Development of a quantitative competitive reverse transcriptase PCR (QCRT-PCR)

The following section describes the methodologies used for the development of a quantitative competitive reverse transcriptase PCR assay (QC RT-PCR) for the detection of HCM V in blood and tissue samples.

2.7.1 Preparation of the gB 149bp insert for cloning into pT3/T718U vector

The original pUC13 plasmids containing the 149bp inserts of either wild type gB or control gB with the mutated Hpa1 restriction site were digested with EcoR1 and Hind III in a double digest to remove the insert from the multiple cloning site. 10/^1 of pUC13 plasmid DNA (500ng//^l) with either sequence of the gB insert was digested with 1.0//I EcoRI (10 units//^l) and Hindlll (10 unitsZ/zl), 3//I 10X buffer B (lO m M Tris-HCI, lOOmM NaCI, 5mM IVIgClg Im M p-mercaptoethanol) made up to a final volume of Z0^\

with S D W . The reaction mixture was digested at 37°C for 2 hours. 2^A of each digestion was removed, 8//I of SD W and 5/^1 of loading dye was added, the products were

then analysed on a 1% agarose gel in 1XTBE buffer together with V g A Hindlll/EcoRI marker and V g PCR marker (Promega 100-1000bp).

2.7.2 Preparation of pT3/T718U vector

The vector pT3/T718U (Pharmacia) was digested with EcoR I and Hind III for 2 hours at 37°C to open the multiple cloning site to allow unidirectional insertion of the gB DNA fragments by cohesive end ligation. 10/^1 of the pT3/T7 18U (lOOng/^il) vector was digested with 1.0//I EcoRI (10 units/^^l) and I.O^^I Hindlll (10 units/;^l), 3/^1 10X buffer B (lOmM Tris-HCI, lOOmM NaCI, 5mM MgClj, Im M P-mercaptoethanol) made up to a final volume of 30/^1 with lO^^I SDW. The reaction mixture was digested at 37°C for 2 hours.

2^À of the digestion reaction was removed together with 2^\ of the uncut vector (diluted 1:10), 8/^1 of S D W and of loading dye was added and the DNA analysed on a 1% agarose gel in 1XTBE buffer together with 1/^g X Hindlll/EcoRI marker and 1/^g PCR marker (Promega 100-1000bp) to verify complete digestion of the vector.

2.7.3 Purification of pT3/T718U and the gB wiid type and control inserts

Once the sizes of the inserts and the vector had been confirmed by gel electrophoresis, the remainder of the digests from section 2.7.1 and section 2.7.2 were purified from a 1% low melting point (1% w/v, Sigma) molecular biology grade low melting point agarose in 1XTBE, and used for ligation. The pT3/T718U vector DNA and inserts were analysed on a 1% low melting point gel at 75 volts in 1XTBE buffer for approximately 45min, or until when visualised the insert fragment had separated sufficiently from the pUC13 vector. The gel fragments of interest were excised from the gel using a scalpel under short wave length U.V to minimise DNA mutagenesis. DNA was purified from the gel using the Gene Clean II kit (Bio, 101, USA) according to the manufacturer’s instructions. Briefly the gel fragm ent was weighed, and V^ volume of TBE modifier buffer added followed by 4.5 volumes of sodium iodide (Nal) stock solution. The fragments were melted at 55°C for 5

minutes, of GLASSMILK suspension was added mixed and incubated on ice for 5 minutes to allow the DNA to bind the silica matrix. The GLASSMILK was pelleted at 13,000g for one minute and the supematant removed. The pellet was washed three times with 700^^1 N EW W ASH and the DNA eluted from the matrix by incubation at 55°C for 3 minutes in T.E (pH 8.0). A 2//I aliqout of each of the purified 149bp gB inserts and the vector DNA was analysed on a 1% agarose with V g A Hindlll/EcoRI marker and l^^g PCR marker (Promega 100-1000bp) to confirm the purity and size of the DNA.

2.7.4 Cohesive end ligation of the 149bp gB Inserts Into pT3/T718U vector

100ng of the 149bp gB inserts were ligated into 10ng of the vector (as a 1:1 ratio) in the presence of 1.25 units of T4 DNA ligase (Northumbria), Im M ATP (Amersham) in 1/^110X ligation buffer (0.5M Tris-HCI pH 7.5, lOOmM MgClg, lOOmM DDT, 500/^g/ml bovine serum albumin) made up to a total volume of lO^^I with SDW . Control ligations of the linearised vector alone and the insert alone with all ligation reaction components were also prepared. Ligation reactions were incubated at 16°C overnight. 10/^1 of each ligation reaction was then used to transform 100/^1 of competent Eschericia coli (E.cofi) JM 109 cells as described.

2.7.5 Preparation of transformation competent E.co/i JM109 cells

JM109 cells (Pharmacia) carrying the P episome with the following genotype were used for all transformation experiments:

e14'(M rcA ) æcA1 endA1 gyrA96 thi-1 supE44 relA1 A{lac-proAB) [F’

traD36 proAB lacPZA M 15\

A single colony from a agar plate (0.5% (w/v) yeast extract (Sigma), 2% (w/v) tryptone (Oxoid), 20m M MgSO^, 1.4% agar (Sigma) the pH of the agar was adjusted to 7.6 with KOH prior to autoclaving) streaked with a stock culture of E.co//JM 109 cells was picked aseptically and inoculated into 7ml YB media (0.5% (w/v) yeast extract (Sigma), 2% (w/v) tryptone (Oxoid), 20mM MgSO^, and the pH of the media adjusted to 7.6 with KOH prior to autoclaving), and incubated for 2-3 hours at 37°C in a shaking incubator (250 rpm) until the O D5 5 0 reached 0.3. 5ml of the culture was used to inoculate 100ml sterile y s

media (pre-warmed to 3 / b ) in a conical flask, the culture was shaken (250 rpm) at 37°C for approximately 2 hours or until the OD5 5 0 reached 0.48. The cells were chilled on ice