Download 2- pcr primer design and reaction optimisation

The Polymerase Chain Reaction
Organized by:
Dr. Abir Adel SAAD
Biotechnology Dept, IGSR, University of Alexandria
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The Polymerase Chain Reaction
(PCR)
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Principle of the PCR
PCR primer design and reaction optimisation
Standard PCR protocol
Reverse transcription PCR: RNA -> lots of DNA
Calculating concentrations for PCR
Troubleshooting
The applications of PCR
PCR Glossary
Real time PCR
Principle of Sequencing
DNA amplification is the cornerstone of modern biotechnology and it is also a key
procedure in numerous basic studies involving DNA and other biomolecules. All methods for
DNA amplification have rested on the concept of DNA strand complementarity discovered by
Watson and Crick fifty years ago.
Polymerase chain reaction (PCR) is still the most popular amplification method,
however alternatives to PCR have successfully invaded the area. The emergence of such
methodologies has significantly widened the range of approaches for DNA amplification and
dramatically improved the technological abilities of basic and applied researchers in various
fields of life sciences. It will not be an exaggeration to say that now no research related to DNA
can be performed without the employment of DNA amplification procedures.
Newly-developed protocols including real-time PCR and other new PCR developments,
plus several powerful non-PCR isothermal DNA amplification techniques are now being used
in the field of molecular biology.
1- Principle of the PCR
The purpose of a PCR (Polymerase Chain Reaction) is to make a huge number of
copies of a gene. This is necessary to have enough starting template for sequencing.
The cycling reactions :
There are three major steps in a PCR, which are repeated for 30 or 40 cycles. This is
done on an automated cycler “Thermocycler”, which can heat and cool the tubes with
the reaction mixture in a very short time.
1.
Denaturation at 94°C :
During the denaturation, the double strand melts open to single stranded DNA,
all enzymatic reactions stop (for example: the extension from a previous
cycle).
2.
Annealing at 54°C :
The primers are jiggling around, caused by the Brownian motion. Ionic bonds
are constantly formed and broken between the single stranded primer and the
single stranded template. The more stable bonds last a little bit longer (primers
that fit exactly) and on that little piece of double stranded DNA (template and
primer), the polymerase can attach and starts copying the template. Once there
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are a few bases built in, the ionic bond is so strong between the template and
the primer, that it does not break anymore.
3.
Extension at 72°C :
This is the ideal working temperature for the polymerase. The primers, where
there are a few bases built in, already have a stronger ionic attraction to the
template than the forces breaking these attractions. Primers that are on
positions with no exact match, get loose again (because of the higher
temperature) and don't give an extension of the fragment.
The bases (complementary to the template) are coupled to the primer on the 3' side
(the polymerase adds dNTP's from 5' to 3', reading the template from 3' to 5' side,
bases are added complementary to the template)
The different steps in PCR.
Because both strands are copied during PCR, there is an exponential increase of the
number of copies of the gene. Suppose there is only one copy of the wanted gene
before the cycling starts, after one cycle, there will be 2 copies, after two cycles, there
will be 4 copies, three cycles will result in 8 copies and so on.
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The exponential amplification of the gene in PCR.
Is there a gene copied during PCR and is it the right size ?
Before the PCR product is used in further applications, it has to be checked if :
1.
There is a product formed.
Though biochemistry is an exact science, not every PCR is successful. There is for
example a possibility that the quality of the DNA is poor, that one of the primers
doesn't fit, or that there is too much starting template
2.
The product is of the right size
It is possible that there is a product, for example a band of 500 bases, but the expected
gene should be 1800 bases long. In that case, one of the primers probably fits on a
part of the gene closer to the other primer. It is also possible that both primers fit on a
totally different gene.
3.
Only one band is formed.
As in the description above, it is possible that the primers fit on the desired locations,
and also on other locations. In that case, you can have different bands in one lane on a
gel.
Verification of the PCR product on gel.
The ladder is a mixture of fragments with known
size to compare with the PCR fragments. Notice
that the distance between the different fragments
of the ladder is logarithmic. Lane 1: PCR fragment
is approximately 1850 bases long. Lane 2 and 4:
the fragments are approximately 800 bases long.
Lane 3: no product is formed, so the PCR failed.
Lane 5: multiple bands are formed because one of
the primers fits on different places.
