Optimization of RAPD Reactions

Optimization of RAPD reactions requires a methodical approach to deterrmne what effect different reagent concentrations as weU as the thermal parameters have upon the number and size of amplification products and if informative RAPD profiles only appear under particular sets of conditions. However, because of the nature of the shorter ohgonucleotide primers the RAPD reactions are more sensitive to very subtle changes in reagent concentrations and particularly the different temperatures between which the reactions are cycled. A wide range of commerciahy available thermal cyclers exist, each differing shghtly in terms of temperature ramping (heating and cooling) and therefore efficiency. RAPD reactions must therefore be optimized according to the thermal cycler used m the researcher's own laboratory. One group recently described the effect the transition interval between annealing, extension and dénaturation steps can have upon amplification (Schweder 1995) whereby extra anq)lification products were observed when the transition time was more than doubled.

As with two-primer PCR there are very many variables that need to be considered Wien using RAPDs for genetic analysis. These include assaying each reaction reagent over a series of different concentrations in combination with differing primer, polymerase and template concentrations; in addition different annealing tenoperatures, and the length of time of each heating step can be used to obtain particular anoplification products and influence the specificity of the reaction. Shown below are examples of the different reaction conditions used to determine the quality and reproducibihty of anq)lification using two different RAPD primers. The effect of different magnesium, deoxynucleotide triphosphates (dNTPs) and primer concentrations were investigated as well as different tenq)eratures.

Primers were obtained from Operon Technologies (kit L and kit K each containing

2 0 random primers; Operon suggest naming of ohgonucleotide primers in the

fohowing manner: the fourth ohgo of kit L for exanple is "OPL-04"; and to specify a particular product one should add the molecular weight of the product m subscript; ie OPL-OOjoo refers to a 300bp product amplified by primer OPL-09). The two primers used in the fohowing optimization reactions were OPL-01 (^GGCATGACCT^) and OPK-07 AGCGAGCAAG^ ). Camne genomic DNA used were 553 (German Shepherd dog) and 603 (Dobermann Pinscher) for OPL-01 and a pitbuh terrier and Jack Russeh with OPK-07 (see below for preparation of canine genomic DNA from blood). Different dogs were included to determine if variations in reaction conditions would generate polymorphisms specific to a particular reaction mix and secondly to determine if markers could be anq)hfied over a range of reaction conditions.

Optimization of OPL-01 Primer: 4.1.1 Concentration

The effect of five different concentrations of Mg^^ on the amplification of the two canine genomic DNAs were examined - 1.0,1.5, 1.8, 2.0 and 3.0mM. Reactions were made in autoclaved 0.5ml eppendorf tubes, in 50.0pl volumes; OPL-01 (20pmol/|il), l.Opl; dNTPs (5mM each dNTP), l.Opl; xlO NH4 buffer (Bioline: 160mM

670mM Tris-HCl, pH8.8, 0.1% Tween-20), 5.0|l i1; MgCl^ (Bioline:50mM) either 1.0, 1.5, 1.8, 2.0, or 3.0pl; genomic DNA template, l.Opl (lOng). Each reaction was made up to 50.0pl with sterile H2O.

3 0 mMMg

0.344 0.298

Figure 4.0 Effect o f different magnesium ion concentrations upon R APD (O PL -01 primer) amplification. Concentrations o f 1.0, 1.5, 1.8, 2 .0 and 3.0m M M gClj w ere used in 50.0pl reactions w ith canine D N A sam ples 553 (German Shepherd) and 603 (Dobermann).

4.1.2 dNTP Concentration

Six different dNTP concentrations were analyzed - IpM and 0.5, 1.0, 3.0, 5.0 and lOmM with genomic DNA samples 553 and 603. The reaction conditions were identical to those used for the magnesium control reactions and the different dNTP concentrations added in l.Opl volumes to each reaction. A standard concentration ofMgCl^ of 1.5mM working concentration and primer concentration of 20pmol/pl were used for each reaction.

1.0 0.5 3.0 5.0 IxlO"^ mMdNTP M Kb 2.0 1.6 M 0.396 0.344 0.298 n > U S r - ci a.

F ig u re 4.1 a. Effect o f changing dNTP concentration on the fidelity o f R APD primer OPL-02. Sam ples: 553 - German Shepherd; 603 - D obennann; M - l k b D N A ladder. Five different concentrations o f dNTP (5m M stock) were used, 1x10'^, 0.5, 1.0, 3.0 and 5.0 mM. Figure b show s the effect o f doubling the previously optimized dNTP concentration to lOmM per reaction using sam ple 553 as template.

4.1.3 Primer OPL-Ql Concentration

Reactions for 5, 10, 20 and 50pmol/|il OPL-01 primer concentrations were made with 1.5mM working concentration ofMgClj and l.Ofil of dNTPs (5mM).

40 pmol/pl

0.396 0.344 0.298

Figure 4 2 Effect o f differing concentrations o f R APD primer (O PL -01). An 8-fold difference in R A PD primer concentration w as investigated and the effect upon amplification products observed using canine D N A sam p les 553 (G ennan Shepherd) and 603 (Dobermann). Primer concentrations 5, 10, 20 and 40pm ol/pl in 50.0pl reactions.

The thermal cycler (Quatro Biosystems) was pre-heated at 95”C and the reactions placed in the block for 2-3 minutes to enable complete dénaturation of the DNA to single stranded molecules. Five units of BioTaq polymerase (Bioline 5units/pl) was then added to each reaction tube, the control tube first. Forty cycles of 95“C 10 seconds, annealing at 40”C for 30 seconds and extension at I T C for 1 minute were then initiated, followed by a final extension step of 7 minutes. Reactions were placed on ice and lOx bromophenol blue loading dye added to 30.Oui of each reaction. The amplification products were visualized on a 1.5%-2% agarose gels (Sigma) stamed with ethidium bromide. All three sets of optimization reactions were run simultaneously.

Optimization for OPK-07

This included another set of magnesium ion titrations as well as observing changes in amplification products at different temperatures of annealing. The differences are only the canine DNA samples (Dog 1 - pitbull. Dog 2 - Jack Russell).

4.1.4 Concentration

Reactions were made up with final concentrations of 0.5, 0.8, 1.0, 2.0, 3.0 and 5.0 mM MgCl^ in 50.0pl reaction volumes with 20pmol OPK-07 primer and l.Opl dNTPs (5mM). Dog 1 Dog 2 M ' 0.5 0.8 1.0 2.0 3.0 S.o'^^O.S 0.8 1.0 2.0 3.0 5.0 M kb 2.0 1.6 1.0 0.5 0.396 0.344 0.298

Figure 4 3 ElBFect o f changing magnesium ion concentration on RAPD primer OPK-07. The amplification products betw een tw o different dogs were compared under different con cen tration s o f MgClj (0 .5 , 0.8, 1.0, 2.0, 3.0, 5.0m M in 5 0.0p l reaction). D og 1 - pitbull terrier; D o g 2 - Jack Russell; M- Ikb D N A ladder.

4.1.5 Annealing Temperature

Temperatures of 36“C, 40^0 and 45“C were used with Dog 1 and Dog 2 DNA samples under the same conditions as above.

36'C 40'C 45'C Kb 3.0 2.0 1.6 1.0 0.5

F igure 4.4 Effect o f changing annealing temperature on RAPD

amplification. Three different temperatures were exam ined to determ ine if any amplification differences could be observed between two different dog breeds. D og 1 - pitbull terrier genom ic D N A ; D og 2 - Jack Russell genom ic D N A ; M -lk b D N A ladder. 1 Ong o f each D N A w as used as template in each reaction.