Principle:
The polymerase chain reaction (PCR) is a method for oligonucleotide primer directed enzymatic amplification of a specific DNA sequence of interest. This technique is capable of amplifying a sequence 105 to 106-fold from nanogram amounts of template DNA within a large background of irrelevant sequences (e.g. from total genomic DNA). A prerequisite for amplifying a sequence using PCR is to have known, unique sequences flanking the segment of DNA to be amplified so that specific oligonucleotides can be obtained. It is not necessary to know anything about the intervening sequence between the primers. The PCR product is amplified from the DNA template using a heat-stable DNA polymerase from Thermus aquaticus (Taq DNA polymerase) and using an automated thermal cycler (Perkin-Elmer/Cetus) to put the reaction through 30 or more cycles of denaturing, annealing of primers, and polymerization. After amplification by PCR, the products are separated by polyacrylamide gel electrophoresis and are directly visualized after staining with ethidium bromide.
Time required:
1.1-2 Days
2.PCR reaction: 3-6 hours or overnight
3.Polyacrylamide gel electrophoresis using "Mighty-small II" gel apparatus: 2.5 hours
4.Ethidium bromide staining and photography: 45 minutes
Special reagents:
1. Synthetic oligonucleotide primer pair flanking the sequence to be amplified
2. 2.5X PCR Buffer (250 mM KCl, 50 mM Tris-HCl pH 8.3, 7.5 mMMgCl2)
3.Mixture of four dNTPS (dGTP, dATP, dTTP, dCTP) each at 2.5 mM (Ultrapure dNTP set, Pharmacia #27-2035-01). The dNTP mixture is made by adding equal volumes of a 10 mM solution of each of the four separate dNTPs together.
4.Taq DNA Polymerase (AmpliTaqTM, Perkin-Elmer/Cetus)
5.Light mineral oil
6.Acrylamide (electrophoresis grade)
7.N,N'-Methylenebisacrylamide (electrophoresis grade, Ultra-Pure/BRL, #5516U
8.Ammonium persulfate (Ultra-Pure/BRL, #5523UA)
9.TEMED (N,N,N'N' Tetramethylethylenediamine, Ultra-Pure/ BRL, #5524U
Special Equipment:
1.Mighty-small II SE-250 vertical gel electrophoresis unit (Hoefer)
2.Perkin-Elmer/Cetus Thermal Cycler
3.Sterile Thin-wall 0.5 ml Thermocycler microfuge tubes: (TC-5, Midwest Scientific)
Instructions for preparation of oligonucleotides, strategies for optimizing the specificity of a PCR reaction, pouring and running polyacrylamide gels using the "Mighty-small II" unit, and additional helpful information appear at the end of this protocol.
Recommendations for choosing oligonucleotide primers :
The aim is to choose oligonucleotide primers complementary to relatively unique sequences flanking the segment to be amplified. Although more rigorous calculations and considerations can be employed to choose optimal primers, a few general guidelines will be given below to supply a good starting point. Primers for PCR are generally 20-30 bp long and are chosen to be complementary to one strand (5' to 3') upstream and complementary to the opposite strand (5' to 3') downstream from the sequence to be amplified. The 5' ends of the primers define the ends of the amplified PCR product. Primers should ideally contain relatively balanced GC vs. AT content (e.g. 45-55% GC), and no long stretches of any one base. Caution should also be taken that the two primers of the primer pair do not contain complementary structures >2 bp to avoid "primer dimer" formation resulting from annealing of the two primers (especially at their 3' ends). The target sequence to be amplified is ideally 200-400 bp in length, with an upper limit probably around 3 kb.
Procedure for polymerase chain reaction:
The PCR reaction can be performed in volumes from 5 祃 to 200 祃 or more. The protocol below is similar to that used by the Center for Genetics in Medicine when screening the YAC library using a specific PCR assay and is carried out in a 5 祃 reaction volume. This volume is recommended when the purpose of the experiment is diagnostic (to visualize whether or not a specific product is generated). A scaled up volume can be used if the PCR product will be recovered from the gel or used for sequencing. The 5 祃 reaction is performed in a 0.5 ml eppendorf tube and covered by a drop of oil before placing in the thermal cycler.
The following components will make up one reaction (5 祃 total volume), but a cocktail of everything except the DNA will be made first:
Cocktail for 10 reactions
1.0 祃 5X PCR Buffer
10 祃 5X PCR Buffer
0.4 祃 dNTP mixture (each at 2.5 mM)
4 祃 dNTPs
0.2 祃* Primer pair (each primer at 25 礛)
2 祃 Primer pair (The primer pair solution is 1:1 mixture of the 50 礛 primer solutions)
0.1 祃 Taq polymerase
1 祃 Taq polymerase
2.3 祃 ddH2O
23 祃 ddH20 plus,
1.0 祃 DNA (100 ng genomic template DNA or < 50 ng cloned template)
*The range of final primer pair concentrations in a normal reaction mix is 0.25 - 2.5 礛 and 0.5 礛 is sometimes ideal.
Because of the small volumes involved, it is convenient to make a cocktail of the first five ingredients for each primer pair to be used. For instance, if 8 PCR reactions are to be performed from 8 different genomic or cloned DNA templates using one primer pair, then a cocktail may be made (including a slight excess) for 10 reactions by mixing together each of the volumes above multiplied by 10. A 4 祃 aliquot of the cocktail will then be added to the 1.0 祃 of DNA in each tube.
Steps: 1.Plan your experiment before adding any reagents (#primer pairs to be used, number of DNA templates, etc.). After doing so, make the appropriate cocktail/s and ensure complete mixing by tapping the tube and quick spinning. (N.B. Caution should be used to avoidcontamination of reactions with even small amounts of DNA. In addition, care should be taken to avoid contamination of pipetmen with carryover amplification products from previous reactions) 2.Pipet 4.0 祃 of the appropriate cocktail directly into the bottom of a sterile microeppendorf tube for each reaction. The tubes should be labeled by placing a round sticker on the cap to prevent smearing by oil in subsequent steps.
3.Add 1.0 祃 of the DNA directly into the drop of cocktail in each tube and ensure adequate mixing. Quick spin to collect the reaction mixture in the bottom of the tube.
4.Overlay each reaction with one drop of light mineral oil using a pasteur pipet. The samples may be quick spun if necessary before placing in the Perkin Elmer/Cetus PCR machine.
5.Place a drop of mineral oil into each well in the thermal cycler temperature block to be used for the samples (this ensures rapid temperature equilibration during cycling)
6.Place the tightly capped tubes in the temperature block and make sure each is firmly seated by pressing on the tubes individually. The PCR machine must now be programmed for the specific
reaction conditions desired (See brief operating instructions for Perkin-Elmer PCR machine). Each cycle in the polymerase chain reaction involves three steps (denaturing, primer annealing, polymerization), and the products are amplified by performing many cycles one after the other with the help of the automated thermal cycler. Refer to the literature citations at the end of this
protocol for detailed explanation of the reaction. The Taq polymerase is heat stable, and remains active despite the high denaturing temperature of each cycle. A representative set of reaction conditions for 25-35 cycles is: 作者: 微笑的海豚 时间: 2011-8-20 15:18
I. Denature
93-94 degrees C 1.5 minutes
II. Anneal 50-65 degrees C 2 minutes
III. Polymerize 72 degrees C 2 minutes
Strategies for optimizing PCR reactions are at the end of the protocol.
7.After completion of the PCR reaction, remove the tubes from the temperature block and wipe the outside free of excess oil before placing in an eppendorf rack.
8.Add 2.0 祃 of 5X Ficoll stop dye directly into the aqueous phase "bubble" at the bottom of each tube, and then add 100 祃 of chloroform:isoamyl alcohol (24:1) to each tube, shake well, and spin briefly.
