Optimization Results and Comments

A 3-fold range of magnesium ion concentrations were investigated with OPL-01. At least 14 products were amplified using ImM and 1.5mM MgCl2 from canine

sample 553 and about 10 products from 603. Further increases in concentration caused the majority of products anoplified to be abohshed, until at 3mM MgCl2 per

reaction each reaction from both sangles yielded only a smear. A product of - l .l k b was observed in the German Shepherd (553) sample but not in the Dobermann (603) at the two favourable concentrations.

Clearer amplification products were generated when magnesium titrations were performed using OPK-07 and the pitbull and Jack Russell DNA sangles. Here, the range of concentrations analyzed was 10-fold. No amplification products were observed at 0.5mM MgCl2 from either sample. A product of 580bp was observed

at 0.8mM from each sample, although the Jack Russell sample amplified three fiirther products, 0.9, 1.5 and 2. Ikb. From reactions using l.OmM to 5.0mM at least 6 products were amphfied from each sample. The l.OmM MgCl2 reactions

amplified the most number of products in the pitbull sample ( 8 products ranging

form 0.4kb to 1.5kb), whereas a concentration of 2.0mM yielded the greater number of products per reaction for the Jack Russell sanple. Several products were observed in one sanple and not the other. For the Jack Russeh sample, three products were amplified that were absent from the pitbuh reactions. These were 2.0kb, 1.4kb and l.lkb products. OPK-O?2kb was amplified best at l.OmM and 2.0mM concentrations and was not anpHfied at 5.0mM. OPK-07^ 4^b was only

amplified using 2.0mM MgCl2 and above and OPK-07^ was amphfied in ah Jack

Russeh reactions except 0.5 and 0.8mM MgCl2. A 580bp product was amplified

from both sanples at 0.8mM and l.OmM but was not amplified at higher concentrations. In the pitbuh a 540bp product was amphfied at l.OmM which was absent from the Jack Russeh reaction of the same concentration. A second pitbuh- pecific product of 660bp was anplified in ah reactions but was best amphfied at l.OmM and 2.0mM. For the optimization for OPK-07 increasing the magnesium concentration did result in an increase in the number of products amphfied up to

a 'threshold' concentration after which level smearing occured. One particular product OPK-OTggo was arq)lified from a pitbull sample consistently over a 5-fold increase in magnesium concentration; a Jack Russell-specific product (OPK-OV^kb) was only anplified at LOmM and 2.0mM MgClj. Products common to both dog sarqples (OPK-OTggo) were also only amplified at certain concentrations - 0 . 8 and

LOmM MgClj.

The effect of a 10,000-fold variation in dNTP concentration with OPL-01 showed that dNTP concentrations below lOOpM dNTP working concentration did not produce successful or clear amplifications from either dog sanple. The effect of doubling the working concentration to 200pM dNTPs increased the intensity of the products generated.

Different concentrations of OPL-Olwere also tested using the standard working concentrations of O.lmM dNTP and 1.5mM MgClj per reaction. Doubling the "normal' concentration of primer to 40pmol/|Lil resulted in smears from both canine sangles. Clearer amplification products were observed at concentrations of primer less than 20pmol/pl and seemed to be 'sanple-specific' at 5 and 10pmol/p,l stocks; ie the Dobermann (603) products were better anplified using 5pmol/|rl OPL-01 than with the 10pmol/|rl stock. The converse was true for the German Shepherd (553) sanq)le. At these low concentrations products OPL-Oli and OPL-Glj 27kb

were arr^)lified from the German Shepherd sanq)le specifically.

The effect of different annealing temperatures was investigated with OPK-07 primer at 40®C and 45®C and using 2 0pmol/p.l primer and the standard working

concentrations for the other reagents as before. At both tenq)eratures, two products were consistently anq)lifred from each dog - OPK-OTpgo and OPK-GTg^o although at the higher ten^erature the amplification of the 6 6Gbp product was

much more intense. Several bands were observed at one particular temperature and not the other and some of these were specific to one of the dog sanq)les. At even less specific arq)lification conditions (annealing temperature = 36°C) some of the

fainter amplification products were more intense (OPK-OVy^g for exan^le from dog 2, the Jack Russell).

A great number of factors need to be taken into consideration and manipulated in order to yield sucessful an^lification products using RAPD PCR Generation of a large number of products does not constitute optimization of a primer or set of conditions, and in this respect the researcher should decide whether one is trying to optimize the amplification of a specific product or the entire RAPD profile as they could require two completely different reaction conditions. Amplification fidelity and reproducibility depends upon the simultaneous interactions and effects of all the conq)onents of a reaction so that by altering one corqponent the entire profile of a reaction, in terms of number and size of products amplified can differ dramatically. For example in the magnesium ion titration reactions a difference of only 0.2mM working concentration of MgClj was suf&cient to increase both the number and intensities of products amplified from both canine samples significantly (pitbull and Jack Russell).

4.3 Segregation of Canine Breeds: Analysis of Polymorphic RAPD Profiles Once the methods for different primers were shown to be successful in generating RAPD profiles, they were applied to the analysis of several different canine breeds.

Canine Breeds

More than 35 dogs from ten different breeds were analyzed. The numbers and letters in brackets represent individual unrelated dog samples from that particular breed. Labrador (357,385, 338, 364); German Shepherd (397, 392, 387, 553, 564, 596, 678); Dobermann (603, 610, 650, 655); Miniature Poodles (43, 44, 74, 85); Rotweiler (413, 365, 381, 410); Boxer (380, 391, 412); Pitbull Terrier (B, C, others); Cross-bred (333, 335, 367); Irish Setter (27, 319, 322, 323); Dobermann- cross (L). The Irish setter group of dogs represent a small pedigree family: 27 and 323 are offspring, 319 is the father and 322 the mother. The cross-bred dogs are mongrels of unknown breeding.