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The polym erase chain reaction (PCR) results in the selective am plification o f a particular portion o f chosen DNA m olecule (Frohman et a l , 1988). Any region can be chosen, so long as the sequences at the borders are known. These border regions provide the sequence to which two short oligonucleotide prim ers hybridise, one to each strand o f the double helix, delineating the region to be am plified. A therm ostable DNA polym erase 1 enzym e extends the prim ers towards each other in a reaction cycle o f three steps:

1) D énaturation - heating to 94-98°C denatures DNA double helix^presenting bases for hybridisation with primers.

2) Prim er annealing - cooling the tem perature to 50-55°C allows prim ers to anneal to their targets on tem plate sequence. The higher the annealing tem perature the m ore specific is the prim er-tem pi ate association. The optim um annealing tem perature depends upon the prim er m elting tem perature (Tm), the tem perature at which a correctly base paired prim er-tem plate hybrid “m elts”, w hich is dictated by prim er length and nucleotide content. Tm can be calculated by the sim ple formula:

Tm = (4 X [G + C]) + (2 X [A + T]) °C

If annealing tem perature is too high, prim ers and tem plate rem ain dissociated. If annealing tem perature is too low prim er m ism atch occurs generating false positives.

3) Extension - Usually occurs at 72°C, the approxim ate optim um tem perature at w hich therm ostable polym erases extend the prim ers tow ards one another by adding dNTPs from the reaction m ixture to their 3 ’ - ends.

The region o f DNA encom passed by the prim ers is exponentially am plified by repetition o f these cycles.

D ifferent therm ostable polym erase 1 molecules have been isolated that can be utilised for different purposes in PCR reactions. Each therm ophilic polym erase has unique characteristics, such as pH and salt optim a that affect the efficacy o f the PC R protocol.

Chapter 2 M aterials & M ethods

The enzym es are usually sold in unit quantities. One unit (lU ) is defined as the am ount of enzym e required to catalyse the incorporation of lOnmol o f dN TP into acid-insoluble m aterial in 30 m inutes at 74°C.

2.2.2.1

Taq polymerase

Taq D N A Polym erase (Chien et al., 1976) is a therm ostable enzym e isolated from therm ophilic bacterium Thermus aquaticus. This unm odified enzym e replicates D N A at 74°C and exhibits a half-life of 40 minutes at 95°C (Chien et a i , 1976). The enzym e catalyzes the polym erization o f nucleotides into duplex D N A in the 5"—>3" direction in the presence o f magnesium. However, Taq has a relatively high error rate (one error ~ every 125,000 nucleotides) since it does not have a 3 '^ 5 ' exonuclease proofreading function. The most com m on m utations caused by Taq are AT to G C transitions (K eohavong and Thilly, 1989) or it can generate deletion m utations if the tem plate DNA has the potential to form secondary structures (Cariello et a i , 1991). H ow ever, the error rate o f Taq polym erase can be greatly reduced by m odifying the reaction conditions (Ling et a i , 1991). If used to am plify a novel D N A fragm ent, sequencing o f products from at least three repeats of the sam e PCR experim ent is required to confirm correct sequence. Taq polym erase reaction m ixtures and PC R protocols are outlined in Table 2.4 and Table 2.5 respectively.

Reagent

cDNA tem plate (25ng/|il)

Gene Specific Prim er 1 (10pmoi/|il) Gene Specific Prim er 2 (lOpmol/pl)

10 X Thermophilic buffer * M gC b (25mM)

Taq polymerase dNTP mix

Sterile deionised H2O (dH20)

Amount per reaction

1-2.5 pi 2.5 pi 2.5 pi 5 pi 2.5 pi Ipl (2.5 U) 0.5 pM To 50pl

Table 2.4 - Standard Taq polymerase reaction mix for a 50pJ PCR reaction

* Stock 10 X Therm ophilic Reaction Buffer without M gC ^: 500mM KCl, lOOmM Tris-HCl (pH 9.0 at 25°C) and 1.0% Triton X-100. If reaction mixtures differed the altered param eter will be stated in the text.

C hapter 2 M aterials & M ethods

Protocol

Taq30 Taq60 Temp Time Cycles Temp Time

Denature 95°C 1 min 1 95°C 1 min

Denature 95°C 30 s

30

95°C 30 s

Anneal 50°C 30 s 50°C 30 s

Extension 72°C 30 s 72°C 1 min

Extension 72°C 10 min 1 72°C 10 min

Cool Hold @ 4°C 1 Hold @ 4°C

Table 2.5 - Taq polymerase protocols

Referred to as Taq30 and Taq60 from this point onwards, these were the standard PCR parameters programmed into thermocycler for reactions employing Taq polymerase.

Taq polym erase possesses a nontem plate-dependent term inal transferase activity that adds a single deoxyadenosine (dA) to the 3 ’ ends of PCR products. These 3 ’ A overhangs can be exploited to insert the fragm ent into linearised vectors containing single deoxythym idine (dT) residue overhangs. This activity can also be exploited to add single 3 ’ A overhangs onto the ends o f blunt ended PC R products or restriction fragm ents, by incubating the cD N A fragm ent with Taq at 72°C for 15 m inutes in buffer containing dATPs.

2.2.2.2

Pfu and Pfu Turbo polymerases

P fu is a high fidelity polym erase identified in Pyrococcus fu rio su s, w hich is very therm ostable (Stratagene, La Jolla, CA). It can tolerate tem peratures exceeding 95°C, enabling it to PCR amplify GC-rich targets and has a very low error rate (~ once every 767,000 nucleotides) because it possesses a 3'—>5' exonuclease proofreading function. H ow ever, Pfu is m uch slow er than Taq, requiring a m inim um extension tim e o f 1.5 - 2 m inutes/kb o f am plified tem plate and it generates blunt ended PC R products.