9.Carefully remove only the aqueous "bubble" with a P20 pipetman set to 7-8 祃 by placing the pipet tip against the bubble and slowly drawing it in. Each sample should then be placed in a separate clean eppendorf tube before loading onto the polyacrylamide gel.
10.The reaction products are conveniently separated according to size by electrophoresis through a 10% polyacrylamide "Mighty-small II" gel at 110 V for 2-2.5 hours, and visualized after staining the gel with ethidium bromide.
ADDITIONAL INFORMATION:
Preparation of Oligonucleotides:
Oligonucleotide primers are synthesized using an automated machine (we currently order primers through the Center for Genetics in Medicine) and are received in a glass vial in an ammomium solution. It is convenient to remove about one half of the total volume for each oligonucleotide and divide this volume further into two 1.5 ml eppendorf tubes (the remainder of the ammonium stock solution is stored at 4 degrees
C). The oligonucleotides must be prepared as detailed below before use in PCR reactions:
1.Incubate each sample in a heating block at 55 degrees C overnight and then dry in a rotary vacuum concentrator for 4-6 hours. (Warning: a cold trap should be used when drying the samples to absorb the ammonia)
2.Resuspend each oligonucleotide (from both eppendorfs) in a total of 500 祃 of TE.
3.Make a 1:200 dilution by diluting 5 祃 of each oligonucleotidewith 1.0 ml of TE and measure absorbance of UV light in a spectrophotometer at 260 and 280 nm. The concentration of the stock of resuspended oligonucleotide can then be calculated:
A260 of 1.0 = 35 礸/祃 for DNA oligonucleotides. If A260 = 0.203 for a oligomer of 21 nucleotides, then 0.203 x 35 x 200 (dilution) = 1421 礸/ml (original solution),or 1.421 x 106 礸/L 21 (#nucleotides) x 330 礸/祄ol = 6930 礸/祄ol 1.421 x 106 礸/L = 205 祄ol/L (礛) ---------------- 6930 礸/祄ol
4.Make 50 礛 solutions in TE of each oligonucleotide for subsequent use in PCR reactions.
Strategies for optimizing the efficiency of PCR reactions:
The conditions required for generation of a specific, essentially unique product (single strong band) will nearly always need to be optimized empirically. In particular, the annealing temperature is important in determining the specificity of the reaction (that is to say, at lower temperatures the primers may anneal to similar irrelevant sequences elsewhere in the genome and prime these, resulting in the formation of multiple products). In general, higher annealing temperatures result in more stringent conditions for primer annealing and more specific products. A good place to start is with a low annealing temperature around 50-55 degrees C, with optimization by testing at 3-5 degrees C increments until maximum specificity is reached. Theoretically, oligonucleotide primers with a high GC content may require a very high annealing temperature to maximize specificity. While this is a good rule of thumb, the optimum temperature may not correspond well to this estimate. Occasionally, specifity will reach a maximum at a certain temperature and at higher annealing temperatures, multiple new products or no products at all will be generated. Although annealing temperature is perhaps the easiest variable to change, specificity may also be increased by reducing the concentration of primers or Taq polymerase, minimizing the times allowed for annealing and extension, or reducing the free Mg++ concentration. An optimum of Mg++ concentration usually exists in the 1-10 mM range. Too low Mg++ concentration may result in no
products and an excess may result in a variety of unwanted products.
Pouring and Running Polyacrylamide Gels using the Hoefer SE-250
"Mighty-small II" gel electrophoresis unit: (Simplified instructions are provided below, for detailed instructions, refer to the Hoefer manual). Multiple identical polyacrylamide gels can be pre-cast in the supplied SE 275 multiple gel caster. Acrylamide is a neurotoxin and should be handled with caution. Wear gloves at all times when handling acrylamide and be careful to avoid spills.
1.Clean the multiple gel caster and place flat on the bench top in front of you. Place the rubber gasket in its groove without stretching it and lubricate with a thin layer of the Cello-seal provided by Hoefer.
2.Build the gel casting units by carefully placing and seating components in the following order from the bottom up: waxed paper, notched alumina plate, T-shaped spacers (0.75 or1.0 mm), glass plate, waxed paper, etc. Approximately 5 complete 0.75 mm gels can be cast at one time with one or two additional glass plates needed to fill extra space.
3.Place the top cover on the multiple gel caster and apply red spring clamps to side grooves, ensuring adequate sealing. Be sure that the port at the bottom of the front plate has a small piece of rubber tubing on it and is clamped off.
4.Mix the ingredients for 50 ml of acrylamide (minus the TEMED) in a clean beaker, as detailed in the recipe below for a 10% polyacrylamide gel. Add the TEMED with thorough mixing just before pouring the gels.
5.Carefully pour the acrylamide evenly into the gel casting units in the multiple gel caster until the multiple gel caster is almost overflowing.
6.Insert the appropriate sized comb (0.75mm for 0.75 mm spacers) into each gel casting unit, and allow the acrylamide to polymerize for at least 1 hour. After complete polymerization, the gels may be wrapped in cellophane and stored at 4 degrees C.
Solutions:
40% Acrylamide/ 2% bis stock
acrylamide: 38 g
N,N'-methylene bisacrylamide 2 g
dH20: to 100 ml
Mini-gel ("Mighty-small II") 10% polyacrylamide (50 ml volume)
12.5 ml 40% Acrylamide/ 2% bis stock
25 ml water
5 ml 10x TBE
7.5 ml glycerol
+ 714 祃 10% APS + 17.2 祃 TEMED
References:
Mullis, K. and F. A. Faloona. (1987). "Specific synthesis of DNA in vitro via a polymerase catalyzed chain reaction."Meth. in Enzymol. 255:335-350.
Mullis, K, Faloona, F., Scharf, S., Saiki, R., Horn, G., and H. Erlich. (1986). "Specific enzymatic amplification of DNA in vitro: The polymerase chain reaction." Cold Spring Harbor Symposia on Quantitative Biology, Volume 51, Cold Spring Harbor Laboratory. p. 263-272.
Williams, J. F. (1989). "Optimization strategies for the polymerase chain reaction." Biotechniques 7(7):762-768.
PCR Technology: Principles and Applications for DNA Amplification. (1989). Erlich, H.A. (ed.), Stockton Press,作者: 微笑的海豚 时间: 2011-8-20 15:19
PCR PRIMER DESIGN AND REACTION OPTIMISATION
Ed Rybicki, Department of Molecular and Cell Biology, University of Cape Town
copyright, 1992, 1996, 2001
Contents
· Factors Affecting the PCR
o Denaturing Temperature and Time
o Annealing Temperature and Primer Design
o Primer Length
o Degenerate Primers
o Elongation Temperature and Time
o Reaction Buffer
o Cycle Number
§ Nested Primer PCR
· Labelling of PCR products with digoxygenin-11-dUTP
· Helix Destabilisers / Additives
· Useful Universal cDNA PCR Primer
· A simple set of rules for primer sequence design
· REFERENCES
Factors Affecting the PCR:
Denaturing Temperature and time
The specific complementary association due to hydrogen bonding of single-stranded nucleic acids is referred to as "annealing": two complementary sequences will form hydrogen bonds between their complementary bases (G to C, and A to T or U) and form a stable double-stranded, anti-parallel "hybrid" molecule. One may make nucleic acid (NA) single-stranded for the purpose of annealing - if it is not single-stranded already, like most RNA viruses - by heating it to a point above the "melting temperature" of the double- or partially-double-stranded form, and then flash-cooling it: this ensures the "denatured" or separated strands do not re-anneal. Additionally, if the NA is heated in buffers of ionic strength lower than 150mM NaCl, the melting temperature is generally less than 100oC - which is why PCR works with denaturing temperatures of 91-97oC.
