DOCTRINA JURISPRUDENCIAL RESPECTO A LA MEDIDA

A restriction map, linear map o f the relative positions o f restriction enzyme recognition sites, is the first step in analysing a piece o f DNA o f unknown structure. This requires a combination o f partial (reduced digestion so that not every site is cut), single and double (cleavage by two different enzymes) digestions and sizing o f the resulting fragments by gel electrophoresis.

Restriction mapping o f the OTR cDNA insert was carried out using the foUowing enzymes in single and double digests:

Restriction Recognition Endonuclease Sequence

Acc I GT|(A or C)(G or T)AC

EcoR I GjAATTC Hinc n GTPylPuAC Hind m A|AGCTT Kpn I GGTACjC Pst I CTGCA|G Sac I GAGCT|C Xho I C|TCGAG

*Py - Pyrimidine; Pu - Purine

This set o f restriction enzymes was chosen on the basis that bluescript SK+ plasmid and M13mpl8/mpl9 phagemid cloning vectors contained a single site for each located exclusively to the poly cloning site. All enzymes selected had 6 base pair recognition sequences (6-cutters).

All enzymes were from Northumbria Biologicals Ltd, Cramlington, Northumberland, U.K. Digests were carried out in the reaction buffers provided by the manufacturer and used according to their instructions. Restricted DNA fragments were analysed on 0.8% agarose/TBE gel containing 1 pg/ml EtBr.

2.2.2.6 Gel purification of restricted fragments ('Gene Clean )

A 1% low melting point (LMP) agarose gel was prepared by heating 0.5 g LMP agarose (Gibco-BRL Life Technologies Ltd) in 50 ml TAE gel running buffer (Appendix A; section 2.2.2.3). The gel band corresponding to the required fragment was excised from the gel, under UV light, using a sterile scalpel blade and transferred to a pre-weighed sterile 1.5 ml Eppendorf tube. The fragment was purified from the gel using the Geneclean® II kit (Stratech Scientific, Luton, Bedfordshire, U.K.). The weight o f the excised band was determined and 3 volumes o f Nal stock solution (4°C)

added. The tube was placed at 50°C in a water bath for 5 min to melt the agarose and the contents mixed at regular intervals. An insoluble silica matrix (GLASSMILK®) was added (5 pi for suspensions containing 5 pg or less o f DNA and an additional microlitre for each 0.5 pg o f DNA above 5 pg) and the tubes placed on ice for 5 min to allow binding o f the DNA to the matrix. The süica matrix-bound DNA was centrifuged (11600g, 5 sec) and the Nal supernatant transferred to another tube, this contained DNA and was put through the binding procedure again if required. The pellet was washed with between 10-50 volumes (350 pi) NEWWASH® (14 ml NEWCONCENTRATE® in 590 ml 50% ethanol) centrifuged as before (11600g, 5 sec) and the supernatant discarded. The wash step was repeated twice. The washed pellet was resuspended in 5 pi 1 x TE buffer and incubated at 50°C for 2-3 min to elute the DNA from the GLASSMILK®. The tube was centrifuged (11600g, 30 sec) and the supernatant, containing the eluted DNA, carefully removed to a fresh sterile tube. Approximately 80% o f the bound DNA eluted in this first step and the elution was repeated to obtain a further 10-20% recovery. The amount o f DNA recovered was determined on a 0.8% agarose gel run alongside 1 pg Hind WJX DNA size marker.

2.1.2.7 Construction of single-stranded templates

2.2.2.7.1 Preparation of bacteriophage M13 vector

MIS is a filamentous bacteriophage specific to Escherichia coli (Hofschneider, 196S) which contains a single-stranded closed circular molecule o f DNA approximately 6400 nucleotides in length. When used as a cloning vector this bacteriophage generates large quantities o f DNA molecules that carry the sequence o f one strand o f the foreign DNA. Such single stranded DNAs were the templates o f choice in this study fo r DNA sequencing by the dideoxy chain-termination method described in section 2.2.4.1 below.

