CONTROL DE ACCESO

Individual clones of the De Jong Human Genomic PAC Library which were identified by library screening were obtained from the UK MRC HGMP-RC. Sequenced clones which were identified by BLAST searches were obtained directly from Pieter de Jong, Roswell Park Cancer Institute, Buffalo NY, USA.

2.2.5(i) PAC DNA mini-prep

The PAC clone was plated on an LB agar plate (Section 2.1.3(ii)) containing 25mg/ml kanamycin (Section 2.1.1) and incubated overnight at 37°C. A single bacterial colony of the PAC clone was picked and used to inoculate 10ml Terrific Broth (Section 2.1.3(iv)) containing 25mg/ml kanamycin (Section 2.1.1) in a 50ml tube, which was incubated overnight at 37°C with shaking (200rpm). The tube was centrifuged at 2000g for 5 minutes. The bacterial pellet was resuspended in 2ml ice-cold DNA Prep Solution 1 (Section 2.1.8(v)) by vortexing and left on ice for 10 minutes. 4ml freshly made DNA Prep Solution 2 (Section 2.1.8(vi)) at room temperature was added, the solutions gently mixed and left on ice for 10 minutes. 2ml ice-cold DNA Prep Solution 3 (Section 2.1.8(vii)) was added, the solutions gently mixed and left on ice for 20 minutes. The tube was centrifuged at 2000g for 10 minutes. The supernatant was transferred to a 15ml tube, mixed with 5ml isopropanol and left at room temperature for 30-45 minutes. The tube was centrifuged at 2000g for 10 minutes. The supernatant was discarded and the DNA pellet was air-dried. The pellet was resuspended in 200jtil TE (Section 2.1.8(H)). 2p\ RNase A (Section 2.1.5) was added and the tube was incubated at 37°C for 15 minutes. The remainder of the protocol utilises the various parts of the Hybaid Recovery Plasmid Mini-prep Kit, supplied by Hybaid Ltd, Teddington, Middlesex (Spin Filter, Catch Tube, Binding Buffer, Wash Solution). The DNA solution was transferred to a Spin Filter held in a Catch Tube. 250jtil Binding Buffer was added, the solutions were mixed with a pipette and the tube was centrifuged at 16000g for 1 minute. The

liquid in the Catch Tube was discarded. 500/xl Wash Solution was added to the Spin Filter and the tube was again centrifuged at 16000g for 1 minute. The liquid in the Catch Tube was again discarded. The tube was centrifuged at 16000g for another minute and the Spin Filter was transferred to a clean Catch Tube. 50/zl TE (Section 2.1.8(h)) was added to the Spin Filter and the tube was incubated in a waterbath at 65°C for 10 minutes. The tube was briefly vortexed and immediately centrifuged at 16000g for 1 minute. The spin filter was discarded, leaving the purified DNA solution in the catch tube.

2.2.5(ii) PAC DNA midi-prep

The PAC clone was plated on an LB agar plate (Section 2.1.3(ii)) containing 25mg/ml kanamycin (Section 2.1.1) and incubated overnight at 37°C. A single bacterial colony of the PAC clone was picked and used to inoculate 50ml Terrific Broth (Section 2.1.3(iv)) containing 25mg/ml kanamycin (Section 2.1.1) in a 250ml conical flask, which was incubated overnight at 37°C with shaking (200rpm). The culture was transferred to a 50ml tube, which was centrifuged at 2000g for 5 minutes. The bacterial pellet was resuspended in 7.5ml ice-cold DNA Prep Solution 1 (Section 2.1.8(v)) and left on ice for 10 minutes. 15ml freshly made DNA Prep Solution 2 (Section 2.1.8(vi)) at room temperature was added, the solutions gently mixed and left on ice for 10 minutes. 7.5ml ice-cold DNA Prep Solution 3 (Section 2.1.8(vii)) was added, the solutions gently mixed and left on ice for 20 minutes. The tube was centrifuged at 2000g for 10 minutes. The supernatant was filtered through gauze into a clean 50ml tube. The volume was made up to 50ml with isopropanol, the solutions were mixed by inverting and left at room temperature for 30-45 minutes. The tube was centrifuged at 2000g for 10 minutes. The DNA pellet was resuspended in 3ml TE (Section 2.1.8(h)), transferred to a 15ml tube and 1ml of lOM NH^OAc (Section 2.1.8(xxxiii)) was added. The tube was left on ice for 20 minutes. The tube was centrifuged at 2000g for 10 minutes. The supernatant was transferred to a clean 15ml tube, 2.5 volumes of ethanol