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2- PCR PRIMER DESIGN AND REACTION
OPTIMISATION
Contents
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Factors Affecting the PCR
Denaturing Temperature and Time
Annealing Temperature and Primer Design
Primer Length
Degenerate Primers
Elongation Temperature and Time
Reaction Buffer
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 singlestranded 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 singlestranded 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.
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.
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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(s). One should aim at using an annealing temperature (Ta)
about 5oC below the lowest Tm of the 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 Ta 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).
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.
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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.
For example, there is a 1⁄4 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 nonspecific 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 (gB) 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(s), 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).
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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.
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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 :
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v/v)
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 Triton X-100 (0.05 - 0.10%
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.
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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 reamplification 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:
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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 (Soiné 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 reused, for periods up to 3 years.
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) :
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dimethyl sulphoxide (DMSO),
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dimethyl formamide (DMF),
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urea
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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
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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):
1. primers should be 17-28 bases in length;
2. base composition should be 50-60% (G+C);
3. primers should end (3') in a G or C, or CG or GC: this prevents "breathing" of
ends and increases efficiency of priming;
4. Tms between 55-80oC are preferred;
5. 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;
6. 3'-ends of primers should not be complementary (ie. base pair), as otherwise
primer dimers will be synthesised preferentially to any other product;
7. 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:
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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.
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3- Standard PCR Protocol
Contents
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Recommended Reagent Concentrations
Recommended Reaction Conditions
Initial Conditions
Temperature Cycling
"Hot Start" PCR
Asymmetric PCR for ssDNA Production
Detecting Products
Labelling PCR Products with Digoxigenin
Cleaning PCR Products
Sequencing PCR Products
Cloning PCR Products
AND ALWAYS REMEMBER:
Recommended Reagent Concentrations:
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Primers: 0.2 - 1.0 uM
Nucleotides: 50 - 200 uM EACH dNTP
Dimethyl sulphoxide (DMSO): 0 - 10% (v/v)
Taq polymerase: 0.5 - 1.0 Units/50ul rxn
Target DNA: 1 ng - 1 ug (NB: higher concn for total genomic DNA; lower for
plasmid / purified DNA / virus DNA target)
Buffer: use proprietary or home-made 10x rxn mix; eg: Cetus, Promega. This should
contain: minimum of 1.5mM Mg2+, usually some detergent, perhaps some gelatin or
BSA. Promega now supply 25mM MgCl2, to allow user-specified [Mg2+] for
reaction optimisation with different combinations of primers and targets.
MAKE POOLED MASTER MIX OF REAGENTS IN ABSENCE OF DNA using
DNA-free pipette, then dispense to individual tubes (using DNA-free pipette), and
add DNA to individual reactions USING PLUGGED TIPS.
NOTE:
USE PLUGGED PIPETTE TIPS: prevents aerosol contamination of pipettes.
Use of detergents is recommended only for Taq from Promega (up to 0.1% v/v, Triton
X-100 or Tween-20). DMSO apparently allows better denaturation of longer target
sequences (>1kb) and more product.
DO NOT USE SAME PIPETTE FOR DISPENSING NUCLEIC ACIDS AS YOU
USE FOR DISPENSING REAGENTS
Remember sample volume should not exceed 1/10th reaction volume, and sample
DNA/NTP/primer concentrations should not be too high as otherwise all available
Mg2+ is chelated out of solution and enzyme reactivity is adversely affected. Any
increase in dNTPs over 200uM means [Mg2+] should be re-optimised.
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Low primer, target, Taq, and nucleotide concentrations are to be favoured as these
generally ensure cleaner product and lower background, perhaps at the cost of
detection sensitivity.
Recommended Reaction Conditions:
Initial Conditions:
Initial denaturation at start: 92 - 97oC for 3 - 5 min. If you denature at 97oC, denature
sample only; add rest of mix after reaction colls to annealing temperature (prevents
premature denaturation of enzyme).
Initial annealing temperature: as high as feasible for 3 min (eg: 50 - 75oC). Stringent
initial conditions mean less non-specific product, especially when amplifying from
eukaryotic genomic DNA.
Initial elongation temperature: 72oC for 3 - 5 min. This allows complete elongation of
product on rare templates.
