PCR实验指导与常见问题分析 - 分子生物学 - 资讯 - 蚂蚁淘

来自 : 发布时间：2025-07-10

CONTENTPCR guide: a discussion of the main parameters influencing the outcome of the PCR and multiplex PCR reaction in 16 pages/sections and using over 45 pictures PCR troubleshooting: some commonly asked questions and likely solutions Standards: provides images of the standard multiplex reactions used during this work. It is useful to consult before reading other pages of the PCR guide Applications: includes examples of some applications of PCR and multiplex PCR. Still under construction. [NextPage]Standard PCR reaction mix Consider the standard PCR reaction mix (25 L reaction) below. All reactions are run for 30 cycles.Table 1. PCR reaction components100 ng/25 L* The PCR buffer used was made after the recommendations of the manufacturer/vendor (Perkin Elmer Cetus). The 10x PCR buffer contains: 500 mM KCl; 100 mM Tris-HCl (pH 8.3); 15 mM MgCl2 (the final concentrations of these ingredients in the PCR mix are: 50 mM KCl; 10 mM Tris-HCl; 1.5 mM MgCl2).It is useful to prepare a larger volume of this buffer (10-15ml), aliquot it and store the vials at -20 C for years.It is best to start pipetting water first, followed by the other ingredients. There was no difference in results when various components of the reaction were pipetted in different orders. To minimize the chance of primer binding to the DNA template and to prevent the polymerase from working (even theoretically) prior to the first denaturing step, it is useful to keep the vials on ice while pipetting the ingredients of the reaction. Depending on the profile of the laboratory (i.e. current DNA probes in use), pipetting can be done under a laminar flow of sterile air (when plasmids are commonly used in the lab ) or at the bench (when the template DNA is genomic DNA or when a larger amount of DNA is used). When plasmids, phages or cosmids are used as templates in PCR, it is very important to use aerosol-resistant pipette tips, otherwise, false positive results are almost always the rule (even trace amounts of these targets provide a sufficient numer of copies to allow amplification to work). When using complex templates like genomic DNA (of which, sometimes, tens or hundreds of nanogrames are taken in one reaction) such precaution may not be necessary. However, to be on the safe side, it is a good idea to use aerosol resistant tips for every PCR reaction. Another problem when pipetting small volumes (1-2 L) of a complex DNA sample (like genomic DNA) is the likelihood of differences in the amount of DNA actually taken in each PCR vial. This is illustrated in Fig. 1 below, where multiplex PCR was performed on two different genomic DNA samples. Fig. 5. Multiplex PCR test reaction for pipetting errors.Two genomic DNA samples (each 100 ng/ml) were used in multiplex PCR reactions with mix J, simultaneously amplifying eleven different loci (between 165 and 85 bp long). Labeling was done by adding radioactive dCTP to the reaction mix and separation of products was done on a sequencing PAA gel.One microliter each of DNA sample A was taken in vials 1-4, and of DNA sample B in vials 5-8. On the left side, the DNA was pipetted separately in each vial. On the right side, the DNA was mixed with all other PCR ingredients and the mixture was split in equal parts in the vials.The uneven amplification on the left side indicates that, even after thourough mixing, 1 microliter of genomic DNA may contain variable amounts of DNA. This may negatively influence interpretation of the data, especially in quantitative PCR and multiplex PCR reactions. On the right side, amplifications are much more consistent (compare 1-4 and 5-8). Small differences may be due to slight temperature differences in various places in the metal block of the thermocycler.First PCR program The requirement of an optimal PCR reaction is to amplify a specific locus without any unspecific by-products. Therefore, annealing needs to take place at a sufficiently high temperature to allow only the perfect DNA-DNA matches to occur in the reaction. For any given primer pair, the PCR program can be selected based on the composition (GC content) of the primers and the length of the expected PCR product. In the majority of the cases, products expected to be amplified are relatively small (from 0.1 to 2-3 kb). (For long-range PCR (amplifying products of 10 to 20-30 kb) commercial kits are available). The activity of the Taq polymerase is about 2000 nucleotides/minute at optimal temperature (72-78o C) and the extension time in the reaction can be calculated accordingly.As the activity of the enzyme may not be always optimal during the reaction, an easy rule applied successfully by the author was to consider an extension time (in minutes) equal to the number of kb of the product to be amplified (1 min for a 1 kb product, 2 min for a two kb product etc.). Later on, after the product(s) become \"known\", extension time may be further reduced. Many researchers use a 2-5 minutes first denaturing step before the actual cycling starts. This is supposed to help denaturing the target DNA better (especially the hard to denature templates). Also, a final last extension time, of 5-10 minutes, is described in many papers (supposedly to help finish the elongation of many or most PCR products initiated during the last cycle). Both these steps have been tested for a numer of different loci, and, based on this experience, neither the first denaturing nor the last extension time changed in any way the outcome of the PCR reaction. Therefore, it is the author\'s habit not to use these steps (light blue in the table below) anymore. An annealing time of 30-60 seconds was sufficient for all primer pairs tested so far. The annealing temperature can be chosen based on the melting temperature of the primers (which can be calculated using othe many applications, freely available for molecular biologists). This may work, but sometimes the results may not match the expectations. Therefore, a simple procedure used many times by the author was to use an initial annealing temperature of 54 o C (usually good for most primers with a length around 20 bp or more). If unspecific products result, this temperature should be inccreased. If the reaction is specific (only the expected product is synthesized) the temperature can be used as is. It is desirable (but not absolutely necessary) that the two primers have a close melting temperature or Tm (say, within 5o C or so). If Tm difference between the two primers is high, the lower Tm can be increased by increasing the length of that primer at the 3\' end (this can also keep the size of the amplified locus constant) or the 5\' end. For the seventy or so primers used during this work, a denaturing time of 30-60 seconds at 94 o C was sufficient to achieve good PCR products. Too long a denaturing time, will increase the time the Taq polymerase is subjected at high temperatures, and increases the percentage of polymerase molecules that lose their activity. Number of cycles. In general, 30 cycles should be sufficient for a usual PCR reaction. An increased number of cycles will not dramatically change the amount of product (see below). Table 2. Designing a first PCR program.Thermocyclers and PCR vials A number of different types of thermocyclers and PCR vials were used and tested in time. Some potentially useful observations were made:The same PCR program will work slightly different on different thermocyclers (temperature and time profiles may be different, depending on the construction), therefore the PCR results using the same primer pair may vary. However, with proper cycling adjustments, the same results can be obtained on most (or any) thermocyclers. Many new PCR machines accomodate the thin-walled 0.2 ml PCR vials (and/or 96 wells microtiter dishes) in their metal block. For this type of vials, the differences in results from vial to vial are usually negligable (contact between the metal and plastic is very good and aided by the downward pressure from the heated lid). Older machine type could accomodate 0.5 or 1.5 ml vials. Because of slight differences in shape and wall thickness among manufacturers, contact between the vials and the metal block of the thermocycler was not always perfect, often resulting in reduced or no amplification. Such a case is exemplified in the figure below. Some manufacturers offer machines controlling the temperature of a small waterbath in which the PCR vials rest during the reaction. In such cases, due to the good thermal change between water and plastic, variations in PCR results