was added and the tube was left on ice for 30 minutes. The tube was centrifuged at 2000g for 10 minutes. The supernatant was discarded and the DNA pellet was resuspended in 300/xl TE (Section 2.1.8(H)). The solution was transferred to a 1.5ml Eppendorf tube, 3/d RNase A (Section 2.1.5) was added and the tube was incubated at 37°C for 15 minutes. 300/tl of 28% PEG 8000/20mM M gClj (Section 2.1.8(xxxiv)) solution was added, the solutions were mixed by inverting and the tube was left at room temperature for 30 minutes. The tube was centrifuged at 16000g for 10 minutes. The supernatant was discarded and the DNA pellet was washed with 1ml 70% ethanol. The tube was centrifuged at 16000g for several minutes, the supernatant was discarded and the pellet was resuspended in lOO/il TE (Section 2.1.8(H)).

2.2.5(iii) PAC DNA maxi-prep

The FAC clone was plated on an LB agar plate (Section 2.1.3(H)) containing 25mg/ml kanamycin (Section 2.1.1) and incubated overnight at 37°C. A single bacterial colony of the FAC clone was picked and used to inoculate 250ml Terrific Broth (Section 2.1.3(iv)) containing 25mg/ml kanamycin (Section 2.1.1) in a 11 conical flask, which was incubated overnight at 37°C with shaking (200rpm). The culture was transferred to a 300ml Sorvall centrifuge bottle and centrifuged at 10700g for 10 minutes. The bacterial pellet was resuspended in 40ml ice-cold DNA Frep Solution 1 (Section 2.1.8(v)) and left on ice for 10 minutes. 80ml of freshly made DNA Frep Solution 2 (Section 2.1.8(vi)) at room temperature was added, the solutions gently mixed and left on ice for 10 minutes. 40ml ice-cold DNA Frep Solution 3 (Section 2.1.8(vii)) was added, the solutions gently mixed and left on ice for 20 minutes. The bottle was centrifuged at 10700g for 10 minutes. The supernatant was filtered through gauze into a clean 300ml Sorvall centrifuge bottle and 100ml isopropanol was added. The solutions were mixed and left at room temperature for 30-45 minutes. The bottle was centrifuged at 10700g for 10 minutes. The supernatant was discarded and the DNA pellet was resuspended in 7.5ml TE (Section 2.1.8(H)). The solution was transferred to a 15ml

tube, 2.5ml lOM NH^OAc (Section 2.1.8(xxxiii)) was added and the tube was left on ice for 20 minutes. The tube was centrifuged at 2000g for 5 minutes. The supernatant containing the DNA was transferred to a 50ml tube, 2.5 volumes of ethanol was added and the tube was left on ice for 30 minutes. The tube was centrifuged at 2000g for 10 minutes. The DNA pellet was resuspended in 500/tl de-ionised water and the solution was transferred to a 1.5ml Eppendorf tube. RNase A (Section 2.1.5) was added to 20jLig/ml and the tube was incubated at 37°C for 15 minutes. The DNA was then further purified using a Qiagen-tip Q-lOO Plasmid M ini-prep Kit, supplied by Qiagen Ltd., Crawley, West Sussex, according to the manufacturer’s instructions.

2.2.6 Genomic DNA Purification

2.2.6(i) From blood

Some genomic DNA samples prepared from peripheral blood were supplied by Dr Chris Jones (M olecular Haematology Unit, Institute of Child Health, London). The others were prepared by the following technique.

