Yeast Artificial Chromosomes (YAC) Methods

2.5.1 Yeast strain and Vector

The ICRF (Larin et al., 1991) and ICI (Anand et al, 1990) human 48, XXXX YAC library were used in these studies. The cloning vector used to construct the libraries is pYAC4 which comprises the ds-acting yeast chromosomal sequences joined to segments of the bacterial plasmid vector pBR322. This allows propagation of the YAC vector in

E. coli., by standard procedures.

2.5.2

Preparation of YAC D NA in plugs

YAC done DNA was prepared according to Vollrath and Davies (1987), with slight modification. Yeast dones were streaked out onto AHC agar plates (section 2.9) and incubated at 30“C overnight. A single large colony was picked into 10ml AHC broth (section 2.9) and grown at 30“C for 24 hours; the culture was subsequently used to seed a 100ml or 200ml culture in AHC broth. This was grown with agitation at 30°C for 40

hours after which the O.D.600 was measured and the number of cells were calculated (O.D^OO of 0.3 corresponds to 3.3 x 10^ cells/ml).

Cells were pelleted by centrifugation for 10 minutes at 4K, and resuspended in 50ml SCE solution (section 2.9). The cells were re-pelleted and resuspended in SCEM (SCE with 30mM p-mercaptoethanol, made fresh each time) to a final concentration of 2.5 X 10^ cells per ml. Lyticase (Sigma) was added to a final concentration of 120jug/ ml

and incubation was performed at 30“C for 1 hour. Low melting point (LMP) agarose (1%) was prepared in IM sorbitol (section 2.9) and kept molten at 55“C. Yeast cells (0.5ml) were mixed with an equal volume of LMP agarose and 200^1 aliquots were dispensed into plastic moulds and left to set at 4“C.

The plugs were placed in proteinase K/sarcosyl solution (section 2.9) and incubated at 50“C for 2 days. Following extensive rinsing with TE buffer, proteinase K was inactivated by treatment with 0.04mg/ ml PMSF (phenyl-methyl-sulphonyl-fluoride) in TE (section 2.9) with incubation at 50“C for 30 minutes. The plugs were then rinsed three times in TE buffer and stored at 4“C.

2.5.3

Pulsed Field Gel Electrophoresis (PFGE)

Pulsed field gel electrophoresis is a powerful technique for resolving DNA molecules in the megabase size range (Schwartz and Cantor, 1984), and enables large regions of genomic DNA to be mapped and analysed. Its wide separation range has bridged the size gap between conventional gel electrophoresis (up to 50kb) and cytogenetic techniques (5Mb). It is hence capable of separating chromosome size DNAs from the yeast genome.

In PFGE, DNA molecules undergo continuous re-orientation caused by periodic changes in alternating electric fields. The duration of the alternating electric field is known as the switch time or pulse time. Each time the electric field is switched, the DNA molecules must re-orientate or change their direction of migration in the gel matrix. Larger DNA molecules require longer times to re-orientate with each change of the electric field whereas smaller molecules that can re-orientate quickly, will spend a larger portion of each switch interval migrating with the field. Hence, as the size of the DNA molecules increases, the pulse time must be increased to resolve these molecules. The experiments described here were conducted on the CHEF-DRII apparatus (Biorad) based on the CHEF (clamped homogeneous electric fields) technique (Carle and Olson, 1984)

2.5.3.1 Casting and loading the gel

100ml of 1% agarose (Molecular Biology Certified; Biorad) in 0.5 X TBE buffer (section 2.9) was poured into the casting stand (standard casting stand. Biorad) and allowed to set for 1 hour at room temperature. Sample plugs were loaded while the gel remained in the casting stand. Care was taken so that samples were less than 90% of the height of the well. They were then sealed in place by filling each sample well with 1% low melting agarose (Biorad). The agarose was allowed to solidify for 15 minutes.

2.5.3.2 Gel electrophoresis

The electrophoresis chamber was rinsed briefly with two litres of pre-cooled distilled water. Two litres of pre-cooled 0.5 X TBE electrophoresis buffer was poured into the chamber and allowed to circulate briefly through the recirculating pump. The gel was placed in the centre of the chamber and the buffer flow through the pump was adjusted to IL/hour so that the gel was not disturbed during electrophoresis. The following electrophoresis parameters and voltage were selected to separate DNA in the range 450kb-225kb (accurate operating instructions can be found in the Biorad CHEF-DR n manual):

a) Pulsewave 760: Initial A Time: 25 seconds. Final A time: 25 seconds (note: this pulse mode is called ramping), b) Model 200/2.0 power supply: Voltage : 175V, Run Time: 20 hours. After the first run a) Pulsewave 760 was set again to:Initial A Time: 45 seconds. Final A Time: 45 seconds, b) Voltage: 175V, Run Time: 20 hours. Gels were run in the cold room to ensure the temperature was maintained at approximately 14°C during electrophoresis.

