Determinación de parentesco

2.2.1. cDNA libraries

Four main cDNA libraries were utilised in this study: JY (B-lymphoblastoid cell line), DX3 (malignant melanoma), y U937 (y-IFN induced macrophage) and NW (B-lymphoblastoid cell line). These were a gift from Dr. David Simmonds (ICRF, Oxford). The libraries were constructed according to the method of Seed (1987) in

the plasmid vector CDM8, and propagated in Escherichia coli MC1061/P3. The

titer of the cDNA library was first determined by preparing a lO^ dilution o f the frozen library stock and plating out dilutions on L-agar plates containing ampicillin and tetracycline at concentrations o f 50 |Xg/ml and 7.5 ixg/ml respectively. These plates were incubated overnight at 37®C and the IG‘2 dilution stored at 4°C.

The following day, the number of colonies obtained for each dilution were counted and used to calculate the volume of the 10*2 dilution required to yield 250 000 colonies (optimal number for a 20 X 20cm filter). The calculated dilution volume was then spread onto four 20 X 20cm Hybond N+ membranes (Amersham) which were on the surface of L-agar (ampicillin and tetracycline selection) 245 X 245mm Nunc plates. These master plates were incubated at 37®C overnight and used the next day to prepare replica filters as follows. 12 fresh L- agar (ampicillin and tetracycline selection) 245 X 245mm Nunc plates were poured and overlaid with Hybond N+ membranes. Two replica filters were prepared from each master. The first master was removed from its agar plate and placed on 2 sheets of Whatman 3MM paper. The prewetted replica filter was carefully placed on top of the master. 2 sheets of Whatman 3MM paper were layered on top of the filters and the filters pressed together. The master filter was marked with a pattern of spots and the same pattern was also marked on the replica. The two filters were pulled apart and the replica filter returned to its plate. This process was repeated with the remaining 3 master filters resulting in 4 duplicated replica filters (8 in total). All 12 plates were incubated at 37°C for 4 hrs, the master plates were then stored at 4°C and the replica plates placed at room temperature overnight to regrow the colonies.

Once the colonies had regrown, the replica filters were processed as follows. Each filter was placed, colony side up, on a pad of Whatman 3MM paper soaked in DS for 9 mins. The filters were blotted dry and then dipped briefly in NS. DNA

was fixed onto the filters by placing them, colony side up, on pads of Whatman 3MM soaked in 0.4M sodium hydroxide (NaOH). The filters were blotted dry then placed on Whatman 3MM pads soaked in NS for 2 X 3 mins. Bacterial colonies stuck to the filters may lead to high background hybridisation levels so these were washed off using a tissue soaked in 5X SSC. The filters were rinsed in 5X SSC then blotted dry and stored at 4°C.

After hybridisation of the filters with the probe of interest, the presence of a positive hybridisation signal on the duplicate filters was used to identify the positive colony on the master plate. A 5mm area around the positive colony was scraped off the master plate using a sterile inoculating loop. This was transferred to 1ml LB containing ampicillin and tetracycline. After mixing, successive 10 fold dilutions were made of the stock tube and plated out onto 140 X 140mm L-agar plates (containing 50pg/ml ampicillin and 7.5pg/ml tetracycline). The stock tube was stored at 4°C and the plates were incubated at 37°C overnight. The next day, filter lifts were taken from those plates with well separated colonies. A Hybond N+ filter was overlaid onto the colonies for 1 min. During this time orientation marks were made on the filter and plate using a needle. The secondary filter was then placed, colony side up, on Whatman 3MM pads soaked in DS for 7 mins, blotted dry and placed on NS for 2 X 3 mins. The DNA was fixed to the filters by placing them on Whatman 3MM pads soaked in 0.4M NaOH. The filters were briefly rinsed in 2X SSC and hybridised again with the probe of interest. The plates were incubated at 37°C until the colonies had regrown and then they were stored at 4°C.

Single colonies positive with the probe were identified by hybridisation and autoradiography. These were picked into 5ml LB (ampicillin and tetracycline selection) and cDNA purified as described in section 2.6.

2.2.2. Cosmid libraries

Two cosmid libraries were utilised in the course of the work described in this report: 1) an enriched cosmid library made from the yeast artificial chromosome (YAC) 11.2 which is 450 kb in size and encompasses the region between the genes DMB and DOB in the class II region of the human MHC; 2) a flow-sorted chromosome 6 cosmid library. The construction and utilisation of these cosmid libraries is discussed, in detail, in chapter 3.

In parallel with the cosmid library constructed from YAC 11.2 and the flow- sorted chromosome 6 cosmid library, clones were also isolated from a PI bacteriophage library.

2.2.3. PI genomic library

The total PI library (Francis et al., 1995) was prepared from DNA derived from the lymphoblastoid cell line GM1416B with a karyotype 48 XXXX. DNA was cloned into pAdlOSacBll vector and clones were recovered in the E. coli strain NS3145. The library was robotically arrayed onto high density grids, each containing 20736 clones (Nizetic et al., 1991) and represents a 1.2 fold genome coverage.