Temperature Cycling:
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92 - 94oC for 30 - 60 sec (denature)
37 - 72oC for 30 - 60 sec (anneal)
72oC for 30 - 60 sec (elongate) (60 sec per kb target sequence length)
25 - 35 cycles only (otherwise enzyme decay causes artifacts)
72oC for 5 min at end to allow complete elongation of all product DNA
NOTE:
"Quickie" PCR is quite feasible: eg, [94oC 30 sec / 45oC 30 sec / 72oC 30 sec] x 30,
for short products (200 - 500 bp).
DON'T RUN TOO MANY CYCLES: if you don't see a band with 30 cycles you
probably won't after 40; rather take an aliquot from the reaction mix and re-PCR with
fresh reagents.
"Hot Start" PCR:
In certain circumstances one wishes to avoid mixing primers and target DNA at low
temperatures in the presence of Taq polymerase: Taq pol is almost as efficient as
Klenow pol at 37oC; consequently, if primers mis-anneal at low temperature prior to
initial template denaturation, "non-specific" amplification may occur. This may be
avoided by only adding enzyme after the initial denaturation, before the reaction cools
to the chosen annealing temperature. This is most conveniently done by putting wax
"gems"TM into the reaction tube after addition of everything except enzyme, then
putting enzyme on top of the gem: the wax melts when the temperature reaches +/80oC, and the enzyme mixes with the rest of the reaction mix while the molten wax
floats on top and seals the mix, taking the place of mineral oil. Information is that
"gems" may be substituted by VaselineTM.
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Asymmetric PCR for ssDNA Production:
Simply use a 100:1 molar ratio of the two primers (eg: primer 1 at 0.5uM, primer 2 at
0.005uM). This allows production of mainly ssDNA of the sense of the more
abundant primer, which is useful for sequencing purposes or making ssDNA probes.
Detecting Products:
Take 1/10th - 1/3rd of the reaction mix CAREFULLY from under the oil or from
under the Vaseline or solidified wax, using a micropipette with plugged tip, IN AN
AREA AWAY FROM YOUR PCR PREPARATION AREA!
Mix this with some gel loading buffer(1:1 - 1:5 mix:loading buffer): this is TBE
containing 10 - 20% glycerol or sucrose and a dash of bromophenol blue (BPB)
tracking dye.
Load 5 - 30ul of sample into wells of 0.8 - 3.0% submarine agarose gel made up in
TBE, preferably containing 50ng/ml ethidium bromide.
Run at 80 -120 volts (not too slow or small products diffuse; not too fast or bands
smear) until BPB reaches end of gel (large products) or 2/3 down gel (small
products). Use DNA markers going from 2kb down to 100 bp or less (recommend
BM PCR markers).
View on UV light box at 254 - 300 nm, photo 1 - 5 sec.
NOTE:
Small products are best seen on 3% agarose gels that have been run fast (eg: 100
volts), with BPB run to 1⁄2 - 2/3 down the gel. It is best to include EthBr in the gel
AND in the gel buffer, as post-electrophoresis staining can result in band smearing
due to diffusion, and if there is no EthBr in the buffer the dye runs backwards out of
the gel, and smaller bands are stripped of dye and are not visible.
NUSIEVE TM gel (FMC Corp) can also be used for small products - better resolution
than agarose.
Polyacrylamide gels can be silver stained by simple protocols for extreme sensitivity
of detection.
Gels can be blotted directly after soaking in 0.5M NaOH / 1.5M NaCl for 10-20 min:
"dry blotting" works well (eg: gel is over- and under-layered with paper towel stacks
and pressed; bands transfer up and down), as does classic "Southern" blotting. Bands
blotted in this way are already covalently fixed onto nylon membranes, and simply
need a rinse in 5xSSPE before prehybridisation.
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The example shown is of detection of Human papillomavirus type 16 (HPV-16) DNA
amplified from cervical biopsy samples (Williamson A-L, Rybicki EP (1991)
Detection of genital human papillomaviruses by polymerase chain reaction
amplification with degenerate nested primers. J Med Virol 33: 165-171). The left
panel is a photo of an EthBR-stained 2% agarose gel; the right is an autoradiograph of
a Southern blot probed with 32P-labelled HPV-16 DNA. Note how much more
sensitive blotting is, and how much more specific the detection is.
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.