are very little (if any). Fig. 6. Variation in amplification due to lack of proper contact between the metal block and some vials. A PCR mixture containing all ingredients was split in nine equal parts in the same typ/brand of vials, and the tubes were placed in different wells of the metal block of a thermocycler. Reactions 2, 4, 5 and 9 were negative. The same aspect was not reproducible: in another experiment, reactions in other positions could become negative. This was explained by slight variations in vial construction (wall shape or thickness) but not by temperature variations in the metal block (when the aspect should have been reproducible).RETURN TO PCR guide[NextPage]Choosing/designing PCR primersIn designing primers for PCR, the following steps/rules were tested and proven to be useful: length of individual primers between 18-24 bases. Longer primers (30-35 bp) seem to work in more similar cycling conditions compared with shorter primers, and can make multiplexing easier (see picuters below). it is desirable (but not absolutely necessary) that the two primers have a close melting temperature or Tm (say, within 5o C or so). If Tm difference between the two primers is high, the lower Tm can be increased by increasing the length of that primer at the 3\' end (this can also keep the size of the amplified locus constant) or the 5\' end. purine:pyrimidine content around 1:1 (maybe 40-60%) if possible, primer sequence should start and end with 1-2 GC pairs each primer pair should be tested for primer-primer interactions. For this purpose a useful Macintosh program is \"CPrimer\", a freeware available at ftp.bio.indiana.edu. This program also provides the melting temperature for the sequences entered, thus helping in designing PCR programs. Very convenient, some web sites offer programs that can be used directly on those sites to do the same functions: (search for optimal primers, melting temperatures). primer sequences should be aligned with all DNA sequences entered in the databases (using BLAST programs) and checked for similarities with repetitive sequences or with other loci, elwhere in the genome. If two loci are very similar (for example across species) it is useful to design the primers so that at least 1-2 bases at the 3\' end are specific for the locus to be amplified cycling conditions and buffer concentrations should be adjusted for each primer pair, so that amplification of the desired locus is specific, with no secondary products (see other pages). If this is not possible, the sequences of the primers should be either elongated with 4-5 bases or simply, changed entirely. Fig. 7. Multiplex PCR using primers 18-24 bp longWhen PCR reaction Eight individual loci are amplified with similar intensities when the primer pairs are used separately. When equimolar amount of these primers are mixed together for a multiplex reaction (Mix K), some of the products are much weaker (#1, #2, #5, #6) than other. In this case, primers had \"usual\" length, between 18-24bp.(primers used in this case amplify polymorphic loci, explaining the \"double\" or \"triple\" bands as seen on a regular agarose gel)Fig. 8. Multiplex PCR using primers 30-35 bp longCompared to the figure above, in this case the primers used for multiplexing were longer than 30 bp (up to 37 bp). Equimolar amounts of primer were used and all loci were amplified with comparable intensities in each reaction.RETURN TO PCR guide[NextPage]Reaction volumeQ: Does the PCR reaction volume (negatively) influence the outcome?A: No, especially since the introduction of the small, thin walled, 0.2 ml plastic vials fitting the 96 well metal blocks of the thermocyclers.A number of observations are worth mentioning:if using older model thermocyclers (without a heated lid), to run small volume PCR reactions, mineral oil is necessary to cover the reaction mixture. Use of oil increases the volume of liquid in the vial and thus can influence somewhat the outcome. Besides, older thermocyclers require the use of larger plastic vials, with thicker walls, that fit less well in the metal blocks and thus may increase the likelihood of variation in PCr outcome. small, thin-walled plastic vials designed for the 96 well metal blocks are ideal for running small-volume PCR reactions. Due to the heated lid of the thermocyclers, there are no mineral oil requirements. When tested, reactions yielded similar results, whether the reaction volume was 100, 25 or 5 microliters. It is important to mention, that small volume PCRs may be very beneficial when using small amounts of DNA template. In general, at a constant amount of template DNA, the yield of PCR product per microliter reaction is higher when the reaction volume is 5 L compared to 100 L. This may allow visualisation of the PCR products, sometimes invisible when larger reaction volumes are used. RETURN TO PCR guide[NextPage]Multiplexing primer pairs Single locus PCR. First step in designing a multiplex PCR is choosing the primer pairs which can be combined. One important requirement is to find a PCR program allowing optimal amplification of all loci when taken individually (Fig. 9). This is achieved by adjusting the annealing and extension time and temperature. Fig. 9. Single-locus PCR with 34 different primer pairs using the same cycling conditions. Arrows indicate position of the specific products in lanes 25, 28 and 33, in which other unspecific products also appear. Such primer pairs are difficult to use both by themself and in multiplex PCR. However, even though some unspecific products still appeared, primer pair 28 was multiplexed in mixture 5 (Figure 1) and used in a microdeletion screening project. The unspecific products did not interfere with data interpretation. Examples of multiplex reactions using these primers are shown in Fig. 1.Multiplexing equimolar primer mixtures. The next step is combining the desired primer pairs in multiplex mixture(s), using equimolar amounts of each primer. PCR amplification of the multiplex mixtures can be performed, first using exactly the same PCR program as with individual primer pairs. Very often, this will results in preferential amplification of some loci. Such a situation will require further adjustment in cycling conditions and primer concentration. Although, sometimes unspecific products can be seen in single-locus PCR (yellow arrow in PCR product # 2), these unspecific products usually become invisible when the multiplex reaction is performed. This is probably due to the concurrent ampification of many specific loci, which overwhelms the unspecific products (although they are probably still present in small quantities). Fig. 7 (duplicate). Single locus PCR and multiplex PCR with equimolar amounts of primers from mixture K, performed in the same cycling conditions. In Some products of mixture K become weak or invisible, requiring further adjustment of primer amount(s) and of cycling conditions. Primers used in mixture K amplify polymorphic loci, explaining the appearence of multiple bands on a nondenaturing agarose gel.Fig. 10. Equimolar amounts of the same primers used for mixture K (see also Fig. 7 above), where amplified in pairs. In lanes 1, 2 and 4, one locus was amplified less efficiently than the other one (arrows). As mentioned before, amplification of the \"weaker\" loci can be improved increasing the amount of primers or adjusting the reaction conditions.RETURN TO PCR guide[NextPage]Adjustment of cycling conditions annealing time and temperature extension time and temperature For example, figure 11 illustrates the influence of the extension temperature. Equimolar primer mixtures A-D were amplified using two different PCR programs, one at 65o C (yellow lanes) and the other at 72o C (green lanes) extension temperature. In general, there is a higher yield of PCR products for A, B and D when program A was used. This shows that the 72o C extension temperature, negtively influenced amplification of some loci (pink arrows),while also making some unspecific products visible (yellow arrows). It is likely that, for the short PCR products used in these examples (below 500 bp), the higher annealing temperature is probably detrimental to the stability of the DNA helix, so less strands of DNA have the chance to become \"copied\" by the polymerase after annealing. Fig. 11. Example of the influence of extension temperature. Multiplex PCR with mixtrues A-B using two different PCR programs. Reactions on the right side (green) were performed in identical cycling conditions with Fig. 9, whereas reactions on the left side (yellow) were