Taurine Levels in Human Aqueous Humour MEDICAL SCIENCES (2000), 2, 11−14 © 2000 SULTAN QABOOS UNIVERSITY Department of Biochemistry, Sultan Qaboos University, P.O.Box: 35, Postal Code: 123, Muscat, Sultanate of Oman *To whom correspondence should be addressed. 11 Effect of single mismatches at 3′– e nd of primers on polymerase chain reaction *Simsek M, Adnan H تأثير عدم التوافق االحادي عند نقطة األنتنهاء الثالثي للمواد التمهيدية في )حافز البلمرة(سلسلة تفاعل البوليمارز .عدنان. سمسك ، ح. م ظهر أن االرتباط : الطريقة والنتائج . وقواعد االنتهاء الثالثي للمحفز ) بيتاجين الهيموجلوبين ( بين قواعد الحمض النووي توافق ال عدم مدى تأثير ثالثة أنواع من معرفة: الهدف: الملخص مض ا قاعدة ح268، وهي منطقة تحتوي على )الهدف( الهيموجلوبين ساعد في عملية التكاثر السريع للمنطقة المنشودة في جين بيتا) ج/ج،أ/ج،ت/ج(الذي حدث بين زوجي القواعد الثالث آان ) ج/ت(تالحم قاعدتي المحفز والهدف . درجة مئوية، وهي ما تعرف بدرجة حرارة التالحم المتماثل65 و 45نووي مزدوج، وذلك باستخدام سلسلة من درجات الحرارة تراوحت بين أي تفاعل يذآر ) ج/أأو ج/ج ( التحام القواعد األخرى لم ينتج من . هذا التالحم في آل درجات الحرارة المذآورة سلفا حدث )ج/س(قاعدتي قويا مثل التالحم الطبيعي الذي يحدث بين . درجة مئوية50 و 45 عند درجتي حرارة ج/جسوى التفاعل الضعيف للقاعدة . وقد تم التفاعل مع المنطقة المنشودة من جين بيتا الهيموجلوبين)ج /ج و ج/أ (ان المحفزين اللذين أمكنهما التفاعل هما زوجى القواعد : الخالصة ABSTRACT: Objective and Method – To investigate the effect of three different mismatches (G/T, G/A or G/G) at the 3•– end of a primer to amplify a 268 bp (base pair) region of the human •–globin gene using different annealing temperatures (45 to 65•C). Results – The primer with the G/T mismatch was as efficient as the normal primer (G/C match) in the amplification of a 268 bp product at all temperatures tested. However, the primers having G/A or G/G mismatches at the 3'- end did not produce any specific polymerase chain reaction (PCR) fragment at all the annealing temperatures used, except a barely detectable 268 bp product for the G/G mismatch at 45 and 50•C. Conclusion – We conclude that our PCR system was refractory to amplification when one of the primers contained a G/A or G/G mismatch at the 3•–end with template DNA. KEY WORDS: PCR, mismatched primers, β−globin gene olymerase chain reaction (PCR) is a sensitive and powerful method by which specific sequen- ces of template DNA can be amplified more than a million fold using 20�30 cycles of DNA synthe- sis.1 Each cycle requires three stages: first, denaturation of DNA at high temperature (90�94ºC), second, annealing of oligonucleotide primers at a lower temp- erature (usually at 40 to 60ºC), and finally, extension of primers at 72ûC by taq DNA polymerase, a heat stable enzyme. Most of the DNA polymerases used in PCR lack an intrinsic 3' to 5' exonuclease activity.2 Thus, oligonucleotides with a mismatch at the 3'�end may be extended and serve as primers in the polymerase chain reaction (PCR). The efficiency of such primers would depend on many factors, including the nature of the mismatch, the kinetics of association and dissociation of primer-template DNA duplex at the annealing and extension temperatures, and the effect of a mismatch on the stability of the duplex DNA formed. Several investigators have begun to evaluate the possible effects of mismatches in their primers.3-5 It has been shown that a single mismatch at or near the terminal 3′ base of a primer affects PCR more dramatically than those single mismatches located internally or at 5′ end.6 Using primers with one or more mismatches near the 3′ end, allele specific ampli- fication (ASA) has been developed7 for the detection of mutations in genetic diseases. In this study, we have evaluated the effect on PCR, of three different mismatches (G/T, G/A and G/G) located at the 3′ �terminal end of primers which were used to amplify a small region in the human β- globin gene. METHOD DNA ISOLATION Miller�s salting-out procedure8 was used to isolate genomic DNA from whole blood with ethylene- diamine tetra acetic acid (EDTA) as anticoagulant. The P 11 S I M S E K E T A L 12 nuclei of white blood cells were isolated after lysis of cells with a non-ionic detergent. The isolated cell nuclei were suspended in a buffer containing an ionic detergent (SDS) and proteinase K which digests the proteins associated with chromosomal DNA. All the proteins were precipitated using high salt concentra- tion (salting out) and the DNA in the supernatant was recovered by precipitation with ethanol. The amount of DNA obtained was around 5�10 µg per 0.5 ml whole blood. DNA amplification by Polymerase Chain Reaction. A small region (286 bp) of human β�globin gene was amplified using a pair of primers which were either completely complementary to the globin gene, or had a single mismatch at the 3′ �end. Genomic