EXAMPLE 5 |
Sequencing of a microbial genome |
The ASIN primer walking approach is appl genome as follows: Total DNA is extracte and three bacterial artificial chromosom ning inserts of an average size of 1.3 K of the low copy number BAC vector offers standard cosmid and M13 based vectors, i of the cloned inserts, thus avoiding ana gements or deletions; and (2) the gene l as the problems associated with toxic or In addition, the use of one vector syste and host cultivation procedures. |
ied to sequencing a complete microbial d from a culture of the microorganism al (BAC) libraries constructed, contai- bp. 5 Kbp, and 50-100 Kbp. The use a number of advantages over the more ncluding: (1) the stable maintenance lysis problems caused by gene re-arran- ibraries are more representative, other unclonable sequences are reduced. m enables the use of standard cloning |
Cloned inserts are obtained from the 1.3 colony PCR (13). Specifically, colonies plates and placed into individual wells The cloned inserts are then PCR amplifie This approach of colony PCR amplificatio the cost and effort of plasmid minipreps libraries can be used without the need t culture, and (3) it provides a template further purification (28). The direct se requires that the concentration of the v at which they are exhausted in the ampli the product yield. |
Kbp and 5 Kbp BAC libraries by direct are picked directly from the agar of a 96-well PCR microtitre plate. d using two BAC vector-specific primers. n has three major advantages: (1) are avoided, (2) small insert BAC o grow large volumes of host-cell that can be sequenced directly without quencing of unpurified PCR product ector primers be adjusted to a level fication process but without affecting |
Dye terminator sequencing is performed u 1.3 Kbp and 5 Kbp inserts to a level of 2000 sequencing reactions per 1 Mbp), us in the colony PCR amplification. This pr with little sequence redundancy. The ASI performed to obtain the remaining genomi are assembled with the commercially avai and editing program, as described by Dec contigs are closed by sequencing approxi insert clones from the 50-100 Kbp BAC li or the ASIN approach. These sequences pr and thus obtain the remaining unknown se annotated and coding regions assigned us EXAMPLE 6 |
sing an initial shotgun phase on the one times genome coverage (approximately ing the vector-specific primers used ovides a large portion of the genome N primer walking strategy is then c sequence. The sequence fragments lable Sequencher sequence assembly kert and coworkers (7). Gaps between mately 200 randomly selected large brary using either standard procedures ovide a scaffold to order the contigs quence. The complete sequence is then ing available computer programs(7.9). |
A universal reverse genetics primer libr |
ary |
In a situation where, for example, the n at least 3 amino acids of a peptide or p procedure can be used to obtain the pept DNA encoding the protein. |
ucleotide sequence is not known but rotein sequence is known, the ASIN ide or protein sequence from template |
Thus, the ASIN procedure may be used as library. This requires, for example, the first degenerate 5' tagged nonamer libra library contains a common 5' tag sequenc 7, 8, and 9 from the 3' end. Positions 1 consist of two degenerate amino acid con 3' end would encode for two degenerate a for the amino acids Glu-Leu-Asp would ha This reverse genetics nonamer library wo it would include every possible two amin and sequencing is performed according to reverse primers. It will be clear to the degenerate nonamer and triplet primers c acid sequence at the 3' end of the gene known. |
a universal reverse genetics primer design of a modified version of the ry exemplified above. This primer e and complete degeneracy at positions to 6 from the 3' end, however, would dons. In other words, each primer mino acids. For example, the primer ve the sequence 5' Tag--NNN CTN GA(T/C). uld have about 400 members, that is, o acid combination. PCR amplification the ASIN procedure using suitable skilled addressee that two sets of ould be used when part of the amino encoding the peptide or protein is |
It will also be clear to the skilled add more convenient when one of the amino ac met or trp. |
ressee that this procedure will be ids is encoded by only one codon eg. |
Further, additional specificity for a pa could be obtained by performing multiple embedded sets of ASIN primers. |
rticular peptide or protein sequence nested amplification reactions using |
It will be appreciated by persons skille and/or modifications may be made to the embodiments without departing from the s broadly described. The present embodimen in all respects as illustrative and not |
d in the art that numerous variations invention as shown in the specific pirit or scope of the invention as ts are, therefore, to be considered restrictive. |
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