performed using cycling conditions in which extension temperature was dropped from 72 o C to 65 o C. Reaction worked more efficiently with the lower extension temperature (pink arrow show missing products, yellow arros show unspecific products).Primer amount and buffer concentration. To improve the amplification of some of the DNA products from Fig. 11 above, the amount of primers was increased 2-5x for those loci. At the same time, the PCR buffer concentration was increased to 2x. These modifications allowed a much more efficient and reproducible amplification, with no unspecific products. RETURN TO PCR guide[NextPage]Primer amount in PCRAbsolute value of primer concentration in multiplex PCR. The amount of DNA primer(s) available during the PCR reaction influences the results. Primer concentration taken in a common PCR reaction (for example when amplifying a single locus) is about 100-500 nM each primer. (Primers can be purchased from various sources at concentrations between 10-25 mM each. Usually, 0.5-1ml primer solution is sufficient for a 25-100 ml PCR reaction) In a multiplex PCR test using equimolar primer mixtures (Fig. 13), individual primer concentrations were varied between 500 and 15 nM each primer. Given that mixture A used 14 primers (7 loci) and mixture B 10 primers (5 loci), the final primer concentration varied between 7000 and 200 nM (mixture A) and between 5000 and 150 nM (mixture B). Although equimolar primer mixtures did not usually provide optimal amplification of all loci, this test allowed the observation that too high and too low primer amounts may need to be avoided.Too high primer concentrations may inhibit the multiplex reaction whereas too low amounts may not be sufficient.Fig. 13. Multiplex PCR with mixtures A and B (see also Fig. 1). Numerical values indicate the concentration of each primer in the final reaction. Mixture A includes 14 primers and mixture B includes 10. Reactions work best at around 200 nM (each primer) in mixture A and 60 nM (each primer) in mixture B.Primer and template concentrations. Within limits, increasing primer concentration may improve the outcome of the PCR reaction, and should be considered as a way to optimize PCR reactions. RETURN TO PCR guide[NextPage]PCR buffersA commonly used PCR buffer, includes only KCl, Tris and MgCl2 (for example, Perkin Elmer Cetus); a somewhat more complex buffer was previously proposed for multiplex reactions of the DMD gene exons (Chamberlain et al. (1988) in Nucleic Ac Res 16: 11141-11156). These buffers were compared in multiplex PCR reactions, for their efficiency in supporting the activity of the Taq polymerase. Figure 15 shows that, PCR reactions on four different genomic DNA templates were consistently more efficient (more PCR product) when performed in 1.6x PCR buffer than 1x DMD buffer. Same amount of template DNA and primer were taken in all reactions, which were run in the same conditions at the same time. The same amount of product was loaded in each lane on the gel.Table 3. Comparison of PCR buffers. Fig 15. Comparison of two differnt PCR buffers. Multiplex mixture F was used in PCR amplification of 4 different genomic DNA templates. Reactions were performed in identical conditions with the exception of the buffers. Results indicate a higher yield of products in reactions performed in 1.6 x PCR buffer.RETURN TO PCR guide[NextPage]Salt (KCl) concentrationFor the successful PCR or multiplex PCR amplification of many loci (especially products between 100-1000 bp) raising the buffer concentration to 1.4x-2x (or only the KCl concentration to about 70-100mM) dramatically improves the efficiency of the reaction. In fact the effect of the KCl concentration was more important than any of the adjuvants tested (DMSO, glycerol or BSA). Generally, many primer pairs producing longer amplification products worked better at lower salt concentrations, whereas many primer pairs producing short amplification products worked better at higher salt concentrations. This is illustrated in the three figures below in which there is a realtive shift in the intensity of the products from the longer one towards the shorter ones as the ionic strength increases. An increase in salt concentration makes longer DNA denature slower than shorter DNA, so shorter molecules will be amplified preferrentially. Some primers, however, worked well over a wide range of buffer/salt concentrations. Examples of multiplex reactions at different buffer concentrations are shown in Fig. 16, 17 and 18.Fig. 16. Multipex PCR amplification of mixture A at increasing buffer (salt) concentrations.In Fig. 16, both primers for locus 6 have a melting point around 58° C whereas primers for locus 5 have a melting point around 52° C. At the same ionic strength (1x buffer) and annealing temperature (54° C), amplification of locus 6 will be favored over 5. To increase binding of primers for locus 5 while keeping the annealing temperature the same, stringency of the PCR buffer needs to be decreased. This can be easily done by increasing the KCl (or buffer) concentration. A different example is locus 7, where both primers have a similar melting point with primers for locus 6 (58° C). They are not as well amplified in 1x buffer, but respond well to increase in the salt concentration. In this case, the explanation may be that the entire product 7 has a lower GC content than product 6. This makes the DNA helix of product 7 less stable when exposed to the extension temperature. Some of the new strands may detach from the template, before the polymerase fully amplifies them. Decreasing the stringency of the buffer (1.6x-2x) might \"stick\" the newly synthesized strands better to the template, allowing the polymerase to finish its task.PCR reactions in which only KCl or Tris-HCl concentrations were varied, showed that the described effect is due to the salt (KCl). Tris-HCl concentration did not influence the outcome of the reactions over a large range of concentrations (from 0.75x to 5x) whereas MgCl2 concetrations have a somewhat different effect.Fig. 17. Multipex PCR amplification of mixtures B and C* at increasing buffer (salt) concentrations.Fig. 18. Multipex PCR amplification of mixtures D and E at increasing buffer (salt) concentrations.RETURN TO PCR guide[NextPage]Designing PCR programsBasic Principles The requirement of an optimal PCR reaction is to amplify a specific locus without any unspecific by-products. Therefore, annealing needs to take place at a sufficiently high temperature to allow only the perfect DNA-DNA matches to occur in the reaction. For any given primer pair, the PCR program can be selected based on the composition (GC content) of the primers and the length of the expected PCR product. In the majority of the cases, products expected to be amplified are relatively small (from 0.1 to 2-3 kb). (For long-range PCR (amplifying products of 10 to 20-30 kb) commercial kits are available). The activity of the Taq polymerase is about 2000 nucleotides/minute at optimal temperature (72-78o C) and the extension time in the reaction can be calculated accordingly.As the activity of the enzyme may not be always optimal during the reaction, an easy rule applied successfully by the author was to consider an extension time (in minutes) equal to the number of kb of the product to be amplified (1 min for a 1 kb product, 2 min for a two kb product etc.). Later on, after the product(s) become \"known\", extension time may be further reduced. Many researchers use a 2-5 minutes first denaturing step befo, re the actual cycling starts. This is supposed to help denaturing the target DNA better (especially the hard to denature templates). Also, a final last extension time, of 5-10 minutes, is described in many papers (supposedly to help finish the elongation of many or most PCR products initiated during the last cycle). Both these steps have been tested for a numer of different loci, and, based on this experience, neither the first denaturing nor the last extension time changed in any way the outcome of the PCR reaction. Therefore, it is the author\'s habit not to use these steps (light blue in the table below) anymore. The annealing time can be chosen based on the melting temperature of the primers (which can be calculated using othe many applications, freely available for molecular biologists). This may work, but sometimes the results may not match the expectations. Therefore, a simple procedure used many times by the author was to use an initial annealing temperature of 54 o C (usually good for most primers with a length around 20 bp