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标题:分享：有关pcr的资料

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分享：有关pcr的资料[转载]

我下了一篇给大家做个示范:

Molecular Biology Techniques Manual
Third Edition
Edited by:

Vernon E Coyne, M Diane James, Sharon J Reid and Edward P Rybicki

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Calculating Concentrations for PCR
Ed Rybicki, copyright, 1992, 1996

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Contents
Primers

Nucleotides

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a) Primers:
i) Oligonucleotide primers are generally supplied as "so many OD units/ml" - but what does this mean, in terms of mg/ml, or mmol/ml, etc?

Given: a primer is Y nucleotides (nt) long;

Given: the MW of ssDNA is (330 daltons per nt) x (length in nt) (Sambrook et al., 1989; p. C.1);

Given: the concentration of primer (=ssDNA) producing an OD of 1 at 254 nm in a 1 cm cuvette, is 37 ug/ml;

Then: the MW of the primer is 330.Y daltons

And: X OD/ml = 37.X ug/ml = 37.X mg/l = 37.X /330.Y mM = 37.X.1000/330.Y uM

For example:
B 88/77 primer - a 17-mer oligodeoxynucleotide - as supplied is 12.6 OD units/ml. We need to make a 5 uM stock solution for PCR.

MW: 17 x 330 = 5610

Concentration: 12.6 OD x 37 ug/ml = 466 ug/ml = 466 mg/l = 0.466 g/l

Molarity: 0.466/5610 = 0.000083 Molar = 83 uM

Therefore: we need 5 ul of oligo stock solution in 83 ul (+78 ul water) to make a 5 uM solution (if 1 ul in 83 ul gives a 1 uM soln...)

ii) Calculation of amounts for PCR reactions: if we need a final concentration of 0.5 uM oligo in the PCR reaction mix (final volume 50 ul), we add 5 ul of 5 uM stock to the reaction mix (1/10 final dilution).

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b) Nucleotides:
Stocks of nucleotides for PCR (or other procedure) are NEARLY ALWAYS dNTPs (deoxynucleotides), and concentrations is almost always given in EACH dNTP: that is, the given concentration is EACH nucleotide in the mix, NOT the total concentration. This means that a 2.5 mM dNTP mix for PCR contains 2.5 mM of EACH dNTP, and 10 mM TOTAL dNTPs.

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Example:
i) Make up a 2.5 mM stock solution of dNTPs from stock 100 mM individual dNTPs, supplied by Promega:

FIRST mix equal volumes of each nucleotide (eg: 50 ul): this gives you 200 ul of 25 mM mixed dNTPs (Remember: concn. expressed in EACH dNTP).
THEN dilute this (or aliquot) 1/10 with WATER - aliquot into 100 ul amounts and freeze.
ii) Prepare a 1 mM stock of dNTPs with dTTP substituted to 10% (w/w) by digoxigenin-11-dUTP (DIG-dUTP) for use as a labelling mix for PCR labelling of PCR products:

GIVEN:

DIG-dUTP supplied (by Boehringer Mannheim) at 25 nmol/25ul = 1 umol/ml = 1mM; final concentration of DIG-dUTP must be 1/10th that of other nucleotides, and [DIG-dUTP] + [dTTP] must = [any other dNTP]. Therefore to get a 1 mM dNTP stock one must dilute DIG-dUTP stock 1/10.
FIRST dilute separate 100 mM dNTP stocks to 10 mM (eg. 5 ul to 50 ul, in water).
THEN mix equal volumes (eg. 10 ul) of 10 mM dCTP, dGTP and dATP stock, and 9/10ths volume of dTTP (9 ul). Add equal volume (eg. 10 ul) of of 1 mM DIG-dUTP.
THEN add water to 10 vol (=100 ul; add 51 ul): final concentration each dNTP = 1 mM; final concn DIG-dUTP = 0.1 mM, and of dTTP = 0.9 mM.
iii) USE mix made above at 50 uM each dNTP in a PCR reaction mix, final volume 25ul:

NEED to dilute mix 1/20; therefore use 1.25 ul dNTP labelling mix per 25 ul reaction volume (1/20 = 5/100 = 1.25/25).
To make mastermix: multiply amount of dNTP per reaction by number of reactions.

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See STANDARD PCR PROTOCOL for example of how to make a master mix.

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Return to PCR Contents Page
Return to Molecular Biology Methods Manual

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PCR and multiplex PCR guide

Designing PCR programs

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Basic Principles (see also Page 01)
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 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.
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).

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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.

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Number of cycles
Primer 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.

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Annealing time and temperature

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Annealing 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)

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Annealing temperature (see also figure 18, page 08)
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.

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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).

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Extension time and temperature

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Extension 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).

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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).

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DNA template

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All 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.

(examples of polymorphism are also shown on page 09)

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