A more detailed treatment of annealing / hybridisation is given in an accompanying page, together with explanations of calculations of complexity, conditions for annealing / hybridisation, etc.
Taq polymerase is given as having a half-life of 30 min at 95oC, which is partly why one should not do more than about 30 amplification cycles: however, it is possible to reduce the denaturation temperature after about 10 rounds of amplification, as the mean length of target DNA is decreased: for templates of 300bp or less, denaturation temperature may be reduced to as low as 88oC for 50% (G+C) templates (Yap and McGee, 1991), which means one may do as many as 40 cycles without much decrease in enzyme efficiency.
"Time at temperature" is the main reason for denaturation / loss of activity of Taq: thus, if one reduces this, one will increase the number of cycles that are possible, whether the temperature is reduced or not. Normally the denaturation time is 1 min at 94oC: it is possible, for short template sequences, to reduce this to 30 sec or less. Increase in denaturation temperature and decrease in time may also work: Innis and Gelfand (1990) recommend 96oC for 15 sec.
Annealing Temperature and Primer Design
Primer length and sequence are of critical importance in designing the parameters of a successful amplification: the melting temperature of a NA duplex increases both with its length, and with increasing (G+C) content: a simple formula for calculation of the Tm is
Tm = 4(G + C) + 2(A + T)oC.
Thus, the annealing temperature chosen for a PCR depends directly on length and composition of the primer. One should aim at using an annealing temperature (Ta) about 5oC below the lowest Tm of ther pair of primers to be used (Innis and Gelfand, 1990). A more rigorous treatment of Ta is given by Rychlik et al. (1990): they maintain that if the Ta is increased by 1oC every other cycle, specificity of amplification and yield of products <1kb in length are both increased. One consequence of having too low a Ta is that one or both primers will anneal to sequences other than the true target, as internal single-base mismatches or partial annealing may be tolerated: this is fine if one wishes to amplify similar or related targets; however, it can lead to "non-specific" amplification and consequent reduction in yield of the desired product, if the 3'-most base is paired with a target.
A consequence of too high a Ta is that too little product will be made, as the likelihood of primer annealing is reduced; another and important consideration is that a pair of primers with very different Tas may never give appreciable yields of a unique product, and may also result in inadvertent "asymmetric" or single-strand amplification of the most efficiently primed product strand.
Annealing does not take long: most primers will anneal efficiently in 30 sec or less, unless the Ta is too close to the Tm, or unless they are unusually long.
An illustration of the effect of annealing temperature on the specificity and on the yield of amplification of Human papillomavirus type 16 (HPV-16) is given below (Williamson and Rybicki, 1991: J Med Virol 33: 165-171).作者: 微笑的海豚 时间: 2011-8-20 15:22
Plasmid and biopsy sample DNA templates were amplified at different annealing temperatures as shown: note that while plasmid is amplified from 37 to 55oC, HPV DNA is only specifically amplified at 50oC.
Primer Length
The optimum length of a primer depends upon its (A+T) content, and the Tm of its partner if one runs the risk of having problems such as described above. Apart from the Tm, a prime consideration is that the primers should be complex enough so that the likelihood of annealing to sequences other than the chosen target is very low. (See hybridn.doc).
For example, there is a ¼ chance (4-1) of finding an A, G, C or T in any given DNA sequence; there is a 1/16 chance (4-2) of finding any dinucleotide sequence (eg. AG); a 1/256 chance of finding a given 4-base sequence. Thus, a sixteen base sequence will statistically be present only once in every 416 bases (=4 294 967 296, or 4 billion): this is about the size of the human or maize genome, and 1000x greater than the genome size of E. coli. Thus, the association of a greater-than-17-base oligonucleotide with its target sequence is an extremely sequence-specific process, far more so than the specificity of monoclonal antibodies in binding to specific antigenic determinants. Consequently, 17-mer or longer primers are routinely used for amplification from genomic DNA of animals and plants. Too long a primer length may mean that even high annealing temperatures are not enough to prevent mismatch pairing and non-specific priming.
Degenerate Primers
For amplification of cognate sequences from different organisms, or for "evolutionary PCR", one may increase the chances of getting product by designing "degenerate" primers: these would in fact be a set of primers which have a number of options at several positions in the sequence so as to allow annealing to and amplification of a variety of related sequences. For example, Compton (1990) describes using 14-mer primer sets with 4 and 5 degeneracies as forward and reverse primers, respectively, for the amplification of glycoprotein B (g from related herpesviruses. The reverse primer sequence was as follows:
TCGAATTCNCCYAAYTGNCCNT
where Y = T + C, and N = A + G + C + T, and the 8-base 5'-terminal extension comprises a EcoRI site (underlined) and flanking spacer to ensure the restriction enzyme can cut the product (the New England Biolabs catalogue gives a good list of which enzymes require how long a flanking sequence in order to cut stub ends). Degeneracies obviously reduce the specificity of the primer, meaning mismatch opportunities are greater, and background noise increases; also, increased degeneracy means concentration of the individual primers decreases; thus, greater than 512-fold degeneracy should be avoided. However, I have used primers with as high as 256- and 1024-fold degeneracy for the successful amplification and subsequent direct sequencing of a wide range of Mastreviruses against a background of maize genomic DNA (Rybicki and Hughes, 1990).
Primer sequences were derived from multiple sequence alignments; the mismatch positions were used as 4-base degeneracies for the primers (shown as stars; 5 in F and 4 in R), as shown above. Despite their degeneracy, the primers could be used to amplify a 250 bp sequence from viruses differing in sequence by as much as 50% over the target sequence, and 60% overall. They could also be used to very sensitively detect the presence of Maize streak virus DNA against a background of maize genomic DNA, at dilutions as low as 1/109 infected sap / healthy sap (see below).
Some groups use deoxyinosine (dI) at degenerate positions rather than use mixed oligos: this base-pairs with any other base, effectively giving a four-fold degeneracy at any postion in the oligo where it is present. This lessens problems to do with depletion of specific single oligos in a highly degenerate mixture, but may result in too high a degeneracy where there are 4 or more dIs in an oligo.作者: 微笑的海豚 时间: 2011-8-20 15:23
Elongation Temperature and Time
This is normally 70 - 72oC, for 0.5 - 3 min. Taq actually has a specific activity at 37oC which is very close to that of the Klenow fragment of E coli DNA polymerase I, which accounts for the apparent paradox which results when one tries to understand how primers which anneal at an optimum temperature can then be elongated at a considerably higher temperature - the answer is that elongation occurs from the moment of annealing, even if this is transient, which results in considerably greater stability. At around 70oC the activity is optimal, and primer extension occurs at up to 100 bases/sec. About 1 min is sufficient for reliable amplification of 2kb sequences (Innis and Gelfand, 1990). Longer products require longer times: 3 min is a good bet for 3kb and longer products. Longer times may also be helpful in later cycles when product concentration exceeds enzyme concentration (>1nM), and when dNTP and / or primer depletion may become limiting.
Reaction Buffer
Recommended buffers generally contain :
10-50mM Tris-HCl pH 8.3,
up to 50mM KCl, 1.5mM or higher MgCl2,
primers 0.2 - 1uM each primer,
50 - 200uM each dNTP,
gelatin or BSA to 100ug/ml,
and/or non-ionic detergents such as Tween-20 or Nonidet P-40 or Triton X-100 (0.05 - 0.10% v/v)
(Innis and Gelfand, 1990). Modern formulations may differ considerably, however - they are also generally proprietary.
PCR is supposed to work well in reverse transcriptase buffer, and vice-versa, meaning 1-tube protocols (with cDNA synthesis and subsequent PCR) are possible (Krawetz et al., 19xx; Fuqua et al., 1990).
Higher than 50mM KCl or NaCl inhibits Taq, but some is necessary to facilitate primer annealing.
