ANÁLISIS DE RESULTADOS DE LA VARIABLE DIAGNOSTICO SITUACIONAL

To determine the sequences of peptides displayed on individual phage clones, PCR primers, flanking the variable region by at least 50 base pairs either side, were designed (Table 2.2) and purchased from Invitrogen. Sequencing primers were designed to be within the amplified region but not overlapping with the PCR primers, to avoid sequencing of PCR artefacts.

NAME SEQUENCE T a n n pCBQF CGCCAAGCCCCGTATTTTAC 70°C pCBQR TGAGGCTTGCAGGGAGTCAA 70°C pCBQseq CCGTTTAATGGAAACTTCCTCATG 70“C fBB-4F GCTCGAGCTTACTCCCCAT 70“C fBB-4R ATTAGGCGGGCTGGGTAT 6 7X fBB-4seq CGGCTCGTATAATGTGTGG 6B“C

TABLE 2.2 PCR and sequencing prim ers: PCR (pC89F, pC89R, f88-4F and f88-4R) and sequencing (pC89seq and f88-4seq) primers that were used for amplification and sequencing of phage inserts of the respective libraries. By choosing a similar G/C content for PCR and sequencing primers, annealing temperatures (Tgnn) were ail approximately 70°C such that the same cycling protocol could be used for both libraries.

2.6.1 Colony PCR

Using the appropriate forward and reverse primers for each library, amplicons of 167 bp, 206 bp and 215 bp would be generated for pC89, pC89-C7C and pC89-C10C templates (Figure 2.2). Wild-type f88-4 would produce a 429 bp fragment, whilst a f88-4/15 PCR product would migrate at 470 bp (Figure 2.3).

Colonies were picked with disposable 5 pi inoculating loops, dissolved in 20 pi

dH20 and 5 pi used as template for a 50 pi reaction mix (0.5 pM of each

primer, 0.2 mM dNTPs, 1.5 mM MgCb, 1.25 U Tag DNA Polymerase, 1 x

PCR buffer; all PCR reagents were purchased from Bioline). All PCR

amplifications were carried out at 95°C for 10 min, 30 x (95°C for 1 min, 65°C

for 1 min, 72°C for 1 min) and 72°C for 10' in a Mastercycler gradient thermal

cycler (Eppendorf). The sizes of amplified products were estimated by

subjecting 5 pi aliquots (to which 1 pi 6 x loading buffer was added) to gel electrophoresis on a 3% agarose gel, alongside 5 pi of Hyperladder IV

(Bioline). Glycerol stocks of individual colonies were prepared using the

TCw: --- — --- --- pC89F ^ pC89seq ^ TTCTAGAGATTACGCCAAGCCCCGTATTTTACCCGTTTAATGGAAACTTCCTCATGAAAAAGTCTTTAGTCCTCAAAGCCTCTGTAGCCGTTGCTACCCTCGTTCCGATGCTGT ^ __________ pC89R CTTTCGCTGCTGAGGGTGAATTCTAGGATCCCGCAAAAGCGGCCTTTGACTCCCTGCAAGCCTCAGCGACCGATATATCGGTTATGCGTGGGCGATGGTTGTTGTCATTGTCGG PC89-C7C: pC89F --- ^ pC89seq ^ TTCTAGAGATTACGCCAAGCCCCGTATTTTACCCGTTTAATGGAAACTTCCTCATGAAAAAGTCTTTAGTCCTCAAAGCCTCTGTAGCCGTTGCTACCCTCGTTCCGATGCTGT ^ --- pC89R CTTTCGCTGCTGAGGGTGAATTCGGCTGCNNKNNKNNKNNKNNKNNKNNKNNKNNKTGCGGCGCGGATCCCGCAAAAGCGGCCTTTGACTCCCTGCAAGCCTCAGCGACCGATA PC89-C10C: pC89F --- ^ pC89seq ^ TTCTAGAGATTACGCCAAGCCCCGTATTTTACCCGTTTAATGGAAACTTCCTCATGAAAAAGTCTTTAGTCCTCAAAGCCTCTGTAGCCGTTGCTACCCTCGTTCCGATGCTGT ^--- pC89R CTTTCGCTGCTGAGGGTGAATTCGGCTGCNNKNNKNNKNNKNNKNNKNNKNNKNNKNNKNNKNNKTGCGGCGCGGATCCCGCAAAAGCGGCCTTTGACTCCCTGCAAGCCTCAG