DNA was amplified in 50 µl reaction volumes with 25 pmoles of each primer, 1 unit of ampliTaq DNA polymerase (Perkin-Elmer Cetus Inc.), 200 µM each of the four deoxynucleoside triphosphates, and a buffer containing 10 mM Tris pH 8.3, 50 mM KCl, 0.01% gelatin, and 1 mM MgCl2 (unless otherwise mentioned). All amplifi- cations were started by the addition of the Taq DNA polymerase at 94ûC (hot start) and continued for 30 cycles in a DNA thermal cycler (Hybaid Inc.) Each cycle of amplification contained the following steps: DNA denaturation: 2 minutes at 94ûC, primer anneal- ing: 2 minutes at 50ûC, and primer extension: 3 min- utes at 72ûC. The PCR products were separated by electrophoresis on 2.5% agarose gels and detected by staining with ethidium bromide (0.5 µg/ml) for 15 minutes in electrophoresis buffer (40 mM tris acetate pH 8.0 and 1mM EDTA). A polaroid land camera (MP4 Kodak) was used to take pictures of the gels over a UV trans-illuminator in dark room. Synthesis of DNA primers Two sets of oligonucleotide primers were synthe- sised using a DNA synthesizer (Pharmacia, Gene As- sembler Special 4). One set completely matched the template DNA while the other had an upstream pri- mer with a mismatch at its 3′ �end. If the A residue in the ATG initiation codon is taken as position 1 in the human globin gene, the down�stream primer (HG� P2) was complementary to the sense strand (positions 54 through 73), and had the following sequence: 5′ � CAACTTCATCCACGTTCACC�3′ . The upstream primer (HG�P1) complemented the anti-sense strand of the β�globin gene (from position �195 to �176) with the following sequence: 5′ �AAGAGCCAAGG- ACAGGTAC�3′ . The three different upstream primers (HG�P1A, HG�P1G, HG�P1T) with a single mismatch were identical to the sequence of the HG� P1, except that they had an A, G or T residues, respectively replacing the terminal C at their 3′ �end. RESULTS The primers used in this work specified the ampli- fication of a 268 bp region of the human β�globin gene. Before studying the effect of mismatches at the 3′ �end of primers, an optimization for MgCl2 was carried out since any PCR amplification is known to be very sensitive to magnesium ion concentration.9 Using the normal primers, it was found that a detectable 268 bp PCR product was amplified at different Mg+2 ion concentrations ranging from 0.5 to 4.0 mM in 0.5 mM increments (data not shown). However, above 1.5 mM concentration, there were too many nonspecific pro- ducts, and furthermore, the desired 268 bp product decreased in quantity with increased Mg+2 ion con- FIGURE 1. The effect of Magnesium concentration on PCR. The reaction conditions were as described in the ‘methods’ section, using normal primers, except the variation in Mg 2+ concentration. After 30 cycles of amplification, the PCR products were separated by electrophoresis on a 2.5% agarose gel (at 90 volts for 60 minutes) and detected by staining with ethidium bromide. The arrow shows the position of a specific PCR product (268 bp). Lane M contained DNA size markers. Lane 1 was negative control (without any template DNA added) in the PCR amplification. The reactions in lanes 2 to 6 contained different concentrations of Mg 2+ ions: lanes 2 & 3 (0.5 mM), E F F E C T O F S I N G L E M I S M A T C H E S O N P C R 13 centration. Figure 1 shows a duplicate titration of magnesium ions at 0.5 to 1.0 mM range. There were no detectable non-specific products and the best amplificaton of the 268 bp product was achieved at 1.0 mM MgCl2 concentration (figure 1, lanes 6 &7). To study the effect of mismatches at the 3′ �end of pri- mers, we attempted to amplify the 268 bp DNA frag- ment at different annealing temperatures, from 45 to 65ºC using a normal downstream primer (HG�P2) together with a single mismatch containing upstream primer (HG�P1A, HGP1T or HG�P1G). Having a G residue in the template DNA at the corresponding position, each of these primers would normally form 19 normal base pairs with the template, but in this case, resulted in a mismatch of G/A, G/T or G/G at the 3′ terminal end, respectively. Figure 2 shows some representative results obtained with different primers at 50 and 65ºC. At both annealing temperatures, when normal primer pairs were used (figure 2, lane 5) the desired 268 bp DNA fragment was amplified satis- factorily without any detectable non-specific products. Interestingly, the primer with a G/T mismatch produced similar results (figure 2, lane 3). However, the results obtained with the primers containing either a G/A or G/G mismatch were entirely different. The primer with the G/A mismatch (figure 2B, lane 2) showed no detectable 268 bp product at 65ºC, a fairly distinct but a smaller PCR product than 268 bp at 50ºC, but was undetectable at 65ºC (Lanes 4 in