[Mg2+] affects primer annealing; Tm of template, product and primer-template associations; product specificity; enzyme activity and fidelity. Taq requires free Mg2+, so allowances should be made for dNTPs, primers and template, all of which chelate and sequester the cation; of these, dNTPs are the most concentrated, so [Mg2+] should be 0.5 - 2.5mM greater than [dNTP]. A titration should be performed with varying [Mg2+] with all new template-primer combinations, as these can differ markedly in their requirements, even under the same conditions of concentrations and cycling times/temperatures.
Some enzymes do not need added protein, others are dependent on it. Some enzymes work markedly better in the presence of detergent, probably because it prevents the natural tendency of the enzyme to aggregate.
Primer concentrations should not go above 1uM unless there is a high degree of degeneracy; 0.2uM is sufficient for homologous primers.
Nucleotide concentration need not be above 50uM each: long products may require more, however.
Cycle Number
The number of amplification cycles necessary to produce a band visible on a gel depends largely on the starting concentration of the target DNA: Innis and Gelfand (1990) recommend from 40 - 45 cycles to amplify 50 target molecules, and 25 - 30 to amplify 3x105 molecules to the same concentration. This non-proportionality is due to a so-called plateau effect, which is the attenuation in the exponential rate of product accumulation in late stages of a PCR, when product reaches 0.3 - 1.0 nM. This may be caused by degradation of reactants (dNTPs, enzyme); reactant depletion (primers, dNTPs - former a problem with short products, latter for long products); end-product inhibition (pyrophosphate formation); competition for reactants by non-specific products; competition for primer binding by re-annealing of concentrated (10nM) product (Innis and Gelfand, 1990).
If desired product is not made in 30 cycles, take a small sample (1ul) of the amplified mix and re-amplify 20-30x in a new reaction mix rather than extending the run to more cycles: in some cases where template concentration is limiting, this can give good product where extension of cycling to 40x or more does not.
A variant of this is nested primer PCR: PCR amplification is performed with one set of primers, then some product is taken - with or without removal of reagents - for re-amplification with an internally-situated, "nested" set of primers. This process adds another level of specificity, meaning that all products non-specifically amplified in the first round will not be amplified in the second. This is illustrated below:
This gel photo shows the effect of nested PCR amplification on the detectability of Chicken anaemia virus (CAV) DNA in a dilution series: the PCR1 just detects 1000 template molecules; PCR2 amplifies 1 template molecule (Soin?C, Watson SK, Rybicki EP, Lucio B, Nordgren RM, Parrish CR, Schat KA (1993) Avian Dis 37: 467-476).作者: 微笑的海豚 时间: 2011-8-20 15:23
Labelling of PCR products with digoxygenin-11-dUTP
(DIG; Roche) need be done only in 50uM each dNTP, with the dTTP substituted to 35% with DIG-11-dUTP. NOTE: that the product will have a higher MW than the native product! This results in a very well labelled probe which can be extensively re-used, for periods up to 3 years. See also here.
Helix Destabilisers / Additives
With NAs of high (G+C) content, it may be necessary to use harsher denaturation conditions. For example, one may incorporate up to 10% (w or v/v) :
dimethyl sulphoxide (DMSO),
dimethyl formamide (DMF),
urea
or formamide
in the reaction mix: these additives are presumed to lower the Tm of the target NA, although DMSO at 10% and higher is known to decrease the activity of Taq by up to 50% (Innis and Gelfand, 1990; Gelfand and White, 1990).
Additives may also be necessary in the amplification of long target sequences: DMSO often helps in amplifying products of >1kb. Formamide can apparently dramatically improve the specificity of PCR (Sarkar et al., 1990), while glycerol improves the amplification of high (G+C) templates (Smith et al., 1990).
Polyethylene glycol (PEG) may be a useful additive when DNA template concentration is very low: it promotes macromolecular association by solvent exclusion, meaning the pol can find the DNA.
cDNA PCR
A very useful primer for cDNA synthesis and cDNA PCR comes from a sequencing strategy described by Thweatt et al. (1990): this utilised a mixture of three 21-mer primers consisting of 20 T residues with 3'-terminal A, G or C, respectively, to sequence inside the poly(A) region of cDNA clones of mRNA from eukaryotic origin. I have used it to amplify discrete bands from a variety of poly(A)+ virus RNAs, with only a single specific degenerate primer upstream: the T-primer may anneal anywhere in the poly(A) region, but only molecules which anneal at the beginning of the poly(A) tail, and whose 3'-most base is complementary to the base next to the beginning of the tail, will be extended.
eg: 5'-TTTTTTTTTTTTTTTTTTTTTTTTT(A,G,C)-3'
works for amplification of Potyvirus RNA, and eukaryotic mRNA
A simple set of rules for primer sequence design is as follows (adapted from Innis and Gelfand, 1991):
primers should be 17-28 bases in length;
base composition should be 50-60% (G+C);
primers should end (3') in a G or C, or CG or GC: this prevents "breathing" of ends and increases efficiency of priming;
Tms between 55-80oC are preferred;
runs of three or more Cs or Gs at the 3'-ends of primers may promote mispriming at G or C-rich sequences (because of stability of annealing), and should be avoided;
3'-ends of primers should not be complementary (ie. base pair), as otherwise primer dimers will be synthesised preferentially to any other product;
primer self-complementarity (ability to form 2o structures such as hairpins) should be avoided.
Examples of inter- and intra-primer complementarity which would result in problems:作者: 微笑的海豚 时间: 2011-8-20 15:24
Screen shots taken from analyses done using DNAMAN (Lynnon Biosoft, Quebec, Canada).
REFERENCES
Compton T (1990). Degenerate primers for DNA amplification. pp. 39-45 in: PCR Protocols (Innis, Gelfand, Sninsky and White, eds.); Academic Press, New York.
Fuqua SAW, Fitzgerald SD and McGuire WL (1990). A simple polymerase chain reaction method for detection and cloning of low-abundance transcripts. BioTechniques 9 (2):206-211.
Gelfand DH and White TJ (1990). Thermostable DNA polymerases. pp. 129-141 in: PCR Protocols (Innis, Gelfand, Sninsky and White, eds.); Academic Press, New York.
Innis MA and Gelfand DH (1990). Optimization of PCRs. pp. 3-12 in: PCR Protocols (Innis, Gelfand, Sninsky and White, eds.); Academic Press, New York.
Krawetz SA, Pon RT and Dixon GH (1989). Increased efficiency of the Taq polymerase catalysed polymerase chain reaction. Nucleic Acids Research 17 (2):819.
Rybicki EP and Hughes FL (1990). Detection and typing of maize streak virus and other distantly related geminiviruses of grasses by polymerase chain reaction amplification of a conserved viral sequence. Journal of General Virology 71:2519-2526.
Rychlik W, Spencer WJ and Rhoads RE (1990). Optimization of the annealing temperature for DNA amplification in vitro. Nucleic Acids Research 18 (21):6409-6412.
Sarkar G, Kapeiner S and Sommer SS (1990). Formaqmide can drrastically increase the specificity of PCR. Nucleic Acids Research 18 (24):7465.
Smith KT, Long CM, Bowman B and Manos MM (1990). Using cosolvents to enhance PCR amplification. Amplifications 9/90 (5):16-17.
Thweatt R, Goldstein S and Reis RJS (1990). A universal primer mixture for sequence determination at the 3' ends of cDNAs. Analytical Biochemistry 190:314-316.
Wu DY, Ugozzoli L, Pal BK, Qian J, Wallace RB (1991). The effect of temperature and oligonucleotide primer length on the specificity and efficiency of amplification by the polymerase chain reaction. DNA and Cell Biology 10 (3):233-238.
Yap EPH and McGee JO'D (1991). Short PCR product yields improved by lower denaturation temperatures. Nucleic Acids Research 19 (7):1713.