or more). If unspecific products result, this temperature shoud be inccreased. If the reaction is specific (only the expected product is synthesized) the temperature can be used as is. For the seventy or so primers used during this work, a denaturing time of 30-60 seconds was sufficient to achieve good PCR products. To long a denaturing time, will increase the time the Taq polymerase is subjected at high temperatures, and increases the percentage of polymerase molecules that lose their activity. Number of cycles. In general, 30 cycles should be sufficient for a usual PCR reaction. An increased number of cycles will not dramatically change the amount of product (see below). Influence of annealing temperature and number of loci amplified Like any other PCR, multiplex reactions should be done at a stringent enough temperature, allowing amplification of all loci of interest without \"background\" by-products. Although many individual loci can be specifically amplified at an annealing temperature of 56°-60° C, experiments showed that lowering the annealing temperature by 4-6° C was required for the same loci to be co-amplified in multiplex mixtures. This is demonstrated in Fig. 19 below, showing the same PCR reactions performed in conditions in which the only parameter changed was the annealing temperature. For the multiplex a PCR amplification of mixtures C and C*, an annealing temperature of 54° C seems the most appropriate, although the individual loci (for example \"Y\") could be amplified at 60° C. At 54° C, although some unspecific amplification probably still occurs in the multiplex reaction, it is overcome by the concurrent amplification of an increased number of specific loci and thus remains invisible.In PCR, due to differences in base composition, length of product or secondary structure some loci are more efficiently amplified than others When many loci are simultaneously amplified (multiplexed), the more efficiently amplified loci will negatively influence the yield of product from the less efficient loci. This phenomenon is due in part to the limited supply of enzyme and nucleotides in the PCR reaction. Therefore, in the multiplex procedure the more efficiently amplified loci compete better and take over the less efficiently amplified products, thus rendering them less visible or invisible.(Figure 19 below, depicts a complex situation in which annealing temperature, number of simultaneously amplified loci and buffer concentration were changed in parallel reactions).Fig. 19. Multiplex amplification of mixture C* (first three lanes in each gel), primer pair \"Y\" (lanes 4 to 6, blue arrows) and mixture C (lanes 7 to 12 in 1x or 2x PCR buffer) on three different template DNAs using three PCR programs differing in annealing temperature (48° C, 54° C or 59° C). Lanes 1-9 on each gel show reactions in 1x PCR buffer. Lanes 10-12 on each gel show reactions in 2x PCR buffer. Lanes 7-12 on each gel (under \"1x\" and \"2x\" ) were with primer mixture C. The unmarked lanes are the marker (1 kb ladder). The five arrows to the left side of the first gel indicate the expected products of mix C* (five products). The longest specific product on each gel is marked by a red arrow. Magenta arrow indicates a strong unspecific product. Yellow arrows indicate the two extra products expected in mix C (total of seven products) compared with C*. Blue arrows indicate position of product Y (either by itself or in the multiplex mixture) in the first gel or the lack of product Y in some of the reactions from the last two gels. Multiplex amplification at 48° C shows many unspecific bands. In 1x PCR buffer, the Y product is stronger when amplified in mixture C* (5 primer pairs) than in mixture C (7 primer pairs) showing that, at least for some products, an increased number of simultaneously amplified loci can influence the yield of some individual loci. Raising the PCR buffer concentration from 1x to 2x allows a more even amplification of all specific products and helps in decrease the intensity of many longer unspecific products (compare lanes 7-9 vs. 10-12). The strong 470-480bp unspecific band (magenta arrow) seen with 2x buffer was eliminated by varying the proportion of different primers in the reaction (compare with C in Fig 1). At 59° C the Y product can be seen only when 2x buffer is used or when the locus is amplified alone.Number of cyclesPrimer mix C* was used to amplify two different genomic DNA templates, stopping the reaction after increasing numbers of cycles (Fig. 20). For the same DNA template, results were reproducible among all vials although one of the two genomic DNAs was better, probably due to the higher quality and/or amount of DNA. The most obvious variation in the amount of products was around 24 cycles (for ethidium bromide stained gels). 28-30 cycles are usually sufficient in a reaction. Little or no quantitative changes (i.e., relative amounts of PCR products) were observed with increasing cycle number up 45. Little quantitative gain was noticed when increasing the number of cycles up to 60 (Fig. 21)Fig. 20. Multiplex amplification of mixture C* using two different DNA templates and increasing the numbers of cycles by units of three.Fig. 21. Multiplex amplification of mixture C* using tthe same PCR program and increasing the number of cycles by units of ten (up to 60). No additional ingredients were added in the reactions.RETURN TO PCR guide[NextPage]Annealing time and temperatureAnnealing time An annealing time of 30-45 seconds is commonly used in PCR reactions. Increase in annealing time up o 2-3 minutes did not appreciably influence the outcome of the PCR reactions. However, as the polymerase has some reduced activity between 45 and 65o C (interval in which most annealing temperature are chosen), longer annealing times may increase the likelihood of unspecific amplification products (data not shown)Annealing temperature is one of the most important parameters that need adjustment in the PCR reaction. Moreover, the flexibility of this parameter allows optimization of the reaction in the presence of variable amounts of other ingredients (especially template DNA). For example, the PCR product depicted in Fig. 22 could be amplified easily at annealing temperatures of 55 o C in the presence of 1-100 ng genomic DNA template. Below this limit, there was no detectable PCR product on agarose gels (this primer pair amplifies a polymorphic locus, explaining the two bands seen on non-denaturing agarose gels). It was observed that the specific product can be detected again, even in the presence of very low DNA template concentrations, if the annealing temperature is also decreased. In the reactions depicted in figure 22, the DNA template amount was decreased to 3.1 pg (which is about half the DNA content of a diploid human cell). Remarkably, only one allele was preferentially amplified when the template DNA was approximately 6.6 pg. To achieve these results, reaction was performed at 45 o annealing temperature (a 10 degrees drop from usual). No unspecific products are seen. However, if the same reaction is performed in the presence of a higher amount of DNA template, the low annealing temperature results in the appearance of many unspecific secondary products. Thus, it appears that by decreasing the amount of DNA template, the number of potentially unspecific sites is also decreased, making possible the drop in annealing temperature.Fig. 22. PCR amplification of a plymorphic locus in the presence of decreasing, low amounts of genomic template DNA and at an annealing temperature 10 o C lower than normal.Lanes A-F show slight variation in the amount of product, when vials with identical reaction mixture were placed in different position in the metal block of a thermocycler. Amount of template was 800pg/reaction.Polymorphisms and annealing temperature Annealing temperature is important in finding and documenting polymorphisms. Slight mismatches, (even 1 base-pair mutations) in one of sequences bound by the two primers used to amplify a DNA locus, can be detected by slight variations in annealing temperature and/or by multiplex PCR. In Fig. 23 such a polymorphism on human Y chromosome is detected in a few DNA samples by amplifying that locus along with other ones using multiplex mixture C (see also Fig. 1). In Fig. 24, same polymorphism is detected by performing PCR reaction only with the specific primer pair, but increasing the stringency of the annealing temperature.Fig. 23. Single-locus PCR on 7 different template DNAs with a primer pair amplifying a polymorphic locus (yelow). Multiplex PCR of the same templates when the primer pair is part of mixture C. Reactions were performed in the same cycling conditions (annealing at 54 o C). The slight mismatch in primer binding (polymorphism) is detected only in the multiplex reaction by the lack of the amplification product (magenta arrows).Fig. 24. Same primer mismatch described above can be detected by single-locus PCR reactions after increasing the stringency of the annealing temperature. Samples 3 and 4 show a decrease of product at 61 o C annealing temperature but have a \"normal\" appearance at 59 o C annealing temperature (magenta arrows).RETURN TO PCR guide[NextPage]Extension time and temperatureExtension time In multiplex PCR, as more loci are simultaneously amplified, the pool of enzyme and nucleotides becomes a limiting factor and more time is necessary for the polymerase molecules to complete synthesis of all the products. Extension time will play an important role in adjusting the outcome of the PCR reaction. This is illustrated in the experiments depicted in two figures below. In one experiment, multiplex mixtures A-D (see also fig. 1) were amplified using PCR programs with 1 and 2 minutes extension times, respectively. Higher yields of PCR products were obtained in all four mixtures when the longer extension time was used. Optimal amplification of all loci will require further adjustments in other factors influencing the reaction (buffer concentration, amount of individual primers). A somewhat lower reproducibility of the results between Fig 11 and Fig 25 was most probably due to a combination of small pipetting differences and the fine balance between buffer, dNTP and MgCl2 concentration (see those topics). Within the same experiment, however, results were reproducible and the effect of various parameters could be studied (Fig. 25).In the other experiment (Fig. 26) increasing the extension time in the multiplex PCR increased the amount of longer products, at the \"expense\" of the shorter ones.Fig. 25. Multiplex PCR of mixtures A-D comparing PCR programs with 2 (green) and 1 (yellow) minute extension time at 54° C annealing temperature. Comparison of equivalent lanes shows an improvement in yield when extension time is 2 minutes. Some faint unspecific bands appear, possibly due to the low buffer concentration (1x).Fig. 26. Same multiplex mixture was amplified on PCR programs differing only in their extension time (1 and 4 minutes). Shorter amplification products are preferentially amplified with short extension times (1 minute) whereas the longer products become more visible as the extension time increases (arrows). At the same time, at 4 minutes, the shorter products lose much of their intensity. Reactions in lanes 1a and 1b are identical (different DNA templates only).Extension temperature Figure 11 illustrates the influence of the extension temperature. Equimolar primer mixtures A-D were amplified using two different PCR programs, one at 65o C (yellow lanes) and the other at 72o C (green lanes) extension temperature. In general, there is a higher yield of PCR products for A, B and D when program A was used. This shows that the 72o C extension temperature, negtively influenced amplification of some loci (pink arrows),while also making some unspecific products visible (yellow arrows). It is likely that, for the short PCR products used in these examples (below 500 bp), the higher annealing temperature is probably detrimental to the stability of the DNA helix, so less strands of DNA have the chance to become \"copied\" by the polymerase after annealing. Fig. 11 (duplicate). Example of the influence of extension temperature. Multiplex PCR with mixtrues A-B using two different PCR programs. Reactions on the right side (green) were performed in identical cycling conditions with Fig. 9, whereas reactions on the left side (yellow) were performed using cycling conditions in which extension temperature was dropped from 72 o C to 65 o C. Reaction worked more efficiently with the lower extension temperature (pink arrow show missing products, yellow arros show unspecific products).RETURN TO PCR guide[NextPage]DNA templateAll multiplex reactions performed in this laboratory used human genomic DNA as a template. From both multiplex and single-locus PCR reactions, results showed that the amount of DNA template strongly influences the outcome of the reaction. In conditions in which the amount of DNA available is very low, reaction or cycling conditions can be adapted and modified to allow reaction to work efficiently.The following five images provide examples illustrating the importance of the DNA template concentration.Fig. 27. PCR amplification of very low amounts of genomic DNA using a degenerate primer. Amount of PCR product decreases with the decreasing amount of template.Fig. 28. Multiplex PCR using primer mixture A in 1x PCR buffer. As the amount of template drops, most products become gradually weaker. Cycling conditions were identical. Arrow indicates the presence of an unspecific product.Fig. 29. Multiplex PCR with mixture C* and single-locus PCR with one of the primer pairs form the same mixture. As the DNA template decreases, some bands become weaker in the multiplex reaction. Over the same range of concentrations, this effect is not so visible when only one primer pair is used.Fig. 30. Multiplex PCR with mixture C* and PCR amplification using only one of the primer pairs from the same mixture. Very low template DNA concentrations were used (0.045 is the amount of DNA from 6 diploid cells). Again, the amount of PCR product decreases with the reduction in template DNA but less so when only one primer pair is used. PCR program used has a lower annealing temperature (about 5o C lower) than the program used for the reactions in Fig. 29.Fig. 31. Multiplex PCR with mixture C* on two genomic DNA temlpates, one (yellow) carrying a polymorphism for one primer binding site and another one (green) with perfect match. As in Fig. 30 above, to amplify such reduced amounts of DNA template, the same program with low annealing temperature had to be used. Arrow indicates that the polymorphism at locus 4 is detected with the decrease in DNA template amount.RETURN TO PCR guide[NextPage]Taq polymeraseDifferent concentrations of a Taq polymerase were tested using primer mixture C (Fig. 32). The most efficient enzyme concentration seemed to be around 0.4 l or 2 Units/25 l reaction volume. Too much enzyme, possibly because of the high glycerol concentration in the stock solution, resulted in an unbalanced amplification of various loci and a slight increase in the background. too little enzyme resulted in the lack of some of the amplification productsFig. 32. Amplification products of mixture C, using 0.5 Units/25 l, 1 Unit/25 l, 2Units/25 l, 4 Units/25 l and 8 Units/25 l reaction volume are shown. Arrows indicate the expected positions of the amplification products. The most appropriate enzyme concentration was between 1-2 Units/25 l.Five native Taq polymerases, from five different sources, were used to amplify multiplex mixture D in 1.6x PCR buffer using 2Units enzyme/25 l reaction (Fig. 33). In the same buffering conditions, all these enzyme performed similarly.Fig. 33. Multiplex PCR of mixture D in 1.6x PCR buffer using Taq polymerases from five sources. Lanes 1 to 5 indicate that all enzymes work similarly at the same concentration. Lane 4* (green) shows the products obtained when the enzyme from lane 4 was used in the buffer provided by the vendor. An unspecific product appeared, indicating that buffer composition influences the results.RETURN TO PCR guide[NextPage]Nucleotides (dNTP)dNTP \"instability\" One important observation, coming from experiments with multiplex PCR, is that dNTP stocks are very sensitive to cycles of thawing/freezing. After 3-5 such cycles, multiplex PCR reactions usually did not work well. To avoid such problems, small aliquots (2-5 l) of dNTP (25 mM each), lasting for only a couple of reactions, can be made and kept frozen at -20o C. However, during long-term freezing, small amounts of water evaporate on the walls of the vial changing the concentration of the dNTP solution. Before using, it is essential to centrifuge these vials at high speed in a microfuge.This low stability of the dNTP is not so obvious when single loci are amplified.Another important observation is that, anytime nucleotides are diluted in water, the solution should be buffered (for example with 10mM Tris pH 7.7-8.0, final concentration).Otherwise, an acid pH will promote hydrolysis of dNTP into dNDP