A 20ml blood sample was frozen at -20°C and then thawed. The blood was poured into a 50ml tube which was filled with Extraction Buffer (Section 2.1.8(xii)). The tube was rotated at room temperature for 10 minutes. The tube was removed from the rotator and centrifuged at 2000g for 10 minutes. The supernatant was removed. The process of adding Extraction Buffer, rotating, centrifuging and pouring o ff supernatant was repeated until most of the red colour had gone. The white blood cell pellet was resuspended in 5ml Extraction buffer. Proteinase K (Section 2.1.5) was added to

100/xg/ml final concentration and incubated overnight at 55°C.

2ml of 5M NaCl (Section 2.1.8(xxx)) and 14ml (2 volumes) of chloroform were added and the tube was rotated at room temperature for 1 hour. The tube was removed from

the rotator and centrifuged at 2000g for 10 minutes. The supernatant was removed, mixed with an equal volume of isopropanol and incubated at room temperature for 30- 45 minutes. The precipitated DNA was lifted out of the tube with a plastic loop and placed in a 1.5ml Eppendorf tube containing 1ml of 70% ethanol, which was left at room temperature for 1 hour. The tube was centrifuged at 11600g for 5 minutes. The DNA pellet was resuspended in 200/xl TE (Section 2.1.8(h)).

2.2.6(ii) From cultured cells

The cells were spun down at 220g for 10 minutes and resuspended in 5ml SE Buffer (Section 2.1.8(xxxix)). 250^1 of 20% SDS (Section 2.1.8(xxxviii)) was added, proteinase K (Section 2.1.5) was added to lOOjUg/ml final concentration and the tube was incubated overnight at 55°C. The solution was mixed with an equal volume of phenol, then centrifuged at 2000g for 10 minutes. The upper phase was transferred to a clean 15ml tube, mixed with an equal volume of chloroform and centrifuged at 2000g for 10 minutes. This step was repeated. The upper phase was transferred to a clean 15ml tube, m ixed with an equal volume of isopropanol and incubated at room temperature for 30-45 minutes. The precipitated DNA was lifted out of the tube with a plastic loop and placed in a 1.5ml Eppendorf tube containing 1ml of 70% ethanol, which was left at room temperature for 1 hour. The tube was centrifuged at 11600g for 5 minutes. The DNA pellet was resuspended in 200/tl TE (Section 2.1.8(h)).

2.2.7 Restriction digests

2.2.7(i) With PAC DNA

500ng of PAC DNA was digested with EcoRI (Section 2.1.5) in a 20/tl reaction volume containing 1/xl (lOU) restriction enzyme and 2/tl restriction enzyme buffer by overnight incubation at 37°C. The digests were resolved on a 1% TAE agarose gel which was

run overnight at 25V.

2.2.7(ii) With Cosmid DNA

500ng o f cosmid DNA was digested with EcoRI (Section 2.1.5) in a 20/xl reaction volume containing 1/xl (lOU) restriction enzyme and 2/xl restriction enzyme buffer by overnight incubation at 37°C. The digests were resolved on a 1% TAE agarose gel which was either run overnight at 25V or for 2-3 hours at lOOV.

2.2.7(iii) With Genomic DNA

Restriction digests of genomic DNA for Southern blotting used 10-15/xg of DNA in a 100/xl digest containing 10/xl (lOOU) restriction enzyme (Section 2.1.5) and 10/tl restriction enzyme buffer, which was incubated overnight at the appropriate temperature for the enzyme. The digests were reduced to a volum e o f 15-20/xl in a DNA Concentrator (Jencons (Scientific) Ltd, Leighton Buzzard, Bedfordshire), before adding 2/xl DNA loading buffer (Section 2.1.8(iv)) and loading on a 1% TAE agarose gel. The gel was run overnight at 25V.

2.2.8 Southern Blotting

The agarose gel to be blotted was first washed in 0.25M HCl (Section 2.1.8(xx)) for 10- 15 minutes, then in Denaturing Solution (Section 2.1.8(i)) for 30 minutes.