DETECTION OF NUCLEIC ACIDS BY HYBRIDISATION
Ed Rybicki, Copyright 1992, 1998
Contents
INTRODUCTION
Why would one want to anneal pieces of nucleic acid?
The complementary association of two strands of polynucleotides
Melting Temperatures
Hybridisation Stringency
Summary
INTRODUCTION
Hybridisation is a term used to describe the specific complementary association due to hydrogen bonding, under experimental conditions, of single-stranded nucleic acids. It should more properly be referred to as "annealing", as this is the physical process responsible for the association: two complementary sequences will form hydrogen bonds between their complementary bases (G to C, and A to T or U) and form a stable double-stranded, anti-parallel "hybrid" helical molecule. One may make ones nucleic acid single-stranded for the purpose of annealing - if it is not single-stranded already, like most RNA viruses - by heating it in 0.01M NaCl to a point above the "melting temperature" of the double- or partially-double-stranded form, and then flash-cooling to +0oC: this ensures the "denatured" or separated strands do not re-anneal.
Alternatively, one may denature DNA reversibly by treatment with 0.5M NaOH: this does not work for RNA, as this hydrolyses under these conditions.作者: 微笑的海豚 时间: 2011-8-20 15:24
Why would one want to anneal pieces of nucleic acid?
The answer is simple: nucleic acid hybridisation on membrane filters is a simple, sensitive, and specific means of detecting nucleic acid sequences of interest. One immobilises "target" nucleic acid - denatured so as to be effectively single-stranded - on an absorptive, porous membrane, and then anneals to it an appropriately "tagged" or "labelled" single-stranded probe nucleic acid. After washing off unannealed probe, one detects the immobilised hybrid by means of the label: this is often 32P incorporated into a nucleotide, which allows autoradiographic or scintillometric detection.
One may also use non-radioactive labels and detection systems, for sensitivities of detection down to picogram levels. The system of choice at the moment appears to be the Boehringer Mannheim DIG (digoxigenin) non-radioactive labelling and detection kit, which uses digoxigenin-11-dUTP as a substituted nucleotide which is enzymatically incorporated into DNA.
The mechanism of immobilisation of nucleic acids on membranes is not fully understood: nitrocellulose strongly binds only ss-nucleic acids (ssNA), under conditions of high salt (>1M NaCl), and has to be heated at 80oC in a vacuum to irreversibly attach the NA; nylon membranes (Hybond-N, GeneScreen) bind all nucleic acids under a wide range of salt concentrations, and irreversible or covalent attachment can be achieved by UV irradiation for 5 min or less, or by treatment with 0.4M NaOH.
The complementary association of two strands of polynucleotides
is the basis for replication of all organisms; the complexity inherent in the sequence of the molecules renders the association extremely specific for any molecule longer than sixteen nucleotides. This is easily understood if one considers the combinatorial possibilities of given lengths of "probe" sequence: there is a ?chance (4-1) of finding an A, G, C or T (U for RNA) in any given DNA sequence; there is a 1/16 chance (4-2) of finding any dinucleotide sequence (eg. AG); a 1/256 chance of finding a given 4-base sequence. Thus, a sixteen base sequence will statistically be present only once in every
416 bases (=4 294 967 296, or 4 billion):
this is about the size of the human genome, and 1000x greater than the genome size of E. coli.
Thus, the association of two nucleic acid molecules - presumed to be at least a few hundred bases long - is an extremely sequence-specific process, far more so than the widely-used specificity of monoclonal antibodies in binding to specific antigenic determinants. The correct annealing of two sequences to each other does, however, depend on the physical and chemical solution conditions under which the reaction takes place.
Melting Temperatures
For example, all double-stranded nucleic acids - whether dsDNA, dsRNA or RNANA hybrids - have specific "melting temperatures", which depend mainly upon their specific guanine+cytosine content, but also upon whether they are DNA, RNA, or a mixture (RNA:RNA hybrids have the highest melting temperatures, followed by DNA:RNA hybrids, then dsDNA), and upon the ionic strength of solution.
The melting temperature is also dependent upon the length of the sequences to be annealed: the shorter the probe sequence, the lower the melting temperature. The degree of sequence mismatch also determines the effective melting temperature of a hybrid: Tm decreases by about 1oC for every 1% of mismatched base pairs. It therefore makes sense to maximise probe length in order to minimise Tm reduction due both to length and degree of sequence mismatch. Under standard conditions of annealing (0.8M NaCl, neutral pH) one may calculate the melting temperature ™ of any given DNA hybrid as shown:
Tm = 81.5oC + 0.41(%G + %C) - 550/n
where n=probe length (no. nucleotides).
One can see that the reduction in Tm becomes negligible for probes of length 200 nt or greater. Thus, one may vary the specificity of association of a specific single-stranded "probe" and a target by varying the incubation temperature of the annealing reaction: the higher the temperature, the higher the specificity of the reaction - and the lower the likelihood of annealing taking place.
Hybridisation Stringency
The successful use of nucleic acids as probes for sequences of interest therefore depends upon certain reaction conditions which are in turn determined by the physical properties (ie. length and sequence) of the probe. This leads to the concept of stringency of hybridisation: one increases the stringency by lessening the likelihood of non-homologous annealing. This can be done by simply increasing the temperature of incubation - bearing in mind that rate of hybridisation/annealing is maximal at about Tm - 25oC, and too high a temperature results in very slow annealing. An acceptable compromise is to anneal at a standard temperature (eg. 65oC), and then wash the annealed and immobilised hybrid molecules to varying degrees of stringency: the extent to which one should wash can be assessed by repeated autoradiography, if the probe is 32P-labelled, or by repeated colour assay of replicates in the case of non-radioactively labelled probe. Washing stringency may be increased by varying the ionic strength (from 1.0M NaCl to 0.02M), or varying the temperature (ambient to 65oC). One may also include SDS or other detergent in wash and in hybridisation buffers in order to decrease non-specific attachment of probe to the adsorptive membrane. For this reason a blocking or prehybridisation buffer is normally used before and during the annealing reaction, to block adsorptive sites on the membrane not occupied by target nucleic acid. This normally consists of buffer salts, detergent, protein, inert polymer material, and DNA.作者: 微笑的海豚 时间: 2011-8-20 15:24
It is possible to include various other constituents in annealing buffers, designed to increase the hybridisation rate, or the stringency, or both. Formamide is a helix destabiliser, and enables one to decrease annealing temperature: the presence of formamide decreases the Tm as shown:
TFm = Tm - 0.61(%formamide, w/v)
It is most often used in annealing reactions using RNA as target or probe, and especially with dsRNA hybrids, as these have high Tms which necessitate elevated reaction temperatures. Standard conditions using formamide would be 42oC with 50% formamide content in the annealing buffer. Formamide also decreases the rate of annealing, so one normally includes substances like dextran sulphate - a polyanionic polymer - as "molecular exclusion agents" to decrease the volume of solvent available to the probe. Polyethylene glycol is a far cheaper and equally effective substitute for increasing reaction rate. Too high a concentration of DS or PEG raises "background" or non-specific probe attachment to unacceptably high levels. Their effectiveness is also directly proportional to probe length, and they are useless when oligonucleotides of less than 50 nt in length are used as probes.
Summary
A standard hybridisation reaction, then, consists of probing an immobilised target sequence on a membrane with a labelled specific probe sequence: this is done by annealing the probe to the target under (usually) standard "hybridisation conditions" of 0.9M NaCl, 65oC, for 4-16 hr. Probes are usually molecules of DNA or cDNA, a few hundred nt to several kilobases long, cloned into and grown up as recombinant plasmids in E. coli, and purified by caesium chloride gradient centrifugation. One may also use nucleic acid directly purified from the organism of interest, but this is only really effective if this is a virus or a plasmid, as otherwise the probe length is too great, and the repeat number is too small to give appreciable signal. In other words, probes should not be too long, as otherwise one needs very high concentrations of nucleic acid in order to guarantee a sufficient number of copies of the sequence in order to give a detectable "signal" for detection purposes.