and dNMP and will render them useless for enzymatic DNA polymerizing reactions.Relationship between MgCl2 and dNTP concentration dNTP concentrations of about 200 M each are usually recommended for the Taq polymerase, at 1.5mM MgCl2 (Perkin Elmer Cetus). In a 25 l reaction volume, theoretically these nucleotides should allow synthesis of about 6-6.5 g of DNA. This amount should be sufficient for multiplex reactions in which 5 to 8 or more primer pairs are used at the same time. To work properly (besides the magnesium bound by the dNTP and the DNA), Taq polymerase requires free magnesium. This is probably the reason why small increases in the dNTP concentrations can rapidly inhibit the PCR reaction (Mg gets \"trapped\")whereas increases in magnesium concentration often have positive effects.The relationship between the concentration of magnesium and that of the dNTPs was investigated by performing PCR with a degenerate primer in reactions that contained 200, 400, 600 and 800 M each dNTP, combined with 1.5, 2, 3, 4 or 5 mM MgCl2 (Fig. 34). This test confirmed that any increase in dNTP concentration requires an increase in the concentration of magnesium ions in order for the reaction to work. At 200 M each dNTP, reaction worked at all magmesium concentrations, but for this primer it worked better at 3 mM (which is about double the recommended magnesium concentration for the amount of dNTP). At 800 M each dNTP, reaction worked only aboove 3 mM magnesium.Fig. 34. PCR with a degenerate primer at different Mg and dNTP concentrations. Each of the Mg concentrations (1.5, 2, 3, 4, 5 mM) were combined with each of the following dNTP concentrations (each): 200 M, 400 M, 600 M and 800 M. Results indicate that increasing dNTP concentrations require increasing Mg concentrations for the PCR reactions to work.Common dNTP use in PCR and multiplex PCR In another test aimed at examining the proper dNTP concentration, a multiplex PCR using primer mixture D was performed. The MgCl2 concentration was kept constant (3mM) while the dNTP concentration was increased stepwise from 50 to 100, 200, 400, 600 and 1200 M each deoxynucleotide (Fig. 35). The best results were achieved at 200 and 400 m dNTP; reaction was rapidly inhibited after these values. Lower than usual dNTP concentrations still allowed PCR amplification, but with somewhat less efficiency (lane \"50\").Fig. 35. Multiplex PCR amplification of mixture D in 2x PCR buffer (3 mM Mg) using increasing concentrations of dNTP (50mM, 100mM, 200mM, 400mM, 600mM and 1200mM each). Most efficient amplification is seen at concentrations of 200-400 M each dNTP. Further increase in the dNTP concentration inhibits the reaction when MgCl2 is kept constant.RETURN TO PCR guide[NextPage]MgCl2 concentrationRelationship between MgCl2 and dNTP concentration dNTP concentrations of about 200 M each are usually recommended for the Taq polymerase, at 1.5mM MgCl2 (Perkin Elmer Cetus). In a 25 l reaction volume, theoretically these nucleotides should allow synthesis of about 6-6.5 g of DNA. This amount should be sufficient for multiplex reactions in which 5 to 8 or more primer pairs are used at the same time. To work properly (besides the magnesium bound by the dNTP and the DNA), Taq polymerase requires free magnesium. This is probably the reason why small increases in the dNTP concentrations can rapidly inhibit the PCR reaction (Mg gets \"trapped\")whereas increases in magnesium concentration often have positive effects.The relationship between the concentration of magnesium and that of the dNTPs was investigated by performing PCR with a degenerate primer in reactions that contained 200, 400, 600 and 800 M each dNTP, combined with 1.5, 2, 3, 4 or 5 mM MgCl2 (Fig. 34). This test confirmed that any increase in dNTP concentration requires an increase in the concentration of magnesium ions in order for the reaction to work. At 200 M each dNTP, reaction worked at all magmesium concentrations, but for this primer it worked better at 3 mM (which is about double the recommended magnesium concentration for the amount of dNTP). At 800 M each dNTP, reaction worked only aboove 3 mM magnesium.Fig. 34. PCR with a degenerate primer at different Mg and dNTP concentrations. Each of the Mg concentrations (1.5, 2, 3, 4, 5 mM) were combined with each of the following dNTP concentrations (each): 200 M, 400 M, 600 M and 800 M. Results indicate that increasing dNTP concentrations require increasing Mg concentrations for the PCR reactions to work.Relationship between MgCl2 and buffer (or salt) concentration Two of the most important ingredients influenceing the results of a PCR reaction are the buffer (especially salt) and the magnesium concentrations. To study their relationship, a multiplex PCr was performed using mixture C (Fig. 36, below). Two sets of reactions were performed at two \"extreme\" concentrations of salt (KCl), 1x (50mM) and 3x (150 mM), and various magnesium concentrations (yellow values). Two other sets of reactions were performed at two \"extreme\" magnesium concentrations, 1.5 and 10.8 mM and various salt (KCl) concentrations (blue values). The dNTP concentration was kept constant, at 200 mM each deoxynucleotide. The following observation can be drawn:at 1x salt concentration and 200 mM each dNTP, reaction worked best at about 1.5 mM magnesium. At higher magnesium concentrations unspecific products appeared, but they gradually decreased in intensity towards 21.6 mM (probably because MgCl2 is a salt, decreasing the stringency of the buffer - same way KCl does). at 3x salt concentration and 200 mM each dNTP, reaction worked best between 1.5 and 3.5 mM magnesium. As the stringency of the buffer was already lower than usual (due to the high KCl concnentration), further increase in MgCl2 increased the \"combined\" stringency of teh reaction even more. Thus, fewer long unspecific products were obtained and the reaction was almost completely inhibited towards 21.6 mM magnesium. at 10.8 mM MgCl2 and 200 mM each dNTP, reaction worked best around 2x salt (KCl) concetration (mostly specific products amplified). However, it is obvious that overall amount of PCR product is reduced compared to the reactions taking place at 1.5 mM magnesium. In this respect, high magnesium concentrations seem to inhibit the reaction more than high KCl (3x) concentrations. Therefore, it is likely that this magnesium inhibition is more than just a reduction in stringency of the reaction mixture. at 1.5 mM magnesium and 200 mM each dNTP, reaction worked best around 2x salt (KCl) concentration (all products amplified, few unspecific products visible). Overall product amount is higher than in the reactions taking place at 10.8 mM magnesium.Fig. 36. Realtionship between magnesium and salt (KCl) concentration in PCR reactions. For a detailed description of the figure, please read text above.Effects of variations in MgCl2 concentration only A recommended MgCl2 concentration in a standard PCR reaction is 1.5mM, at dNTP concentrations of around 200 M each. To test the influence of MgCl2, a multiplex PCR with mixture C was performed, keeping dNTP concentration at 200 M each and gradually increasing MgCl2 from 1.8 to 10.8 mM (Fig. 37). The overall amplification became gradually more \"specific\" (unspecific bands disappeared) and the products acquired comparable intensities (at 10.8mM). However, higher concentrations of MgCl2 appeared to inhibit the polymerase activity, decreasing the amount of all products. Taking into consideration the amount of PCR products, the best magnesium concentration should be between 1.8 and 3.6 mM. The large unspecific product (arrow) appeared due to the lower annealing temperature at which the reaction took place.Fig. 37. Multiplex PCR amplification with mixture C at 2x KCl and increasing magnesium concentrations. Overall reaction becomes more specific at 10.8 mM magnesium, but the products are reduced in intensity. The most optimal magnesium concentration is somewhere between 1.8 and 3.6 mM where the PCR product amount is higher. The unspecific product (arrow) appears due to a lower than usual annealing temperature used for this reaction.RETURN TO PCR guide[NextPage]Gel electrophoresisComparison of agarose type (non-polymorphic loci) Two types of agarose from the same manufacturer (both in use in this laboratory) were compared for their efficiency in separating the multiplex PCR products (Fig. 38). Multiplex PCR with primer mixtures A (one sample) and F (4 samples) was performed. Same amount of each reaction was loaded on a 3% agarose gel of each type. Electrophoresis time