To set up the blot 3MM paper was wrapped around an inverted gel tray and placed in a larger plastic tray. 500ml of 0.4M NaOH (Section 2.1.8(xxxi)) was poured over the paper and a plastic 10ml pipette was used to iron out any bubbles. The agarose gel was then placed face down on the 3MM paper. Extra 0.4M NaOH was added around the edges of the gel and any bubbles were ironed out. N+- hybond membrane (wetted in

Denaturing Solution) was placed over the gel and any bubbles were ironed out. Three sheets o f 3MM paper (wetted in Denaturing Solution) were laid over the membrane one at a tim e in the same way. Dry paper towels were then piled on top of the wet 3MM paper. Another plastic tray containing a heavy weight was placed on top of the paper towels. Strips of Parafilm were placed around the edges of the gel to prevent the NaOH soaking straight up into the paper towels. The blot was left overnight.

The paper towels and 3MM paper were removed from on top of the membrane. The membrane and gel were turned over together and the positions of the loading wells in the agarose gel were marked onto the filter with a pencil. The membrane was rinsed in Neutralising Solution (Section 2.1.8(xxxii)), then 2xSSC (Section 2.1.8(xli)), each for several minutes. The membrane was dried for 15-30 seconds (depending on size) in a 600W microwave oven on full power.

2.2.9 Dot blotting

Dot blots of DNA from YAC, FAC and cosmid clones were made using the Bio-Dot M icrofiltration Apparatus supplied by BioRad Laboratories Ltd., Hemel Hempstead, Hertfordshire, according to the manufacturer’s instructions.

2.2.10 Radioactive Probe labelling

2.2.10(i) With Genomic DNA

Genomic DNA was radioactively labelled with a^^P-dCTP by incorporation with Klenow fragment as described for PAC clones below, although 1/xg of DNA was used rather than lOOng.

2.2.10(ii) With YAC DNA

YAC DNA was radioactively labelled with a^^P-dCTP by incorporation with Klenow fragment. 300ng of yeast DNA from the YAC clone was made up to 39^1 in a 1.5ml Eppendorf tube with de-ionised water. The tube (the cap having been pierced with a needle) was placed in a heating block at 95 °C for 5 minutes, then placed on ice. 12/xl of Hexamer Mix (Section 2.1.8(xxii)), 3/tl (15U) Klenow fragment (Section 2.1.5) and 6/xl a^^P-dCTP (Section 2.1.4) were added and the tube was incubated at room temperature for 2-3 hours.

lOOfJil o f TE (Section 2.1.8(h)) was added to the labelled probe, which was then

transferred to the top of a Sephadex G50 column held in a 15ml tube. The column was spun at 350g for 3 minutes. The amount of incorporation of the radionucleotide into the probe was determined by comparing the counts from the column and from the catch tube.

The probe was transferred to a 1.5ml Eppendorf tube containing 300/xl 20xSSC (Section 2.1.8(xlii)) and 150/xl human Cot-1 DNA (Section 2.1.4) and incubated at 95°C for 2-5 m inutes, then left on ice for 2 m inutes. The tube was then incubated in the hybridisation oven for 1.5-2 hours at 65°C.

2.2.10(iii) With PAG DNA

PAC DNA was radioactively labelled with a^^P-dCTP by incorporation with Klenow fragment. lOOng of PAC DNA was made up to 13^1 in a 1.5ml Eppendorf tube with de-ionised water. The tube (the cap having been pierced with a needle) was placed in a heating block at 95°C for 5 minutes, then placed on ice. 4fi\ of Hexamer Mix (Section 2.1.8(xxii)), 1/xl (5U) Klenow fragment (Section 2.1.5) and 2/xl a^^P-dCTP (Section 2.1.4) were added and the tube was incubated at room temperature for 2-3 hours.

lOOjLil of TE (Section 2.1.8(H)) was added to the labelled probe, which was then transferred to the top of a Sephadex G50 column held in a 15ml tube. The column was spun at 350g for 3 minutes. The amount of incorporation of the radionucleotide into the probe was determined by comparing the counts from the column and from the catch tube.

The probe was transferred to a 1.5ml Eppendorf tube containing 100/xl 20xSSC (Section 2.1.8(xlii)) and 50/xl human Cot-1 DNA (Section 2.1.4) and incubated at 95°C for 2-5 m inutes, then left on ice for 2 m inutes. The tube was then incubated in the hybridisation oven for 1.5-2 hours at 65°C.