The MIS vectors used in this study were M lSm plS and M lS m pl9 (both 72500 nucleotides in length). These contain cloning regions with IS different cleavage sites that can accept DNA fragments produced by digestion with many different restriction

enzymes. The m pl8 and m pl9 vectors differ from each other only in the orientation o f the asymmetrical polycloning site. Hence, a restrictedfragment o f double stranded foreign DNA can be inserted in opposite orientations in the two vectors. One o f the two strands o f the fragment will be attached to the (+) strand o f m pl8 and the complementary strand w ill be attached to the (+) strand o f mpl9. Vectors o f the mp series are derived from a recombinant MIS bacteriophage (M lSmpl) and as such confer ampiciUin resistance to the host bacterial cells and contain the regulatory sequences and coding information for the first 146 amino acids o f the ^-galactosidase gene (lacZ). The F'-plasmid o f the host cell, in this case E coli strain JMIOI, lacks coding information fo r amino acids 11-41 o f the same gene. Therefore, unsubstituted vectors a-compliment the F'-plasmid to produce Lac-^ bacteria which form blue

plaques on plates containing X-gal (5-bromo-4-chloro-3-indoyl-^-D-

galactopyranoside; a substrate for (3 -galactosidase) and IPTG (isopropylthio-^-D- galactosidase; a gratuitous inducer o f ^-galactosidase). Insertion o f foreign DNA into the polycloning site in the lacZ region usually eliminates this 'complementation' by disrupting the protein coding region o f the 5' end o f the lacZ gene and gives rise to pale blue or colourless plaques. This represents a simple test to score for recombinants and by using m pl8 and mpl9, as dual vectors, it is possible to use a single primer ('universal primer') to determine the sequence o f nucleotides on opposite strands from each end o f the inserted DNA.

M13mpl8 and M13mpl9 (500 ng; Boehringer Mannheim, Lewes, East Sussex, Brighton) were digested with the appropriate restriction enzymes (equivalent to the enzymes used to generate the restricted fragment of OTR cDNA) as described in sections 2.2.2.2 and 2.2.2.5 An aliquot o f the digest was checked on an agarose gel (Section 2.2.2.3). The remaining DNA was extracted with phenol:chloroform:isoamyl alcohol (24:24:1; section 2.2.2.1) and recovered by ethanol precipitation. When restriction enzymes were used that generated blunt ends the vector DNA was dephosphorylated after digestion, to increase the efficiency o f ligation, using calf intestinal alkaline phosphatase (NBL Gene Science Ltd) according to the method reported by Sambrook et al. (1989).

2.2.2.7.2 Ligation of OTR cDNA fragments and M13 vector

'Gene cleaned' OTR cDNA fragments were combined with restricted M13 vector DNA in a 3-10:1 (insertivector) molar ratio (10 gl final reaction volume). The vector and insert were added to a sterile 1.5 ml Eppendorf tube and the volume made up to 8 pi with sterile water. The DNA was heat-denatured at 65°C for 10 min and then snap- cooled on ice for 2-5 min. T4 ligase buffer (lOx; NBL Gene Sciences Ltd) was added (1 pi; NBL Gene Sciences Ltd) and fhck mixed, followed by T4 DNA ligase (Ipl at

>10 units/pl) gently mixed using a Güson pipette. The ligation reaction was pulsed

in a microfuge and incubated at room temperature for 10 min and then left at 4-8“C overnight.

Positive and negative control ligations were set up containing restricted vector alone plus or minus ligase respectively. Ligations were either used immediately or stored at -20°C until required.

2.2.2.T.3 Preparation of competent cells

E. coli (JMlOl) overnight (o/n) cultures were diluted 1:100, 0.5 ml culture in 50 ml 2TY medium (Appendix A), in a sterile 250 ml baffled conical flask (Phillip Harris Scientific). A fiuther 100 pi o/n culture was used to inoculate 20 ml 2TY in a universal tube (Phillip Harris Scientific) and both dilutions were grown in an orbital shaker for 2 hours to late log phase (A550 0.4-0.6). The universal culture was transferred to a 37®C incubator until required for transformation. The cells fi*om the 50 ml culture were recovered by centrifiigation (2000g, 10 min) at 4®C and gently resuspended in 0.5 volume (25 ml) ice cold freshly prepared 50 mM calcium chloride and incubated on ice for 20 min. The cells were recovered as before (2000g, 10 min) resuspended in 0.1 volume (5 ml) ice cold 50 mM CaClz then incubated on ice for 4 hours. Competent cells were either used immediately, kept on ice or stored at 4°C o/n until required.

The transformation method described was modified fiom that previously reported by Sambrook et al. (1989).

E. coli (JMlOl) competent cells (100 pi) were transformed with half o f the ligation