2.5.3.3 Gel staining

After the 40 hour run the pulse field gel was placed into 0.5^zg/ ml ethidium bromide solution (freshly prepared) and stained for 30 minutes. It was then destained in distilled water for 15 minutes, visualised on a UV transilluminator and photographed.

2.5.4

Isolation of YAC clones from Pulse Field gels

YAC DNA which needed to be separated and isolated from Pulse Field gels was run in 1% low melting point agarose (Molecular Biology Certified; Biorad). Casting, loading and gel electrophoresis was carried out as described in section 2.5.3.1 and section 2.5.3.2 respectively. The required YAC band was then cut out of the ethidium bromide

stained gel under UV light. The gel slice was then put into a small petri-dish containing a little TE buffer pH8.0 and stored at 4“C for further use.

2.6

The Polymerase Chain Reaction (PCR)

2.6.1

General considerations

Primer design, thermal cycling conditions, dNTP concentration and magnesium concentration were initially based on parameters reviewed by Innis and Gelfand (1990) and Saiki (1990). Different sets of primers were individually optimised, based upon their 3' complementarity and primer melting temperatures (Tm) calculated by using the formula 4(G + C) + 2(A + T) = Tm (melting temperature)

then reducing the annealing temperature by 2“-5°C (for primers up to 20 bases in length), or, more accurately for longer primers by using computer software to analyse primer pairs (see section 2.8.4).

Cycle length and number varied between 30 and 40 cycles, depending upon the template, particular methodology, and the model of thermocycler used. These will be noted where necessary. Once optimised, reactions were generally performed on the same machine, it being one of the following models: a Hybaid Thermal Cycler, a Hybaid OmniGene, or a Perkin Elmer GeneAmp PCR system 9600.

2.6.2

Standard parameters for a typical PCR

Unless otherwise stated, the following constitute a 'standard' PCR reaction in a SOjLil volume. PCR amplifications were performed on 150ng of genomic DNA in 1 X Taq reaction buffer (NBL, Bioloine, Promega: lOmM Tris-HCl, 50mM KCl, 1.5mM-2.0mM MgC12 and 0.1% non-ionic detergent), 0.2mM of each dNTP, 25 pico moles of each primer, and 0.3-0.5 units of Taq polymerase (NBL, Bioloine, Promega). The reaction mixtures were overlaid with one drop of mineral oil to prevent evaporation. After a routine initial dénaturation step of 95°C for 3 minutes the cycling conditions were:- denaturation at 94°C for 5-30 seconds, annealing at the appropriate temperature for 30 seconds and extension at 72°C allowing for approximately 1 minute per kilobase extension time.

2.6.2.1 PCR from a bacteriophage plaque

Heat dénaturation of a phage lysate destroys the bacteriophage protein coat releasing DNA into the surrounding media, facilitating the use of PCR to amplify DNA inserts of interest. Isolated plaques were stabbed into separate eppendorf tubes which contained a few ml of SM buffer and a small drop of chloroform and stored at 4°C. An aliquot (3jul) of the phage lysate was used as a template for PCR as described in section

2.6.2.

2.6.2.2 PCR from a bacterial colony

Heat dénaturation of a bacterial colony destroys the bacterial cell wall releasing plasmid DNA into the surrounding media. The DNA insert of interest can then be amplified by PCR. Half of the bacterial colony of interest was picked into the PCR reaction tube with a toothpick and PCR was carried out as described in section 2.6.2. The rest of the bacterial colony was stored on L-agar plates at 4“C for future use.

2.6.2.3 PCR from YAC plug DNA

YAC DNA isolated in plugs (see section 23.2) was used in PCR by cutting a small piece of the plug DNA using a clean scalpel and transferring it into a 0.5/il microfuge tube. The agarose plug was then melted at 95“C for 10 minutes. An equal volume of water was added and an aliquot of 3^zl was used as template DNA in the PCR (section 2.6.2).