This technique was being used routinely in the laboratory to generate yeast artificial chromosome end clones but also has a potential application to genomic DNA.
5.3.1 The first genomic walk upstream of the pdeb coding region
Vectorette libraries were constructed as detailed in section 2.7.8.1, initially using enzymes which were known not to cut within known sequence upstream of a gene specific primer (GSPl) situated just 3' to W85 (figure 3.19). Aliquots were used for rounds of amplification using the Vectorette PCR 1 primer and GSPl. The gene specific primer was designed with thermal characteristics which matched the Vectorette PCR 1 primer as closely as possible to allow a very stringent annealing/extension temperature of 72®C to be used. Figure 5.13 shows the results of the first and second (nested) rounds of amplification of the Vectorette libraries. Although the only products obtained from the first round of amplification in are barely visible (from the Haelll and PvuII libraries), specific bands of the expected size were obtained upon the second PCR. Therefore, the PvuII product, which was the larger of the two was reamplified from a gel-purified band using the original primers and cloned into a plasmid vector (section 2.7.8.2).
The nucleotide sequence information obtained from three clones extended approximately 120bp upstream into unknown upstream sequence and terminated at a PvuII half site ligated to the Vectorette. This PvuII site is conserved in the human PDEB upstream region.
5.3.2 Use of additional Vectorette libraries to aid genomic walking
An additional Sau3Al library was made (section 2.7.8.1) and was subjected to amplification under the same conditions as the other libraries. Initially, a very faint band could be visualised at a larger size than that generated by the PvuII control and upon a semi- nested amplification, this was confirmed (figure 5.14). The negative controls appear to be contaminated with a band which is the same size as the PvuII product, but there is a discrete.
1 lu’ p d i ’h p r o m o t e r rcf^iori arui nuif)pini> o f t r a n s i n p t i o m i l s t a r t s it e s
(A) 0 1 2 3 (B] 1 2 3 4 5 6 7 8
^ 2 1 0 B P
Figure 5.13a First round of PCR front vectorette libraries Haelll (lane 1 ) and Pvull (lane 3) showing the very faint, specific bands obtained with the vectorette PCRl primer and a pdeb gene specific primer mapping just downstream of primer W85. Lane 2. no DNA control. O = PhiX
174/Haelll markers. A reverse image is presented.
Figure 5.13b Nested amplification from dilutions of the first round PCRs using primers W109/W85 (lanes 1 ,2 .3 and 8) and Vectorette PCRl primer/W85 (lanes 4. 5. 6 and 7). Lane 2 no DNA control for W109/W85. lane 6 genomic DNA negative control for vectorette PCR1/W85. lane 7 no DNA control, lane 8 genomic DNA positive control for W109/W85.
4= P <4= p
Figure 5.14 First round (left) and second round semi-nested (right) PCR amplifications from additional vectorette libraries to extend the upstream genomic sequence of pdeb. Primers used: first round - vectorette PCRl/pdeb gene specific primer (GSP) downstream of W85; second round vectorette PCR 1/W85. The lane order in both is identical. P = Pvull librar>'. S = Sau3A 1 library. H = Hindlll library'. R = Rsal library. HA = Haelll library. PP = original Pvull vectorette
PC Rl/G SP product generated in figure 5.13a (positive control), N = no DNA. G = genomic DNA control. <t> = PhiX 174/Haelll markers.
Figure 5.15 Second round PCR amplification from dilutions of the first round reactions which were perfomied using an upstream primer (GSP2) which was synthesised using sequence infomiation gained from the Pvull product (figure 5.13b). A nested primer GSP2Nalso generated from new sequence inlbmiation was used in combination with vectorette PCR 1 primer. Lanes 1 ,2 .7 Vectorette PCR primer only, lanes 3. 4, 8 GSP2N only, lanes 5.6. 9. 10 both primers. T = TaqI library. R = Rsal library. N = no DNA. G = genomic DNA. PhiX 174/Haelll markers.
Figure 5.16 Contlmiation of the continuity of primers -265 and W85 in the 5' region of the pdeb gene. The PCR amplification from genomic DNA (G) generated a band of the expected 475bp si/e. N = no DNA. = PhiX 174/Haelll markers.
The p d eb prom oter region an d m apping o f transcriptional sta rt sites
single band in the Sau3Al lane. Cloning and sequencing of this new product confirmed the presence of the PvuII site and extended the nucleotide information by 68 base pairs.
Using the sequence information generated from the PvuII PCR product, a second specific primer (GSP2) was synthesised and used on two new vectorette libraries, Taql and Rsa 1. Neither of these enzymes cut in a region of known sequence upstream of the GSP2 primer binding site but are four-cutters and were therefore more likely to cut at a point upstream which is able to be amplified under the conditions used.
No visible bands were generated upon the first round of amplification, but on semi nested PCR using combinations of Vectorette PCR 1 primer and/or the GSP2 nested primer, bands were generated which were specific to the combination of primers (figure 5.15). These bands were initially sequenced directly (sections 2.8.3.1 to 2.8.3 3), then agarose-purified, reamplified and cloned into a plasmid vector before sequencing multiple clones (sections 2.4.6, 2.6.4.1 and 2.8.2 respectively).
The extent of the sequence was increased to just over 270bp upstream of the ATG translational start codon. This represents the farthest point for which the sequences of the Taql and Rsal were in complete agreement. After this point, the nucleotide sequences diverged in all three PCR products (data not shown). This divergence was present in clones each of the differenct Vectorette Taql and Rsal products and in the directly sequenced bands, suggesting that this phenomenon is not an artefact generated in cloning the PCR products.
To confirm the validity of the sequence already obtained, a sense strand PCR primer was designed extending from -265, and used in conjunction with W85 on a template of genomic DNA from an unrelated dog. Figure 5.16 shows that the expected band size o f 465bp is obtained. The PCR fragment was not itself sequenced, or tested by genomic Southern blotting to absolutely confirm this. The nucleotide sequence will be shown later.
The p d eb prom oter region a n d mapping o f transcriptional sta rt sites