2.2.10(iv) With Cosmid DNA

Cosmid DNA was radioactively labelled with a^^P-dCTP by incorporation with Klenow fragment exactly as described in Section 2.2.10(iii).

2.2.10(v) With pU C DNA

DNA of pUC clones was radioactively labelled with a^^P-dCTP by incorporation with Klenow fragment exactly as described in Section 2.2.10(iii).

2.2.lO(vi) With PCR Products

PCR products larger than 200bp were radioactively labelled with a^^P-dCTP by incorporation with Klenow fragment exactly as described in Section 2.2.10(iii). PCR products smaller than 200bp were radioactively labelled with y^^P-ATP by end-labelling with PNK exactly as described for oligonucleotides in Section 2.2.10(vii).

2.2.10(vii) With Oligonucleotides

Oligonucleotides were radioactively labelled with y^^P-ATP by end-labelling with PNK (polynucleotide kinase). 50ng of oligonucleotide, 1/d of lOx PNK buffer, 1/d (lOU) of PNK (Section 2.1.5) and 5/xl y^^P-ATP (Section 2.1.4) were mixed in a 1.5ml Eppendorf tube and made up to lO/xl with de-ionised water. The tube was incubated at 37“C for 30 minutes.

lOO/il of TE (Section 2.1.8(h)) was added to the labelled probe, which was then transferred to the top of a Sephadex G50 column held in a 15ml tube. The column was spun at 350g for 3 minutes. The amount of incorporation of the radionucleotide into the probe was determined by comparing the counts from the column and from the catch tube.

2.2.11 Hybridisation of Radioactive Probes

2.2.1 l(i) With Genomic DNA Probes

The blotted membrane was rinsed in 2xSSC (Section 2.1.8(xli)), rolled up, inserted into a hybridisation bottle (which had also been rinsed with 2xSSC) and unrolled. 20ml Hybridisation Mix (Section 2.1.8(xxiii)) was added to the bottle, which was placed in the hybridisation oven at 65°C for at least 15 minutes. The probe was added to the bottle, which was incubated overnight in the hybridisation oven at 65 °C.

The m em brane was washed (in hybridisation oven at 65°C) for 30 m inutes in H ybridisation Wash 1 (Section 2.1.8(xxiv)) and 30 minutes in Hybridisation Wash 2 (Section 2.1.8(xxv)). The membrane was blotted dry with paper towel before being wrapped in Saran wrap, placed in autoradiograph cassettes, overlaid with Kodak X-

OMAT XAR-5 film (Section 2.1.6) and left overnight to expose the film. The film was developed.

Membranes carrying genomic DNA were stripped of radioactive probe by incubation for several minutes in boiling 0.1xSSC/0.1% SDS solution (Section 2.1.8(xliii)). Membranes carrying PAC, cosmid or pUC DNA were stripped o f radioactive probe by washing in 0.4M NaOH (Section 2.1.8(xxxi)) at 65°C for 30 minutes.

2.2.11(ii) With YAC DNA Probes

Hybridisation bottles were rinsed with 2xSSC (Section 2.1.8(xli)). The seven filters of the PAC library were rinsed in 2xSSC, rolled up, inserted into bottles three hybridisation bottles (which had also been rinsed with 2xSSC) and unrolled. 20ml Hybridisation Mix (Section 2.1.8(xxiii)) was added to each of the bottles, which were placed in the hybridisation oven at 65“C for at least 15 minutes.

The probe was divided equally between the hybridisation bottles, which were incubated overnight in the hybridisation oven at 65°C.

The filters were washed (in hybridisation oven at 65°C) for 30 minutes in Hybridisation W ash 1 (Section 2.1.8(xxiv)) and 30 minutes in H ybridisation W ash 2 (Section 2.1.8(xxv)). The filters were blotted dry with paper towel before being wrapped in Saran wrap, placed in autoradiograph cassettes, overlaid with Kodak X-OMAT XAR-5 film (Section 2.1.6) and left overnight to expose the film. The film was developed.

The filters were stripped of radioactive probe by incubation for several minutes in boiling 0.2xSSC/0.1% SDS solution (Section 2.1.8(xliii)) in order to be reused.