HYBRIDIZATION USING SHORT OLIGONUCLEOTIDES

3.1 INTRODUCTION

Rare cutter restriction enzymes that are used for the generation of long range pulsed-field maps cut mammalian genomic DNA infrequently because they usually contain in their recognition sites the unmethylated dinucleotide CpG (Brown and Bird, 1986). This dinucleotide which is under-represented in the human genome, is present in excess in specific regions of the genome known as CpG islands (Bird, 1986, 1987). As described in the introduction DNA segments containing CpG islands also have a high probability of containing or being adjacent to coding sequences (Bird, 1986; Lindsay and Bird, 1987; Gardiner-Garden and Frommer, 1987). Thus selection of clones containing such sites would be useful, not only for linking fragments in long range restriction maps (Poutska and Lehrach, 1986; Smith et al, 1988) but also for the detection of coding regions (Rommens et al, 1989).

A rapid method for the detection of such clones involves the use of a number of 8-mer

oligonucleotides based on rare cutter restriction sites, as probes for hybridization (Estivill and Williamson, 1987; Melmer et al, 1990). A clone identified in this manner which hybridizes with several of these oligos has a high probability of containing a CpG island and thus may contain a coding region.

A similar method has also been developed for the detection of coding regions by hybridization of short and degenerate oligonucleotides based on consensus sequences for splice junctions (Melmer and Buchwaid, 1992). Since most genes contain introns, the consensus regions around splice junctions are very suitable targets for detecting genes.

3.1.1 AIMS

Preliminary work for the testing of the hypothesis that short oligonucleotides could be used for hybridization against coding regions was carried out, in order to understand more about their capabilities for annealing to target DNA. Optimum annealing conditions were first established for hybridization of the short oligonucleotides so that they could then be used as a basis for PCR.

3.1.2 PROBLEMS USING SHORT OLIGONUCLEOTIDES FOR HYBRIDIZATION

i. Hybridization temperature: temperature for oligo annealing is not only much lower, but also much more sensitive than for larger oligos. For instance a change in temperature of a few degrees could significantly affect duplex stability and if too low might allow annealing to non-complementary target sequence and if too high, might prevent annealing to the correct target. Such duplex instabilities are employed in allele-specific oligomelting experiments, where short oligos corresponding to the wild type and to the mutant sequence are annealed to the target sequence and then the mismatched oligo is melted off at a slightly higher temperature (Tybjoerg-Hansen et al, 1990). The formula of Suggs et al (1981) is often used as a rule of thumb for oligonucleotide hybridization:

Tj = {4x(G-kC) + 2x(A-hT)}

where Tj is the temperature at which 50% of oligonucleotides dissociate from the DNA. This formula has been determined empirically and holds true for oligos as short as 1 1-mers

(Wallace et al, 1979), but whether this formula holds true for oligos as short as 8-mers is not

known. The predicted Tj for a GC 8-mer using this formula is 32°C, so hybridization would

need to be performed 5-8”C below this temperature (Suggs et al, 1981). In the experiments performed by Melmer et al (1990), hybridization with the GC 8-mers was performed at 27*’C.

ii. Hybridization stringency: stringency of the hybridization would need to be increased to prevent non-specific hybridization by the formation of stable heteroduplex. Stringency can be increased in several ways, namely by increasing hybridization temperature or decreasing salt concentration.

iii. Self-annealing. With the palindromic nature of rare-cutter enzyme sites one would expect oligos based on these sequences to undergo self annealing.

iv. Degenerate oligos: much larger concentrations of oligo would be required to balance out the degeneracy, in order to achieve a sufficient oligo:target sequence ratio.

3.2 TEST HYBRIDIZATIONS OF OLIGONUCLEOTIDES TO MODEL SYSTEMS 3.2.1 HYBRIDIZATION TEMPERATURE OPTIMIZATION

Slot blot filters were prepared using varying amounts of DNA from the pWE15 vector, which contains two Notl sites (Wahl et al, 1987), Also the vector pMBGPT (constructed by M. Lu, Toronto), which contains one Notl site, was used. As controls the pWE15 vector digested by Notl to destroy the sites, the vector pCVlOS from which pWE15 was derived and which contains no Notl site (Lau and Kan, 1984) and the unrelated vector pUClS were used.

Each filter was prehybridized for 30 mins at 2"C below the hybridization temperature and then hybridized with 5'-end labelled Notl 8-mer (5’GCGGCCGC 3’) for one hour at the

specified temperature (23, 25, 27, 29, 31 and 33^0) using a refrigeration controlled circulating waterbath (Heto Lab Equipment). All filters were washed down at room temperature ( ~ 2 0 ‘*C).

Hybridization occurred up to 33"C, beyond the tem perature predicted by the form ula of Suggs et al (1981), see Fig. 3.1.

Figure 3.1 Slot blot hybridization of Notl 8-mer oligonucleotide to model vectors at

different temperatures. Lanes 1-6 contained Sail digested pW ElS at 0, 10, 50, 100, 200 and SOOng. Lane 7 had SOOng of Notl digested pWE15. Lane 8 had SOOng of EcoRI

digested pMBgpt. Lane 9 had SOOng of Notl digested pMBgpt. Lane 10 had 166ng of EcoRI digested pUC13 and lane 11 had SOOng of EcoRI digested pCV108.

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In order to demonstrate that the control pWE15 was being effectively digested by Notl and that the hybridization could discriminate between the Notl target sites and other sites- such as EagI sites- within pWE15 (with high homology to the Notl 8-mer) and to further study the

effect of temperature on hybridization, a similar experiment was performed using the vectors as before, either linearized with Sail or EcoRI or digested with Notl or Eagl. The DNA was run on an agarose gel to show whether the digestion had worked efficiently (see Fig. 3.2) and then Southern blotted onto nylon filters. A number of filters were produced in this way, all using the same digestion samples. As before the end-labelled Notl 8 -mer was used as probe,

hybridizing at temperatures between 8®C and 42“C (8 , 21, 23, 27, 30, 32, 35 and 42®C) for

16 hours and then washing the filters at 2xSSC at room temperature.

Hybridization occurred at all temperatures used, although specificity was low and background hybridization high at temperatures below 2TC . Specificity and strength of signal was optimum for the 35®C hybridization (3°C higher than the calculated T J.

Figure 3.2 a. Model vector digests, b. Southern hybridization with Notl 8-mer oligo at

27-27.5"C. Lanes numbered as follows: 1-3 pWElS/Sall digests at 10,100 and SOOng, 4-6 pW ElS/N otl digests at 10, 100 and SOOng, 7-9 pWEIS/ Eagl digests at 10, 100 and SOOng, 10-12 pCVlOS/EcoRI digests at 10, 100 and SOOng, 13-lS pCVlOS/EagI digests at 10, 100 and SOOng, 16-18 pUC13/EcoRl at 3.3, 33 and 167ng, M is X/Hindlll ladder (see 2.24).

a.