Labelling PCR Products with Digoxigenin
PCR products may be very conveniently labelled with digoxigenin-11-dUTP (Boehringer-Mannheim) by incorporating the reagent to 10-35% final effective dTTP concentration in a nucleotide mix of final concentration 50-100uM dNTPs (Emanual, 1991; Nucleic Acids Res 19: 2790). This allows substitution to a known extent of probes of exactly defined length, which in turn allows exactly defined bybridisation conditions. It is also the most effective means of labelling PCR products, as it is potentially unsafe and VERY expensive to attempt to do similarly with 32P-dNTPs, and nick-translation or random primed label incorporation are unsuitable because the templates are often too small for efficient labelling.
Make a DIG-dNTP mix for PCR as follows:
DIG NUCLEOTIDE MIX CONCENTRATIONS
Dig-11-dUTP 350 uM
dTTP 650 uM
dATP 1 mM
dCTP 1 mM
dGTP 1 mM作者: 微笑的海豚 时间: 2011-8-20 15:25
For each 50 ul of probe synthesized, a 1/10 dilution is made of the DIG-nucleotide mix when added to the other reagents as described above. The products may be analyzed by agarose gel electrophoresis - NOTE: PRODUCTS ARE LARGER THAN NON-SUBSTITUTED PRODUCT - and detected directly on blots immunologically. Probes can be used as 5-10 ul aliquots directly from PCR product mixes, mixed with hybridisation mix and denatured. Probes can be re-used up to 10 times, stored frozen in between experiments and boiled to denature.
DETECTION OF NUCLEIC ACIDS BY HYBRIDISATION
Ed Rybicki, Copyright 1992, 1998
Contents
INTRODUCTION
Why would one want to anneal pieces of nucleic acid?
The complementary association of two strands of polynucleotides
Melting Temperatures
Hybridisation Stringency
Summary
INTRODUCTION
Hybridisation is a term used to describe the specific complementary association due to hydrogen bonding, under experimental conditions, of single-stranded nucleic acids. It should more properly be referred to as "annealing", as this is the physical process responsible for the association: two complementary sequences will form hydrogen bonds between their complementary bases (G to C, and A to T or U) and form a stable double-stranded, anti-parallel "hybrid" helical molecule. One may make ones nucleic acid single-stranded for the purpose of annealing - if it is not single-stranded already, like most RNA viruses - by heating it in 0.01M NaCl to a point above the "melting temperature" of the double- or partially-double-stranded form, and then flash-cooling to +0oC: this ensures the "denatured" or separated strands do not re-anneal.
Alternatively, one may denature DNA reversibly by treatment with 0.5M NaOH: this does not work for RNA, as this hydrolyses under these conditions.
Why would one want to anneal pieces of nucleic acid?
The answer is simple: nucleic acid hybridisation on membrane filters is a simple, sensitive, and specific means of detecting nucleic acid sequences of interest. One immobilises "target" nucleic acid - denatured so as to be effectively single-stranded - on an absorptive, porous membrane, and then anneals to it an appropriately "tagged" or "labelled" single-stranded probe nucleic acid. After washing off unannealed probe, one detects the immobilised hybrid by means of the label: this is often 32P incorporated into a nucleotide, which allows autoradiographic or scintillometric detection.
One may also use non-radioactive labels and detection systems, for sensitivities of detection down to picogram levels. The system of choice at the moment appears to be the Boehringer Mannheim DIG (digoxigenin) non-radioactive labelling and detection kit, which uses digoxigenin-11-dUTP as a substituted nucleotide which is enzymatically incorporated into DNA.
The mechanism of immobilisation of nucleic acids on membranes is not fully understood: nitrocellulose strongly binds only ss-nucleic acids (ssNA), under conditions of high salt (>1M NaCl), and has to be heated at 80oC in a vacuum to irreversibly attach the NA; nylon membranes (Hybond-N, GeneScreen) bind all nucleic acids under a wide range of salt concentrations, and irreversible or covalent attachment can be achieved by UV irradiation for 5 min or less, or by treatment with 0.4M NaOH.作者: 微笑的海豚 时间: 2011-8-20 15:25
The complementary association of two strands of polynucleotides
is the basis for replication of all organisms; the complexity inherent in the sequence of the molecules renders the association extremely specific for any molecule longer than sixteen nucleotides. This is easily understood if one considers the combinatorial possibilities of given lengths of "probe" sequence: there is a ?chance (4-1) of finding an A, G, C or T (U for RNA) in any given DNA sequence; there is a 1/16 chance (4-2) of finding any dinucleotide sequence (eg. AG); a 1/256 chance of finding a given 4-base sequence. Thus, a sixteen base sequence will statistically be present only once in every
416 bases (=4 294 967 296, or 4 billion):
this is about the size of the human genome, and 1000x greater than the genome size of E. coli.
Thus, the association of two nucleic acid molecules - presumed to be at least a few hundred bases long - is an extremely sequence-specific process, far more so than the widely-used specificity of monoclonal antibodies in binding to specific antigenic determinants. The correct annealing of two sequences to each other does, however, depend on the physical and chemical solution conditions under which the reaction takes place.
Melting Temperatures
For example, all double-stranded nucleic acids - whether dsDNA, dsRNA or RNANA hybrids - have specific "melting temperatures", which depend mainly upon their specific guanine+cytosine content, but also upon whether they are DNA, RNA, or a mixture (RNA:RNA hybrids have the highest melting temperatures, followed by DNA:RNA hybrids, then dsDNA), and upon the ionic strength of solution.
The melting temperature is also dependent upon the length of the sequences to be annealed: the shorter the probe sequence, the lower the melting temperature. The degree of sequence mismatch also determines the effective melting temperature of a hybrid: Tm decreases by about 1oC for every 1% of mismatched base pairs. It therefore makes sense to maximise probe length in order to minimise Tm reduction due both to length and degree of sequence mismatch. Under standard conditions of annealing (0.8M NaCl, neutral pH) one may calculate the melting temperature ™ of any given DNA hybrid as shown:
Tm = 81.5oC + 0.41(%G + %C) - 550/n
where n=probe length (no. nucleotides).
One can see that the reduction in Tm becomes negligible for probes of length 200 nt or greater. Thus, one may vary the specificity of association of a specific single-stranded "probe" and a target by varying the incubation temperature of the annealing reaction: the higher the temperature, the higher the specificity of the reaction - and the lower the likelihood of annealing taking place.
Hybridisation Stringency
The successful use of nucleic acids as probes for sequences of interest therefore depends upon certain reaction conditions which are in turn determined by the physical properties (ie. length and sequence) of the probe. This leads to the concept of stringency of hybridisation: one increases the stringency by lessening the likelihood of non-homologous annealing. This can be done by simply increasing the temperature of incubation - bearing in mind that rate of hybridisation/annealing is maximal at about Tm - 25oC, and too high a temperature results in very slow annealing. An acceptable compromise is to anneal at a standard temperature (eg. 65oC), and then wash the annealed and immobilised hybrid molecules to varying degrees of stringency: the extent to which one should wash can be assessed by repeated autoradiography, if the probe is 32P-labelled, or by repeated colour assay of replicates in the case of non-radioactively labelled probe. Washing stringency may be increased by varying the ionic strength (from 1.0M NaCl to 0.02M), or varying the temperature (ambient to 65oC). One may also include SDS or other detergent in wash and in hybridisation buffers in order to decrease non-specific attachment of probe to the adsorptive membrane. For this reason a blocking or prehybridisation buffer is normally used before and during the annealing reaction, to block adsorptive sites on the membrane not occupied by target nucleic acid. This normally consists of buffer salts, detergent, protein, inert polymer material, and DNA.