FIGURE 2.2 PCR and sequencing strategy for pC89 clones: Annealing sites for PCR primers (pC89F and pC89R) and the sequencing primer (pC89seq; red) within pC89, pC89-C7C and pC89-C10C (5’ - 3’ shown). The variable regions encoding 0 70 and 0100 libraries are highlighted in blue, as is the substituted two-base stuffer in the wild-type vector.

f88-4: f88-4F ^ f88-4seq ^ A G C T C G A G C T T A C T C C C C A T C C C C C T G T T G A C A A T T A A T C A T C G G C T C G T A T A A T G T G T G G A A T T G T G A G C G G A T A A C A A T T T C T T A A T G G A A A C T T C C T C A T G A A A A A G T C T T T A G T T C T T A A A G C A T C T G T T G C T G T T G C G A C T C T T G T T C C T A T G C T A A G C T T T G C C A A C G T C C C T G C A G A A G G T G A T G A C C C G G C T A A A G C T G C T T T T G A C T C T C T T C A G G C T T C T G C T A C T G A A T A C A T C G G C T A C G C T T G G G C T A T G G T G G T T G T T A T C G T T G G T G C T A C T A T T G G C A T C A A A C T T T T C A A A A A A T T C A C T T C T A A A G C G T C T T A A T G A A C T C A G A f884R TACCCAGCCCGCC TAATGA G C G G G C T t T T T T T T A A G C T A G C T T A T G A T G A f88-4/15: f88-4F ► f88-4seq ^ A G C T C G A G C T T A C T C C C C A T C C C C C T G T T G A C A A T T A A T C A T C G G C T C G T A T A A T G T G T G G A A T T G T G A G C G G A T A A C A A T T T C T T A A T G G A A A C T T C C T C A T G A A A A A G T C T T T A G T T C T T A A A G C A T C T G T T G C T G T T G C G A C T C T T G T T C C T A T G C T A A G C T T T G C C N N K N N K N N K N N K N N K N N K N N K N N K N N K N N K N N K N N K N N K N N K N N K C C T G C A G A A G G T G A T G A C C C G G C T A A A G C T G C T T T T G A C T C T C T T C A G G C T T C T G C T A C G C T G C T T T T G A C T C T C T T C A G G C T T C T G C T A C T G A A T A C A T C G G C T A C G C T T G G G C T A T G G T G G T T G T f884-F T A T C G T T G G T G C T A C T A T T G G C A T C A A A C T T T T C A A A A A A T T C A C T T C T A A A G C G T C T T A A T G A A C T C A G A T A C C C A G C C C G C C T A A T G A G C G G G C T T T T T T T T A A

FIGURE 2.3 PCR and sequencing strategy for f88-4 clones: Annealing sites for PCR primers (f88-4F and f88-4R) and the sequencing primer (f88-4seq; red) within f88-4 and f88-4/15 (5’ - 3’). The variable regions encoding the 15-mer library is highlighted in blue, as is the substituted six-base stuffer in the wild-type vector.

2.6.2 Clean-up of PCR reactions

Prior to sequencing, PCR reactions were treated with either ExoSAP-IT (Amersham-Pharmacia) or microCLEAN (Microzone) to remove un­

incorporated dNTPs and residual primers. The former contains both

exonuclease I and shrimp alkaline phosphatase enzymes for the digestion of free dNTPs and de-phosphorylation of 5’ single stranded DNA respectively. 2

pi and 5 pi PCR reaction were used for treatment with ExoSAP-IT and

microCLEAN respectively, and applied according to the manufacturer’s instructions. Final volumes were made up to 20 pi with dHaO.

2.6.3 DNA sequencing

5 pi purified PCR reaction was used for sequencing which corresponded to

100 - 200 ng DNA. Template DNA was added to 0.25 pM sequencing

primers (pC89seq or f88-4seq) and 8 pi DYEnamic™ ET Dye Terminator

(Amersham-Pharmacia) premix in a total volume of 20pl dH20. Following

thermal cycling at 25 x (95°C for 20 sec, 55 °C for 1 min 15 sec), sequencing

reactions were purified by standard isopropanol or ethanol precipitation (see

appendix A) and re-suspended in 12 pi loading buffer (supplied with the

DYEnamic kit). Solutions were then transferred to skirted 96-well plates

(Advanced Biosystems) and analysed using the MegaBACE 500 automated

capillary sequencer (Amersham-Pharmacia). Raw sequencing data was

sequences using the BioEdit analysis programme (http://www.mbio.ncsu.edu/ BioEdit/bioedit.htmh. A typical gel trace is shown in Figure 2.4.

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