was about 1.6-1.7x longer for the regular (SeaKem LE) agarose gel.In accordance to the manufacturer\'s specifications, the NuSieve agarose separates short products better than the regular agarose, and in a reduced amount of time. Although the gels had the same thickness, results also indicate that the \"special\" NuSieve agarose is more transparent than the regular agarose. Although NuSieve agarose is much more expensive, it provides some cost reduction by requiring less amount of agarose for the same separation power and by requiring less amount of separation time. These particular advantages can make such \"specialized\" agaroses useful for particular applications.It is worth mentioning that other agaroses (from different manufacturers) used, perform similarly.Fig. 38. Separation of the same multiplex products of mixtures A and C (four lanes) on two different agaroses. Arrow indicates a few unspecific products in lane 2 and circle indicates primers (or primer-dimers), both of these being stronger on the NuSieve gel. This shows tthat NuSieve gels have a higher transparency. Also, separation on NuSieve gels was achieved in les amount of time, over a shorter gel length. The unmarked lane(s) is the 1 kb ladder (GIBCO).Agarose gel (running time) Agarose gels can be run at various voltages, depending on the separation desired and the available time. It was noticed that, at least for PCR products smaller than 600 bp, separation is better and bands are sharper if gels are run very fast (3-4 hours for a 15-20 cm long 2-3% agarose gel). When the same gel runs at a low voltage overnight (14-16 hours) the products become less separable or \"puffy\" due to the diffusion in the gel (compare Fig. 39 below with lane C in Fig. 1).Fig. 39. Multiplex PCR with mix C was performed on 9 DNA samples to screen for microdeletions (chromosome Y loci. Gel separation was performed overnight (14 hours). Products appear diffuse, less intense, and less separable (product 1 and 2 are \"fused\" together). Green and magenta arrows indicate lack of loci #1 and # 4 (microdeletions) in some of the DNA samples tested. he unmarked lane(s) is the 1 kb ladder (GIBCO).Agarose gels and polymorphic loci As depicted also in Fig. 7, agarose gels can be used to separate PCR products of plymorphic loci. In most cases, two or three bands appear, due to heteroduplex formation between the long and short alleles. However, separation of multiplex PCR reaction products of many polymorphic loci (for example mixture K) coud become a problem for nondenaturing agarose gels. In mixture K, products were chosen so they differ by no less than 5 bp and no more than 45 bp. As depicted in Fig. 7 and Fig. 40 (below), agarose gels do not have sufficient separation power. Bands become to overlap and it is difficult or impossible to find and label each band. Denaturing polyacrylamide gels are recommended in such cases (see below).Fig. 40. Multiplex PCR amplification of mixture K, using four different DNA samples. A 2.5% agarose gel was used to separate the products. As depicted also in Fig. 7, 2-3 bands become visible for each product. When together, many of these bands start overlapping, making identification of individual products/alleles impossible. The unmarked lane(s) is the 1 kb ladder (GIBCO).Non-denaturing PAA gels To separate PCR products differing in only a few bp in length (for example, microsatellite markers), 6-10% PAA gels need to be used. Whereas non-denaturing PAA gels work very well for non-polymorphic loci, unusual bands appear when microsatellites are separated on this type of gels. For example, in an analysis of two polymorphic loci from two hybridomas, each carrying one copy of a human chromosome, for each locus tested there were 2 bands on the non-denaturing PAA gel (Fig. 41). It is unclear where the extra band originates when only one allele is amplified.Fig. 41. Multiplex amplification of two loci on DNA from two human-rodent cell lines, each with a different copy of a human chromosome, and their combination (a+b). Although in lanes a and b each locus yields only one allele (i.e. one band), on a non-denaturing polyacrylamide gel each of the two expected products (arrows) was acompanied by another one running slower on the gel (oblique arrows). A similar aspect persisted in lane A+B. Lanes 1 and 2 show separation of products of mixture K on two different genomic template DNAs. The unmarked lane(s) is the 1 kb ladder (GIBCO).Denaturing PAA gels Denaturing 6% PAA/7M urea sequencing gel can be easily used to separate radiolabeled multiplex PCR products, whether these are polymorphic or unique. Denaturing PAA gels, however, are more expensive, time consuming and might prove technically more difficult. Figure 3 below, shows separation of the unique products of multiplex mixture J; double bands are visible for some of the loci. Figure 4 below shows separation of the polymorphic loci (microsatellites) of multiplex mixture K; two distinct alleles are visible for many of loci in the 8 DNA samples tested.Fig. 3 (duplicate). Multiplex PCR with mix J on four different genomic DNA templates, separated on an a denaturing, 6% polyacrylamide gel. Primers amplify nonpolymorphic loci. The sizes of the longest and the shortest product are also indicated. Fig. 4 (duplicate). Multiplex PCR with mix K on eight different genomic DNA templates, separated on an a denaturing, 6% polyacrylamide gel. These primers amplify polymorphic loci, and alleles of different sizes can be observed. The shortest alleles of locus 6 and the longest alleles of locus 7 can, sometimes, overlap, making it difficult to assign precisely their origin. The sizes of the longest and the shortest product are also indicated.RETURN TO PCR guide[NextPage]Adjuvants in PCR reactionsVarious authors recommend DMSO and glycerol to improve amplification efficiency (higher amount of product) and specificity (no unspecific products) of PCR, when used in concentrations varying between 5-10% (v/v). In the multiplex reaction, however, these adjuvants gave conflicting results. For example, 5% DMSO (Fig. 42) improved the amplification of some products, decreased the amount of others whereas some loci were not influenced at all. Similar results were obtained with 5% glycerol (data not shown). Therefore, the usefulness of these adjuvants needs to be tested in each case. BSA, in conc, entrations of up to 0.8 g/ l (higher than previously described) appeared to increase the efficiency of the PCR reaction much more than either DMSO or glycerol. It should be noted that BSA did not have an inhibitory effect on any of the loci amplified (data not shown).Fig. 42. Comparative multiplex PCR using mixtures A to D with 5% DMSO (superscript D) and without DMSO, in 1x buffer. Some loci from mixture A (blue arrows) are stronger when no DMSO is used. However, DMSO helps amplify (magenta arrows) one locus in mix B and one locus in mixture D. Amplification of PCR products of mixture C* were unaffected by DMSO.RETURN TO PCR guide[NextPage]Troubleshooting for PCR and multiplex PCRTroubleshooting discussion is based on the PCR protocol as described in the table below. All reactions are run for 30 cycles.100 ng/25 L* The 10x PCR buffer contains: 500 mM KCl; 100 mM Tris-HCl (pH 8.3); 15 mM MgCl2 (the final concentrations of these ingredients in the PCR mix are: 50 mM KCl; 10 mM Tris-HCl; 1.5 mM MgCl2). Decrease annealing timeIncrease annealing temperatureDecrease extension timeDecrease extension temperature to 62-68 CIncrease KCl (buffer) concentration to 1.2x-2x, but keep MgCl2 concentration at 1.5-2mM.Increase MgCl2 concentration up to 3-4.5 mM but keep dNTP concentration constant.Take less primerTake less DNA templateTake less Taq polymeraseIf none of the above works: check the primer for repetitive sequences (BLAST align the sequence with the databases) and change the primer(s)Combine some/all of the above Increase annealing temperatureIncrease annealing timeIncrease extension timeIncrease extension temperature to 74-78 CDecrease KCl (buffer) concentration to 0.7-0.8x, but keep MgCl2 concentration at 1.5-2mMIncrease MgCl2 concentration up to 3-4.5 mM but keep dNTP concentration constantTake less primerTake less DNA templateTake less Taq polymeraseIf none of the above works: check the primer for repetitive sequences (BLAST align the sequence with the databases) and change the primer(s)Combine some/all of the above Make sure all PCR ingredients are taken in the reaction (buffer, template, Taq, etc)Change the dNTP solution (very sensitive to cycles of thawing and freezing, especially in multiplex