, of course, the required ch end of the desired product). |
The first (degenerate) primer library ha nonamer primer library but, of course, n and may be, for example, octamers or dec primer. The length of the primer require entation by the skilled addressee. Equal not have to be based on trimers, as exem for example, be based on doublets or fou primer. Again, the length of the primer by the skilled addressee by simple exper first (degenerate) primer library can he nonamer primer library could be reduced 400 primers by omission of 90% of the 40 |
s been exemplified as a 5' tagged eed not necessarily be based on nonamers amers or any other useful length of d can be established by simple experim- ly, the second primer library does plified above, but could instead, rmers or any other useful length of required can be easily ascertained imentation. Further, the size of the reduced. For example, a degenerate from 4096 primers to approximately 96 primers. |
Any template which is amplifiable can be genomic DNA, plasmid clones, cDNA, etc. |
used in this invention including |
It would also be possible, although more a nucleotide sequence of interest, using contain a common 5' tag sequence (eg. th all possible 8 or 9 bp 3' ends to amplif of interest of which, for example, at le In this method, a second primer as descr when the nucleotide sequence of interest primer may be designed based on the vect otide sequence of interest. |
cumbersome, to amplify and sequence a library of primers which simply e M13 reverse primer) and, for example, y any desired nucleotide sequence ast 8 or 9 bp are known, respectively. ibed above is not required. Again, is included in a vector, a reverse or sequence at the 3' end of the nucle- |
This amplification-based primer walking over other primer-library, or hexamer-li approach can avoid problems associated w tion as the walking amplification can be ture after the first cycle, that is, onc to the template for the first time, the ent cycles, both the 5' tag sequence plu other words, the 5' tagged primer only h fication reaction to proceed. Thus, if t poorly, because of secondary structure c ensures efficient amplification. Secondl can be used as the same common 5' tag se cation product. Thirdly, labelled sequen this approach compatible with all sequen complex enzymatic steps required for oth SPEL-6 hexamer libation approach, can be |
approach may have a number of advantages gation based, approaches. First, this ith template secondary structure forma- performed at a high annealing tempera- e the 5' tagged primer has annealed annealing site becomes, on the subsequ- s the original 8 or 9 bp site. In as to "catch" once to enable the ampli- he original 5' tagged primer anneals onstraints, the primer 5' tag sequence y, standard sequencing conditions quence is introduced into each amplifi- cing primers may be used, thus making cing chemistries (eg. Licor). Fourthly, er sequencing technologies, such as avoided. |