It is possible to include various other constituents in annealing buffers, designed to increase the hybridisation rate, or the stringency, or both. Formamide is a helix destabiliser, and enables one to decrease annealing temperature: the presence of formamide decreases the Tm as shown:
TFm = Tm - 0.61(%formamide, w/v)作者: 微笑的海豚 时间: 2011-8-20 15:26
It is most often used in annealing reactions using RNA as target or probe, and especially with dsRNA hybrids, as these have high Tms which necessitate elevated reaction temperatures. Standard conditions using formamide would be 42oC with 50% formamide content in the annealing buffer. Formamide also decreases the rate of annealing, so one normally includes substances like dextran sulphate - a polyanionic polymer - as "molecular exclusion agents" to decrease the volume of solvent available to the probe. Polyethylene glycol is a far cheaper and equally effective substitute for increasing reaction rate. Too high a concentration of DS or PEG raises "background" or non-specific probe attachment to unacceptably high levels. Their effectiveness is also directly proportional to probe length, and they are useless when oligonucleotides of less than 50 nt in length are used as probes.
Summary
A standard hybridisation reaction, then, consists of probing an immobilised target sequence on a membrane with a labelled specific probe sequence: this is done by annealing the probe to the target under (usually) standard "hybridisation conditions" of 0.9M NaCl, 65oC, for 4-16 hr. Probes are usually molecules of DNA or cDNA, a few hundred nt to several kilobases long, cloned into and grown up as recombinant plasmids in E. coli, and purified by caesium chloride gradient centrifugation. One may also use nucleic acid directly purified from the organism of interest, but this is only really effective if this is a virus or a plasmid, as otherwise the probe length is too great, and the repeat number is too small to give appreciable signal. In other words, probes should not be too long, as otherwise one needs very high concentrations of nucleic acid in order to guarantee a sufficient number of copies of the sequence in order to give a detectable "signal" for detection purposes.作者: 微笑的海豚 时间: 2011-8-20 15:26
General Notes on Primer Design in PCR*
By Vincent R. Prezioso, Ph.D.
Perhaps the most critical parameter for successful PCR is the design of Primers. All things being equal, a poorly designed primer can result in a PCR reaction that will not work. The primer sequence determines several things such as the length of the product, its melting temperature and ultimately the yield. A poorly designed primer can result in little or no product due to non-specific amplification and/or primer-dimer formation, which can become competitive enough to suppress product formation. This application note is provided to give rules that should be taken into account when designing primers for PCR. More comprehensive coverage of this subject can be found elswhere1.
Primer selection
Several variables must be taken into account when designing PCR Primers. Among the most critical are:
Primer length
Melting Temperature (Tm)
Specificity
Complementary Primer Sequences
G/C content and Polypyrimidine (T, C) or polypurine (A, G) stretches
3’-end Sequence
Each of these critical elements will be discussed in turn.作者: 微笑的海豚 时间: 2011-8-20 15:26
Primer length
Since both specificity and the temperature and time of annealing are at least partly dependent on primer length, this parameter is critical for successful PCR. In general, oligonucleotides between 18 and 24 bases are extremely sequence specific, provided that the annealing temperature is optimal. Primer length is also proportional to annealing efficiency: in general, the longer the primer, the more inefficient the annealing. With fewer templates primed at each step, this can result in a significant decrease in amplified product. The primers should not be too short, however, unless the application specifically calls for it. As discussed below, the goal should be to design a primer with an annealing temperature of at least 50°C.
The relationship between annealing temperature and melting temperature is one of the “Black Boxes” of PCR. A general rule-of-thumb is to use an annealing temperature that is 5°C lower than the melting temperature. Thus, when aiming for an annealing temperature of at least 50°C, this corresponds to a primer with a calculated melting temperature(Tm) ~55°C. Often, the annealing temperature determined in this fashion will not be optimal and empirical experiments will have to be performed to determine the optimal temperature. This is most easily accomplished using a gradient thermal cycler like Eppendorf Scientific's Mastercycler® Gradient.
Melting Temperature (Tm)
It is important to keep in mind that there are two primers added to a PCR reaction. Both of the oligonucleotide primers should be designed such that they have similar melting temperatures. If primers are mismatched in terms of Tm, amplification will be less efficient or may not work at all since the primer with the higher Tm will mis-prime at lower temperatures and the primer with the lower Tm may not work at higher temperatures.
The melting temperatures of oligos are most accurately calculated using nearest neighbor thermodynamic calculations with the formula:
where H is the enthalpy and S is the entropy for helix formation, R is the molar gas constant and c is the concentration of primer. This is most easily accomplished using any of a number of primer design software packages on the market3. Fortunately, a good working approximation of this value (generally valid for oligos in the 18-24 base range) can be calculated using the formula:作者: 微笑的海豚 时间: 2011-8-20 15:27
Tm = 2(AT) + 4(GC).
The table below shows calculated values for primers of various lengths using this equation, which is known as the Wallace formula, and assuming a 50% GC content4.
Primer Length Tm = 2 (AT) + 4(GC) Primer Length Tm = 2 (AT) + 4(GC)
4 12 °C 22 66 °C
6 18 °C 24 72 °C
8 24 °C 26 78 °C
10 30 °C 28 84 °C
12 36 °C 30 90 °C
14 42 °C 32 96 °C
16 48 °C 34 102 °C
18 54 °C 36 108 °C
20 66 °C 38 114 °C
The temperatures calculated using Wallace's rule are inaccurate at the extremes of this chart.
In addition to calculating the melting temperatures of the primers, care must be taken to ensure that the melting temperature of the product is low enough to ensure 100% melting at 92°C. This parameter will help ensure a more efficient PCR, but is not always necessary for successful PCR. In general, products between 100-600 base pairs are efficiently amplified in many PCR reactions. If there is doubt, the product Tm can be calculated using the formula:
Under standard PCR conditions of 50mM KCL, this reduces to(3):
Tm = 59.9 + 0.41 (%G+C) – 675/length
Specificity
As mentioned above, primer specificity is at least partly dependent on primer length. It is evident that there are many more unique 24 base oligos than there are 15 base pair oligos. That being said, primers must be chosen so that they have a unique sequence within the template DNA that is to be amplified. A primer designed with a highly repetitive sequence will result in a smear when amplifying genomic DNA. However, the same primer may give a single band if a single clone from a genomic library is amplified.
Because Taq Polymerase is active over a broad range of temperatures, primer extension will occur at the lower temperatures of annealing. If the temperature is too low, non-specific priming may occur which can be extended by the polymerase if there is a short homology at the 3' end. In general, a melting temperature of 55 °C - 72 °C gives the best results (Note that this corresponds to a primer length of 18-24 bases using Wallace's rule above).
Complementary Primer Sequences
Primers need to be designed with absolutely no intra-primer homology beyond 3 base pairs. If a primer has such a region of self-homology, “snap back”, partially double-stranded structures, can occur which will interfere with annealing to the template.
Another related danger is inter-primer homology. Partial homology in the middle regions of two primers can interfere with hybridization. If the homology should occur at the 3' end of either primer, Primer dimer formation will occur which, more often than not, will prevent the formation of the desired product via competition.
G/C content and Polypyrimidine (T, C) or polypurine (A, G) stretches
The base composition of primers should be between 45% and 55% GC. The primer sequence must be chosen such that there is no PolyG or Poly C stretches that can promote non-specific annealing. Poly A and Poly T stretches are also to be avoided as these will “breath” and open up stretches of the primer-template complex. This can lower the efficiency of amplification. Polypyrimidine (T, C) and polypurine (A, G) stretches should also be avoided. Ideally the primer will have a near random mix of nucleotides, a 50% GC content and be ~20 bases long. This will put the Tm in the range of 56oC - 62oC1.
