PCR仪

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).

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:

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.

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.

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

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.

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)

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.

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.

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:

Tmprimer = (delta)H [(delta)S+ R ln (c/4)] -273.15°C + 16.6 log 10 [K+]

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: