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DNA fragments, for gel retardation or footprinting assays, were radiolabelled at one

end to produce high specific activity probes for each strand (Goodwin, 1990). One of two methods were employed, either the T4 polynucleotide kinase reaction (5' end) or end-filling

with reverse transcriptase (3' end), in o rd er to locate the labelled nudeotide(s) at the most

favourable position relative to the proposed nuclear protein binding site. An overview of

the procedure followed for the production of each DNA probe specifically labelled at one

end is given in Table 2.3 (p74) and th e region that it spans is given in Figure 2.1 (p75). Double-stranded oligonucleotides w ere labelled only by the T4 polynucleotide kinase

reaction, on both 5' termini for gel retardation assays.

i). T4 polynucleotide kinase reaction

Fifty microgrammes of the relevant plasmid DNA, this was either pBSII-LTRF or

-LTRfl which contain specific restriction fragments from the 5' or 3' regions of the HIV-1 LTR (see Figure 2.3 and Chapter 3, Section 3.4), was digested with the first restriction

C hapter 2 71

enzyme in a final volume of 100/4l. An aliquot was removed and the extent of digestion

checked by electrophoresis through a 1% agarose gel [see Section 2.83, i)] before proceeding to add 20 units of CIAP to the incubation buffer. This was incubated at 37°C

for a further 30 min to remove 5' terminal phosphates from the linearized DNA. After addition of 2 n\ 0.5 Af EDTA (pH 8.0) and 5 n\ 20% (w /v) SDS the solution was extracted twice with 100 ¿<1 of phenol/chloroform/iso-amyl alcohol (25:24:1) and then once with chloroform/iso-amyl alcohol (24:1). The DNA was precipitated with two volumes of

ethanol and centrifuged at 20,00Gg for 10 min. The pellet was washed once with 70% ethanol and the remaining ethanol removed by brief centrifugation and aspiration with a

micropipette before resuspension in 20/41 TE buffer. A portion (10/4g) of the DNA was

labelled with ^2p by mixing the following components: 4/41 of the digested and phosphatased DNA, 5/41 of kinase buffer (500 mAf Tris-HCl [pH 7.6), 100 mAf MgCl2, 50 mM DTT, lm M Spermidine. 1 mM EDTA), 12.5 n \ 32P-,dATP (5000 Ci/mmol; 125 /4Ci), 27.5/41 distilled water and 2/41 (20 units) T4 polynucleotide kinase in a final volume of 50/41. This was incubated at 37°C for 45 min before EDTA and SDS were added

to stop the reaction and the solution phenol extracted and ethanol precipitated (as before). The pellet was resuspended in 200/41 TE, then re-precipitated by the addition of 20/413 Af

sodium acetate (pH 7.0) and 2.5 volumes of ethanol. The D NA was centrifuged and washed

twice with 70% ethanol as described earlier, before being redissolved in 20/41 distilled water and digested with the second restriction enzyme in a volume of 50/41 for 1-2 hrs. The

following were then added: 1 /4l 0.5 M EDTA (pH 8.0), 2.5 /4l 20% (w/v) SDS and 12/41 20% (w/v) Ficoll, and the sample loaded into two wells o f a 5% native polyacrylamide gel

and electrophoresed at 150 V [see Section 2.83, iii)J. Formamide load dye (80% [v/vj deionized formamide, 10 mM NaOH. 1 mAf EDTA, 0.1% [w/v] Xylene Cyanol, 0.1% [w/v] Bromophenol blue) was placed in adjacent wells to m onitor the electrophoresis which was

continued until the bromophenol blue reached the bottom (2V4-3 hrs). The gel plates were separated from the apparatus, one glass plate removed and the gel face covered with Saran

Chapter 2 72

This was developed to locate the band of interest which was excised with a clean scalpel.

The DNA was extracted from the gel slice by the 'crush and soak' method [see Section 2.8.3, iv)] and resuspended in 100 /¿I TE to give a solution of 15,000-45,000 cp m /jd (2.4 - 7.2 x 10^ cpm/pg).

Double-stranded oligonucleotides were labelled in a similar procedure: oligonucleotide (5 pmol) was mixed with 2 pi kinase buffer, 2.5 p i 32P 7dATP (5000 Ci/mmol; 25 pC\), 1 p i T4 polynucleotide kinase and distilled water to give 20 pi

final volume and incubated at 37°C for 45 min. The reaction was stopped by the addition of 0.8 p\ 0.5 M EDTA (pH 8.0) and 1 pi 20% (w/v) SDS, mixed with 5 pi 20% (w /v ) Ficoll, and loaded into one well of a 10% non-denaturing polyacrylamide gel [see Section

2.8.3, in)]. This was electrophoresed at 150 V for approximately 1 hr (with formamide load dye in adjacent wells) and the labelled oligonucleotide located by autoradiography for

2 min as described earlier. After elution from the gel slice and removal of polyacrylamide

fragments [see Section 2.8.3, iv)] the solution was extracted once with

phenol/chloroform/iso-amyl alcohol (25:24:1) and ethanol precipitated in the presence of carrier tRNA (5 pg). The oligonucleotide was recovered by centrifugation a t 20,00Cfc for 25 min, washed twice with 70% ethanol and resuspended in 100 p\ TE. This gave a solution of 50,000-100,000 cpm/^1 (3.5 - 7.0 x 10^cpm/^g).

ii). End-filling with reverse transcriptase

The protocol described above for the labelling of DNA fragments w as followed

except the end-filling reaction was performed in place of the kinase reaction. Ten

microgrammes of DNA (4 p\), digested with the first restriction enzyme, was mixed with 5*1 RT buffer (100 mA/ [pH 8.3], 800 mA/ KCI, 100 mA/ MgCl2. 20mA/ dG T P , 20mA/ d r IF), 2 p\ 300 mA/ 0-mercaptoethanol, 12.5 pi 32P-adATP (6000 Ci/mmol; 125 *Ci), 12.5*11 32P-adCTP (6000Ci/mmol; 125*iCi), 12*il distilled water and 2*il (40 units)

AMV reverse transcriptase. This was incubated for 1 hr at 37 °C. The reaction was

C hapter 2 73

incorporation of two labelled nucleotides into each DNA molecule the exposure time for

autoradiography of the gel was reduced to 5-20 seconds and the final LTR fragment resuspended in 200 /d TE. This gave a solution of 135,000-175,000 cpm/iil (4.4 - 5.7 x 107 cpm/^g).