3’-end Sequence
It is well established that the 3' terminal position in PCR primers is essential for the control of mis-priming5. We have already explored the problem of primer homologies occurring at these regions. Another variable to look at is the inclusion of a G or C residue at the 3' end of primers. This “GC Clamp” helps to ensure correct binding at the 3' end due to the stronger hydrogen bonding of G/C residues. It also helps to improve the efficiency of the reaction by minimizing any “breathing” that might occur.作者: 微笑的海豚 时间: 2011-8-20 15:27
Conclusion
It is essential that care is taken in the design of primers for PCR. Several parameters including the length of the primer, %GC content and the 3' sequence need to be optimized for successful PCR. Certain of these parameters can be easily manually optimized while others are best done with commercial computer programs. In any event, careful observance of the general rules of primer design will help ensure successful experiments.
References
Dieffenbach, C.W., Lowe, T.M.J., Dveksler, G.S., General Concepts for PCR Primer Design, in PCR Primer, A Laboratory Manual, Dieffenbach, C.W, and Dveksler, G.S., Ed., Cold Spring Harbor Laboratory Press, New York, 1995, 133-155.
Innis, M.A., and Gelfand, D.H., Optimization of PCRs, in PCR protocols, A Guide to Methods and Applications, Innis, M.A., Gelfand, D.H., Sninsky, J.J., and White, T.J., Ed., CRC Press, London, 1994, 5-11.
Sharrocks, A.D., The design of primers for PCR, in PCR Technology, Current Innovations, Griffin, H.G., and Griffin, A.M, Ed., CRC Press, London, 1994, 5-11.
Suggs, S.V., Hirose, T., Miyake, E.H., Kawashima, M.J., Johnson, K.I., and Wallace, R.B., Using Purified Genes, in ICN-UCLA Symp. Developmental Biology, Vol. 23, Brown, D.D. Ed., Academic Press, New York, 1981, 683.
Kwok, S., Kellog, D.E. McKinney, N., Spasic, D., Goda, L., Levenson, C., and Sninsky, J.J., Effects of primer-template mismatches on the polymerase chain reaction: Human Immunodeficiency Virus 1 model studies. Nucleic Acids Res. 18:999-1005, 1990.
* The Polymerase Chain Reaction (PCR) is protected by patent. The patent is held by Hoffmann-La Roche.作者: 微笑的海豚 时间: 2011-8-20 15:28
PCR
Polymerase Chain Reaction
1) Add the following to a microfuge tube:
10 ul reaction buffer
1 ul 15 uM forward primer
1 ul 15 uM reverse primer
1 ul template DNA
5 ul 2 mM dNTP
8 ul 25 mM MgCl2 or MgSO4 (volume variable)
water (to make up to 100 ul)
2) Place tube in a thermocycler. Heat sample to 95 °C, then add 0.5 -1 ul of enzyme (Taq, Tli, Pfu etc.). Add a few drops of mineral oil.
3) Start the PCR cycles according the following schemes:
a) denaturation - 94 ° C, 30-90 sec.
b) annealing - 55 °C (or -5° Tm), 0.5-2 min.
c) extension - 72 °C, 1 min. (time depends on length of PCR product and enzyme used)
repeat cycles 29 times
4) Add a final extension step of 5 min. to fill in any uncompleted polymerisation. Then cooled down to 4- 25 °C.
Note:
Most of the parameters can be varied to optimise the PCR (more at Tavi's PCR guide):
a) Mg++ - one of the main variables - change the amount added if the PCR result is poor. Mg++ affects the annealing of the oligo to the template DNA by stabilising the oligo-template interaction, it also stabilises the replication complex of polymerase with template-primer. It can therefore also increases non-specific annealing and produced undesirable PCR products (gives multiple bands in gel). EDTA which chelate Mg++ can change the Mg++ concentration.作者: 微笑的海豚 时间: 2011-8-20 15:28
b) Template DNA concentration - PCR is very powerful tool for DNA amplification therefore very little DNA is needed. But to reduce the likelihood of error by Taq DNA polymerase, a higher DNA concentration can be used, though too much template may increase the amount of contaminants and reduce efficiency.
c) Enzymes used - Taq DNA polymerase has a higher error rate (no proof-reading 3' to 5' exonuclease activity) than Tli or Pfu. Use Tli, Pfu or other polymerases with good proof-reading capability if high fidelity is needed. Taq, however, is less fussy than other polymerases and less likely to fail. It can be used in combination with other enzymes to increase its fidelity. Taq also tends to add extra A's at the 3'end (extra A's are useful for TA cloning but needs to be removed if blunt end ligation is to be done). More enzymes can also be added to improve efficiency (since Taq may be damaged in repeated cycling) but may increase non-specific PCR products. Vent polymerase may degrade primer and therefore not ideal for mutagenesis-by-PCR work.
d) dNTP - can use up to 1.5 mM dNTP. dNTP chelate Mg++, therefore amount of Mg++ used may need to be changed. However excessive dNTP can increase the error rate and possibly inhibits Taq. Lowering the dNTP (10-50 uM) may therefore also reduce error rate. Larger size PCR fragment need more dNTP.
e) primers - up to 3 uM of primers may be used, but high primer to template ratio can results in non-specific amplification and primer-dimer formation (note: store primers in small aliquots).
f) Primer design - check primer sequences to avoid primer-dimer formation. Add a GC-clamp at the 5' end if a restriction site is introduced there. One or two G or C at the 3' end is fine but try to avoid having too many (it can result in non-specific PCR products). Perfect complementarity of 18 bases or more is ideal. See Guide.
g) Thermal cycling - denaturation time can be increased if template GC content is high. Higher annealing temperature may be needed for primers with high GC content or longer primers (calculate Tm). Using a gradient (if your PCR machine permits it) is a useful way of determining the annealing temperature. Extension time should be extended for larger PCR products; but reduced it whenever possible to limit damage to enzyme. Extension time is also affected by the enzymes used e.g for Taq - assume 1000 base/min (also check suppliers' recommendations, actual rate is much higher). The number of cycle can be increased if the number of template DNA is very low, and decreased if high amount of template DNA is used (higher template DNA is preferable for PCR cloning - lower error rate in the PCR).
h) Additives -
Glycerol (5-10%), formamide (1-5%) or DMSO (2-10%) can be added in PCR for template DNA with high GC content (they change the Tm of primer-template hybridisation reaction and the thermostability of polymerase enzyme). Glycerol can protects Taq against heat damage, while formamide may lower enzyme resistence.
0.5 -2M Betaine (stock solution - 5M) is also useful for PCR over high GC content and long stretches of DNA (Long PCR / LA PCR). Perform a titration to determine to optimum concentration (1.3 M recommended). Reduce melting temperature (92 -93 °C) and annealing temperature (1-2°C lower). It may be useful to use betaine in combination with other reagents like 5%DMSO. Betaine is often the secret (and unnecessarily expensive) ingredient of many commercial kits.
>50mM TMAC (tetramethylammonium chloride), TEAC (tetraethylammonium chloride), and TMANO (trimethlamine N-oxide) can also be used.作者: 微笑的海豚 时间: 2011-8-20 15:29
BSA (up to 0.8 µg/µl) can also improve efficiency of PCR reaction.
See also Dan Cruickshank's PCR additives and Alkami Enhancers for more.
i) PCR buffer
Higher concentration of PCR buffer may be used to improve efficiency.
This buffer may work better than the buffer supplied from commercial sources.
16.6 mM ammonium sulfate
67.7 mM TRIS-HCl, pH 8.89
10 mM beta-mercaptoethanol
170 micrograms/ml BSA
1.5-3 mM MgCl2作者: 微笑的海豚 时间: 2011-8-20 15:31
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