figure 2B and 2A respectively). DISCUSSION We have studied here the priming efficiency of a 20 nucleotide long primer set which provided three different single mismatches (G/T, G/A and G/G) with the template DNA used. The G residue was lo- cated in the template (β-globin gene) while T, A or G were at the 3′ �end of the primers. Our results showed that the G/T mismatch primed the synthesis of a 268 pb specific PCR product, almost as efficiently as the normal primer (HG�P1) which contained a G/C match at the 3′ �end. However, the priming efficiency of the primers with G/A or G/G mismatches was extremely poor, and resulted in the production of no specific PCR fragment that was detectable by staining with ethidium bromide. The results obtained with all three mismatches were essentially the same as in figure 2 at different annealing temperatures tested (45 to 65ºC) except that the specific PCR product (268 bp) was barely detect- able for the G/G mismatch at 45, 50 and 55ºC but for the G/A mismatch, no specific product was detectable FIGURE 2. The effect, on PCR, of single mismatches between the 3’ base of a primer and template DNA at two temperatures The arrows indicate the position of the specific 268 bp PCR product. In both panels, lane M contained DNA sized markers. Lane 1 was negative control and lane 5 the positive control containing normal pair of primers. Lanes 2, 3 and 4 contained the mismatched primers, ending with G/A, G/T and G/G, respectively. S I M S E K E T A L 14 at all at any of the tested annealing temperatures. The G/A mismatch, however, produced a fairly strong and distinct product at 45ºC, sized <200 bp and was not produced at all at higher annealing temperatures (figure 2B, lane 2). We concluded that it must be a non-speci- fic PCR product formed possibly due to dimerization of the primers.2 The priming efficiency of mismatched primers has already been studied by various groups. Our result with G/T mismatch agrees with that of Kwok11 who showed that a single mismatch of T/T, T/C or T/G at 3′ �end had no effect on the priming efficiency of their primers. The same group also studied a A/G mis- match, and our results agree with theirs. However, our results with the G/G mismatched primer (figure 2, lane 4) contradict some of the published work. For example, Newton10 reported that a single G/G mis- match had very little effect on the priming efficiency of their primers, while we could get only a barely detect- able specific PCR product at 45�55ºC, and none at 60 or 65ºC. One potential application of mismatched primers has been towards identification of point mutations in genetic diseases. Using ASA primers which differed only by one base at 3′ �end, like ours, Okayama7 was able to detect three different point mutations in pati- ents with an α1-antitrypsin deficiency. However, their results were contradictory to ours, as well as many other studies reported10,11 in that 10 of the 12 possible mismatches, including a G/T mismatched primer, were all refractory to any detectable amplification. However, it is difficult to make a direct compari- son of the results reported with various 3′ mismatched primers, due to differences in the PCR amplification conditions, especially the length and concentration of primers. In some studies, the primers were too short (12�16 bases)12 and hence a 3′ �mismatch may have more dramatic effects both on the stability of primer- template dublex and the extension of mismatched primer by DNA polymerase. On the other hand, if the primers are too long, like 30 bases used by Ehlen and Dubeau,13 the effect of 3′ �mismatch on PCR may be minimal. The primers used in this work were inter- mediate in size (20 bases long), a size commonly used by many research groups. CONCLUSION In summary, our upstream primers resulting in an A/G or G/G mismatch at the 3′ �end with template DNA were refractory to amplification. But the same primer, when ended with a T/G mismatch, showed no refractoriness, and was as efficient as the normal primer for the amplification of a 268 bp DNA of the human β-globin gene. REFERENCES 1. Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, Mullis KB, Erlich HA. Primer-directed enzymatic amplification of DNA by a thermostable DNA polymerase. 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Allele specific enzymatic amplification of β-globin genomic DNA for diagnosis of sickle cell anaemia. Proc Natl Acad Sci USA 1989, 86, 2757–60. 13. Ehlen T, Dubeau L. Detection of ras point mutations by PCR using mutations-specific, inosine containing oligonucleotide primers. Biochem Biophys Res Commun 160, 441. Effect of single mismatches at 3(–end of primers �on polymerase chain reaction METHOD DNA isolation DNA amplification by Polymerase Chain Reaction. Synthesis of DNA primers