PCR)If you just bought new primers, check for their reliability (bad primer synthesis ?)Increase primer amountIncrease template amountDecrease annealing temperature by 6-10 C and check if you get any product. If you don\'t, check all your PCR ingredients. If you do get products (including unspecific ones) reaction conditions as described above.Combine some/all of the above Gradually decrease the annealing temperature to the lowest possible.Increase the amount of PCR primerIncrease the amount of DNA templateIncrease the amount of Taq polymeraseChange buffer (KCl) concentration (higher if product is lower than 1000bp or lower if product is higher than 1000bp)Add adjuvants. Best, use BSA (0.1 to 0.8 g/ L final concentration). You can also try 5% (v/v, final concentration) DMSO or glycerol.Check primer sequences for mismatches and/or increase the primer length by 5 nucleotidesCombine some/all of the above 5. My two primers have very different melting temperatures (Tm) but I cannot change their locus. What can I do to improve PCR amplification? An easy solution is to increase the length of the primer with low Tm. If you need to keep the size of the product constant, add a few bases at the 3\' end. If size is not a concern, add a few bases at either the 3\' or the 5\' end of that primer.6. I have a number of primer pairs I would like to use together. Can I run a multiplex PCR with them?. How? Very likely, yes.Try amplify all loci seaprately using the same PCR program. If one of the primer pairs yields unspecific products, keep the cycling conditions constant and change other parameters as mentioned above (#1 and #2).Mix equimolar amounts of primers and run the multiplex reaction either in the same cycling conditions or by decreasing only the annealing temperature by 4 C.If some of the loci are weak or not amplified, read below !! Difficult to say. The author has routinely amplified from 2 to 14 loci.Literature describes up to 25 loci or so. 8. One or a few loci in my multiplex reaction are very weak or invisible. How can amplify them? The first choice should be increasing the amount of primer for the \"weak\" loci at the same time with decreasing the amount of primer for all loci that can be amplified. The balance between these amounts is more important than the absolute values used !!.Check primer sequences for primer-primer interactions 9. Short PCR products in my multiplex reaction are weak. How can I improve their yield? Increase KCl (buffer) concentration to 1.2x-2x, but keep MgCl2 concentration at 1.5-2mMDecrease denaturing timeDecrease annealing time and temperatureDecrease extension time and temperatureIncrease amount of primers for the \"weak\" loci while decreasing the amount for the \"strong\" loci.Add adjuvants. Best, use BSA (0.1 to 0.8 g/ L final concentration). You can also try 5% (v/v, final concentration) DMSO or glycerolCombine some/all of the above 10. Longer PCR products in my multiplex reaction are weak. How can I improve their yield? Decrease KCl (buffer) concentration to 0.7-0.8x, but keep MgCl2 concentration at 1.5-2mMIncrease MgCl2 concentration up to 3-4.5 mM but keep dNTP concentration constant.Increase denaturing timeIncrease annealing timeDecrease annealing temperatureIncrease extension time and temperatureIncrease amount of primers for the \"weak\" loci while decreasing the amount for the \"strong\" lociAdd adjuvants. Best, use BSA (0.1 to 0.8 g/ L final concentration). You can also try 5% (v/v, final concentration) DMSO or glycerolCombine some/all of the above 11. All products in my multiplex reaction are weak. How can I improve the yield? Decrease annealing time in small steps (2 C)Decrease extension temperature to 62-68 CIncrease extension timeIncrease template concentrationIncrease overall primer concentrationAdjust Taq polymerase concentrationChange KCl (buffer) concentration, but keep MgCl2 concentration at 1.5-2mMIncrease MgCl2 concentration up to 3-4.5 mM but keep dNTP concentration constant.Add adjuvants. Best, use BSA (0.1 to 0.8 g/ L final concentration). You can also try 5% (v/v, final concentration) DMSO or glycerolCombine some/all of the above 12. Unspecific products appear in my multiplex reaction. Can I get rid of them somehow? If long: increase buffer concentration to 1.2-2x, but keep MgCl2 concentration at 1.5-2mMIf short: decrease buffer concentration to 0.7-0.9x, but keep MgCl2 concentration at 1.5-2mMGradually increase the annealing temperatureDecrease amount of templateDecrease amount of primerDecrease amount of enzymeIncrease MgCl2 concentration up to 3-4.5 mM but keep dNTP concentration constantAdd adjuvants. Best, use BSA (0.1 to 0.8 g/ L final concentration). You can also try 5% (v/v, final concentration) DMSO or glycerolIf nothing works: run PCR reactions for each (multiplexed) locus individually, using an annealing temperature lower than usual. Compare the unspecific products for each locus tested with the unspecific products seen when running the multiplex PCR. This may indicate which primer pair yields the unspecific products in the multiplex reaction.Combine some/all of the above(Note: primer-primer interactions in multiplex PCR are usually translated into lack of some amplification products rather than the appearance of unspecific products) RETURN TO CONTENT[NextPage]Standard multiplex mixturesOver 75 primer pairs were chosen and a number of multiplex mixtures were designed and used for different purposes. Examples of all multiplex mixes are presented below.(All unlabeled gel lanes are the marker: 1 kb DNA ladder (GIBCO). At the bottom of each image, the PCR buffer concentration used is also indicated)Fig. 1. Mixtures A-E on an a 2.5% agarose gel. Arrow indicates the presence of an unspecific product. Although not desirable, this product did not interfere with the use of mix E in a microdeletion screening project. Occasionally, mix C was used without the primers for loci 4 and 5. In such cases, this mix is called mix C*.Fig. 2. Mixtures F-I on a 2.5% agarose gel.Fig. 3 (duplicate). Multiplex PCR with mix J on four different genomic DNA templates, separated on an a denaturing, 6% polyacrylamide gel. Primers amplify nonpolymorphic loci. The sizes of the longest and the shortest product are also indicated. Fig. 4 (duplicate). Multiplex PCR with mix K on eight different genomic DNA templates, separated on an a denaturing, 6% polyacrylamide gel. These primers amplify polymorphic loci, and alleles of different sizes can be observed. The shortest alleles of locus 6 and the longest alleles of locus 7 can, sometimes, overlap, making it difficult to assign precisely their origin. The sizes of the longest and the shortest product are also indicated.RETURN TO CONTENT[NextPage]Microdeletion screeningOne application of multiplex PCR is microdeletion screening. This can be applied to the X and Y chromosomes (male genomic DNA) or to hybrid cell lines (rodent-human) containing one copy only of a human chromosome of interest. Figures below show a few examples of multiplex PCR screening reactions for microdeletions on human chromosome Y.Fig. 43 shows microdeletion screening reactions with multiplex mixtures A and B Fig. 44 shows microdeletion screening reactions with mixture D (gel separation in 4 hours at high voltage) Fig. 45 shows microdeletion screening reactions with mixture D but in conditions in which gel separation was performed overnight (16 hours) at low voltage. PCR products are visible but more diffused. See also page 15. Fig. 43. Y-chromosome microdeletion screening reactions of 12 male genomic DNA samples (yellow) using multiplex mixture B (5 loci) and of 11 DNA samples (green) using multiplex mixture A (7 loci). DNA samples 1, 2, 9, 10 and 12 (yellow) and 1 and 2 (green) show deletion of some loci tested (lack of amplification products).Fig. 44. Y-chromosome microdeletion screening reactions of 16 male genomic DNA samples (yellow) using multiplex mixture D (5 loci). DNA samples 10, 11, 12, and 13 show deletion of some loci tested. Gel separation was done at high voltage, in about 3-4 hours. See also Fig. 45 for comparisons.Fig. 45. Y chromosome deletion screening using esentially the same DNA samples and primer mixture D as in Fig. 44 above. Only the order of the samples on the gel was somewhat changed. Gel separation was done overnigtht (16 hours) at low voltage. Products appear much more diffuse but result interpretation can be easily done. RETURN TO CONTENTPCR方法相关产品:电泳设备紫外设备普通PCR仪定量PCR仪PCR/RT-PCR/qPCR试剂PCR引物 PCR试剂PCR对照特异性PCR试剂盒PCR克隆试剂盒RNA RNase检测/去除 RT-PCR试剂 RT-PCR标准品 定量PCR试剂 定量PCR标记 总RNA分离纯化盒PCR产物纯化核酸酶 聚合酶 反转录酶