Pfu Turbo polym erase is m erely an enhanced version o f Pfu polym erase, sold as a blend of cloned Pfu and a novel therm ostable factor added by the suppliers (Stratagene) that enhances P C R product yields w ithout altering D N A replication fidelity. The error rate of Pfu Turbo is the same as for Pfu and significantly low er than that o f Taq. The main advantage o f using this enzym e over Pfu is that its enhanced perform ance allow s the use of shorter extension times, w hich dram atically shortens a PC R protocol if am plifying a large target. M oreover, Pfu and Pfu Turbo both have m inim al activity at 40-50°C resulting in few er m ispaired prim er-extension reactions than occur w ith Taq.

C hapter 2 M aterials & M ethods Pfu and P fu Turbo reaction mixes and PCR protocols are detailed in Table 2.6 and Table 2.7 respectively. W here stated, some am plification reactions additionally contained 20% DM SO in the reaction mix. This served to reduce the energy requirem ents to m elt G-C rich regions and disrupt any m R N A secondary structure when the am plification of a particular target proved difficult using standard param eters.

Reagent

cDNA template (25ng/pl)

Gene Specific Primer 1 (lOpmol/pl) Gene Specific Primer 2 (10pmol/|il)

10 X Pfu Thermophilic buffer *

Pfu/ Pfu turbo polymerase dNTP mix

Sterile deionised H2O (dHzO)

Amount per reaction

1-2.5 pi 2.5 pi 2.5 pi 5 pi 1 pi (2.5 U) 0.5 pM To 50pl

Table 2.6 - Standard Pfu and Pfu Turbopolymerase reaction mix for a 50pl PCR reaction

* Stock 10 X Pfu Thermophilic Reaction Buffer: lOOmM KCl, 20mM MgSO^, lOOmM (NH4)2S0 4, 200mM Tris-HCl

(pH 8.8 at 25°C), 1.0% Triton X-100 and 1 mg/ml nuclease free BSA. If reaction mixtures differed the altered parameter will be stated in the text.

Table 2.7 - Pfu and Pfu Turbo polymerase protocols

These were the standard PCR parameters programmed into thermocycler for reactions employing Pfu and Pfu Turbo polymerases.

Protocol

Pfu Pfu Turbo Temp Time Cycles Temp Time

Denature 98°C 1 min 1 98°C 1 min

Denature 98°C 30 s

30

98°C 30 s

Anneal 55°C 30 s 55°C 30 s

Extension 72°C 2 min 72°C 1 min

Extension 72°C 10 min 1 72°C 10 min

Cool Hold @ 4°C 1 Hold @ 4°C

2.2.2.3

“Touchdown” PCR

T ouchdow n PC R involves decreasing the annealing tem perature every second cycle to a 'touchdow n' annealing tem perature, which is then used for 10 or so cycles. It was originally intended to simplify the com plicated process o f determ ining optimal annealing tem peratures. A nnealing takes place at approxim ately 15°C above the calculated Tm. D uring the following cycles, the annealing tem perature is gradually reduced by 1° C until it has reached a level o f approxim ately 5°C below Tm. The concept is that any differences in Tm between correct and incorrect annealing gives a 2- fold difference in product am ount per cycle (4-fold per °C). This allow s a critical concentration o f the correct product to build, therefore increasing the ratio betw een correct and incorrect products (Don et a i , 1991).

Chapter 2 M aterials & M ethods

2.2.2.4 “Spiice-overiap” PCR

Splice-overlap PCR is a m ethod of am plifying a single cD N A fragm ent from two or m ore cD N As with overlapping sequence. This technique was used in this investigation to assem ble com plete cD N As of putative y subunits where restriction digest and ligation was not feasible because o f a lack of unique restriction sites. PC R param eters are m uch the same as for a normal PCR, however, two or m ore tem plate cD N A s are included in the mix with a GSP designed to the 5 ’ and 3 ’ extrem ities of the intended final cD N A fragment. If we consider the sim plest case scenario with two tem plate fragm ents, the GSPs anneal to their specific targets and extend each fragm ent during the first cycle. H ow ever if sufficient overlap betw een the target sequences exists, they also prim e one another resulting in their extension in the 5 ’ —>■ 3 ’ direction. This generates a tem plate for the GSPs in the second round of PCR from which the intended final cD N A fragm ent can be am plified Figure 2.3.

Figure 2.3 - The splice overlap PCR principle Top) The two cDNA template fragments are primed in the PCR reaction by a pair of gene specific primers (GSPl & GSP2), one designed to a specific region of either fragment. The regions of sequence overlap between the two template strands also act as primers and during the first round of PCR (middle) the templates are extended as well as each individual short fragment. Bottom) The next PCR cycle begins to amplify the intended cDNA following creation of the extended templates to which GSPl and GSP2 can anneal. This fragment is exponentially amplified because GSPs are able to extend sense and antisense strands in each cycle whereas amplification of the short original templates is linear, only amplifying the complementary strands to each template, resulting in greatly enhanced levels of “spliced” product over amplified templates.

S e q u e n ce overlap PCR 1®’ cycle PCR 2"'* cycle •<GSP2l^ Com plete

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C hapter 2__________________________________________________M aterials & M ethods

2.2.3

DNA is o la tio n a n d p u rific atio n