17
Cleaning PCR Products
*
Getting rid of mineral oil: simply add 50ul of chloroform to the reaction vial,
vortex and centrifuge briefly, and remove the "hanging droplet" of AQUEOUS
solution with a micopipette.
*
Getting rid of wax or Vaseline: simply "spear" wax gem and remove; do as for
oil or bottom-puncture tube and blow out aqueous drop for Vaseline.
*
Cleaning-up DNA: 3 options
*
a protocol which gives DNA that is clean enough for sequencing is the
following: increase volume to 100ul with water, add 10M ammonium acetate soln. to
0.2M final concentration (1/5th volume), add equal volume of isopropanol (propan-2ol), leave on bench 5 min, centrifuge 20 min at 15 000 rpm, remove liquid using
pipette, resuspend in 100ul water or TE, repeat precipitation.
*
Simply do a phenol-CHCl3 extraction (add 20ul phenol to aqueous phase,
vortex, add 50ul CHCl3, vortex, centrifuge, remove UPPER aqueous phase, repeat
CHCl3 extraction).
*
Make aqueous phase up to 400ul, and spin through Millipore Ultrafree-MC
NMWL 30 000 cartridges (at 6000 rpm in microcentrifuge), wash through with
2x400ul water, collect +/-20ul sample: this is pure enough for sequencing.
NOTE:
Product is clean enough for restriction digest immediately after reaction; however,
phenol-chloroform clean-up is recommended as a minimum.
Sequencing PCR Products:
This is best done using ssDNA generated by asymmetric PCR, and the "limiting"
primer for sequencing. However, efficient sequencing of dsDNA generated by normal
PCR is possible using the modification to the SequenaseTM protocol published by
Bachmann et al. (1990) (NAR 18: 1309). CLEAN DNA is resuspended in sequencing
buffer containing 0.5% Nonidet P-40 and 0.5% Tween-20 and sequencing primer,
denatured by heating to 95oC for 5 min, snap-cooled on wet ice, and sequenced by the
"close-to-primer" protocol (eg: dilute extension mixes).
Cloning PCR Products
T-A Cloning Strategy: Taq and other polymerases seem to have a terminal transferase
activity which results in the non-templated addition of a single nucleotide to the 3'ends of PCR products. In the presence of all 4 dNTPs, dA is preferentially added;
however, use of a single dNTP in a reaction mix results in (relatively inefficient)
addition of that nucleotide. This complicates cloning, as the supposedly blunt-ended
PCR product often is not, and blunt-endedcloning protocols often do not work or are
very inefficient. This can be remedied by incubation of PCR products with T4 DNA
pol or Klenow pol, which "polishes" the ends due to a 3'->5' exonuclease activity (Lui
and Schwartz, 1992; BioTechniques, 20: 28-30). However, this terminal transferase
activity is also the basis of a clever cloning strategy: this uses Taq pol to add a single
dT to the 3'-ends of a blunt-cut cloning vector such as pUC or pBluescriptTM, and
simple ligation of the PCR product into the now "sticky-ended" plasmid (Marchuk et
al., 1990; NAR 19: 1156).
Incorporation of Restriction Sites in Primers: Although this may be rendered simple
by incorporating the same or different restriction sites at the 5'-ends of PCR primers which allows generation of sticky ends and straightforward cloning into appropriate
18
vectors - these should have AT LEAST two additional bases 5' to the recognition
sequence to ensure that the enzymes will in fact recognise the sequence - and it is
often found that even when this is done, the efficiency of cutting of fresh product is
next to zero. This can sometimes be remedied by incubating fresh product with
Proteinase K (to digest off tightly-attached Taq pol), but often is not. A solution to the
problem is to use the "Klenow-Kinase-Ligase" (KKL) method: this involves
"polishing" products with Klenow, kinasing them to get 5'-phosphorylation (NB:
OLIGONUCLEOTIDE PRIMERS NORMALLY HAVE NO 5'-PHOSPHATES!!!),
ligating the fragments together to get concatemers, then restricting these with the
appropriate restriction enzymes to generate the sticky-ended fragments suitable for
cloning (Lorens, 1991; PCR Methods and Applications, 1: 140-141).
AND ALWAYS REMEMBER:
*
WORK CLEAN
*
TITRATE MAGNESIUM
*
DON'T USE TOO MUCH TEMPLATE DNA
*
DON'T USE PCR PRODUCTS IN PCR PREPARATION AREAS
*
ALWAYS, ALWAYS INCLUDE WATER AND VERY DILUTE POSITIVE
CONTROLS IN EVERY EXPERIMENT
*
WEAR GLOVES
*
USE PLUGGED TIPS
19
4-Troubleshooting
1. I get (many) longer unspecific products. What can I do?
 Decrease annealing time
 Increase annealing temperature
 Decrease extension time
 Decrease extension temperature to 62-68º C
 Increase KCl (buffer) concentration to 1.2x-2x, but keep MgCl2 concentration at
1.5-2mM.
 Increase MgCl2 concentration up to 3-4.5 mM but keep dNTP concentration
constant.
 Take less primer
 Take less DNA template
 Take less Taq polymerase
 If none of the above works: check the primer for repetitive sequences (BLAST
align the sequence with the databases) and change the primer(s)
 Combine some/all of the above
2. I get (many) shorter unspecific products. What can I do?
 Increase annealing temperature
 Increase annealing time
 Increase extension time
 Increase extension temperature to 74-78º C
 Decrease KCl (buffer) concentration to 0.7-0.8x, but keep MgCl2 concentration at
1.5-2mM
 Increase MgCl2 concentration up to 3-4.5 mM but keep dNTP concentration
constant
 Take less primer
 Take less DNA template
 Take less Taq polymerase
 If none of the above works: check the primer for repetitive sequences (BLAST
align the sequence with the databases) and change the primer(s)
 Combine some/all of the above
3. Reaction was working before, but now I can't get any product.
 Make sure all PCR ingredients are taken in the reaction (buffer, template, Taq,
etc)
 Change the dNTP solution (very sensitive to cycles of thawing and freezing,
especially in multiplex PCR)
 If you just bought new primers, check for their reliability (bad primer synthesis ?)
 Increase primer amount
 Increase template amount
 Decrease annealing temperature by 6-10º C and check if you get any product. If
you don't, check all your PCR ingredients. If you do get products (including
unspecific ones) reaction conditions as described above.
 Combine some/all of the above
20
4. My PCR product is weak. Is there a way to increase the yield?
 Gradually decrease the annealing temperature to the lowest possible.
 Increase the amount of PCR primer
 Increase the amount of DNA template
 Increase the amount of Taq polymerase
 Change buffer (KCl) concentration (higher if product is lower than 1000bp or
lower if product is higher than 1000bp)
 Add adjuvants. Best, use BSA (0.1 to 0.8 µg/µL final concentration). You can also
try 5% (v/v, final concentration) DMSO or glycerol.
 Check primer sequences for mismatches and/or increase the primer length by 5
nucleotides
 Combine some/all of the above
5. My two primers have very different melting temperatures (Tm)
but I cannot change their locus. What can I do to improve PCR
amplification?
An easy solution is to increase the length of the primer with low Tm. If you need to
keep the size of the product constant, add a few bases at the 3' end. If size is not a
concern, add a few bases at either the 3' or the 5' end of that primer.
6. I have a number of primer pairs I would like to use together. Can I
run a multiplex PCR with them?. How?
 Very likely, yes.
 Try amplify all loci seaprately using the same PCR program. If one of the primer
pairs yields unspecific products, keep the cycling conditions constant and change
other parameters as mentioned above (#1 and #2).
 Mix equimolar amounts of primers and run the multiplex reaction either in the
same cycling conditions or by decreasing only the annealing temperature by 4º C.
 If some of the loci are weak or not amplified, read below !!
7. How many loci can I amplify in multiplex PCR at the same time?
Difficult to say. The author has routinely amplified from 2 to 14 loci.
Literature describes up to 25 loci or so.
8. One or a few loci in my multiplex reaction are very weak or
invisible. How can amplify them?
 The first choice should be increasing the amount of primer for the "weak" loci at
the same time with decreasing the amount of primer for all loci that can be
amplified. The balance between these amounts is more important than the absolute
values used !!.
 Check primer sequences for primer-primer interactions
9. Short PCR products in my multiplex reaction are weak. How can I
improve their yield?
21
Increase KCl (buffer) concentration to 1.2x-2x, but keep MgCl2 concentration at 1.52mM
 Decrease denaturing time
 Decrease annealing time and temperature
 Decrease extension time and temperature
 Increase amount of primers for the "weak" loci while decreasing the amount for
the "strong" loci.
 Add adjuvants. Best, use BSA (0.1 to 0.8 µg/µL final concentration). You can also
try 5% (v/v, final concentration) DMSO or glycerol
 Combine some/all of the above
10. Longer PCR products in my multiplex reaction are weak. How
can I improve their yield?
 Decrease KCl (buffer) concentration to 0.7-0.8x, but keep MgCl2 concentration at
1.5-2mM
 Increase MgCl2 concentration up to 3-4.5 mM but keep dNTP concentration
constant.
 Increase denaturing time
 Increase annealing time
 Decrease annealing temperature
 Increase extension time and temperature
 Increase amount of primers for the "weak" loci while decreasing the amount for
the "strong" loci
 Add adjuvants. Best, use BSA (0.1 to 0.8 µg/µL final concentration). You can also
try 5% (v/v, final concentration) DMSO or glycerol
 Combine some/all of the above
11. All products in my multiplex reaction are weak. How can I
improve the yield?
 Decrease annealing time in small steps (2º C)
 Decrease extension temperature to 62-68º C
 Increase extension time
 Increase template concentration
 Increase overall primer concentration
 Adjust Taq polymerase concentration
 Change KCl (buffer) concentration, but keep MgCl2 concentration at 1.5-2mM
 Increase MgCl2 concentration up to 3-4.5 mM but keep dNTP concentration
constant.
 Add adjuvants. Best, use BSA (0.1 to 0.8 µg/µL final concentration). You can
also try 5% (v/v, final concentration) DMSO or glycerol
 Combine some/all of the above
12. Unspecific products appear in my multiplex reaction. Can I get
rid of them somehow?
22
 If long: increase buffer concentration to 1.2-2x, but keep MgCl2 concentration at
1.5-2mM
 If short: decrease buffer concentration to 0.7-0.9x, but keep MgCl2 concentration
at 1.5-2mM
 Gradually increase the annealing temperature
 Decrease amount of template
 Decrease amount of primer
 Decrease amount of enzyme
 Increase MgCl2 concentration up to 3-4.5 mM but keep dNTP concentration
constant
 Add adjuvants. Best, use BSA (0.1 to 0.8 µg/µL final concentration). You can also
try 5% (v/v, final concentration) DMSO or glycerol
 If nothing works: run PCR reactions for each (multiplexed) locus individually,
using an annealing temperature lower than usual. Compare the unspecific products
for each locus tested with the unspecific products seen when running the multiplex
PCR. This may indicate which primer pair yields the unspecific products in the
multiplex reaction.
 Combine some/all of the above
 (Note: primer-primer interactions in multiplex PCR are usually translated into lack
of some amplification products rather than the appearance of unspecific products)
5-The applications of PCR
PCR is such a straightforward procedure that it is sometimes difficult to
understand how it can have become so important in modern research. First we will
deal with its limitations. In order to synthesize primers that will anneal at the correct
positions, the sequences of the boundary regions of the DNA to be amplified must be
known. This means that PCR cannot be used to purify fragments of genes or other
parts of a genome that have never been studied before. A second constraint is the
length of DNA that can be copied. Regions of up to 5 kb can be amplified without too
much difficulty, and longer amplifications - up to 40 kb - are possible using
modifications of the standard technique. However, the >100 kb fragments that are
needed for genome sequencing projects are unattainable by PCR.
What are the strengths of PCR? Primary among these is the ease with which
products representing a single segment of the genome can be obtained from a number
of different DNA samples. We will encounter one important example of this when we
look at how DNA markers are typed in genetic mapping projects. PCR is used in a
similar way to screen human DNA samples for mutations associated with genetic
diseases such as thalassemia and cystic fibrosis. It also forms the basis of genetic
profiling, in which variations in microsatellite length are typed.
23
The use of microsatellite analysis in genetic profiling. In this example, microsatellites located on the
short arm of chromosome 6 have been amplified by the polymerase chain reaction. The PCR products
are labeled with a blue or green fluorescent marker and run in a polyacrylamide gel, each lane showing
the genetic profile of a different individual. No two individuals have the same genetic profile because
each person has a different set of microsatellite length variants, the variants giving rise to bands of
different sizes after PCR. The red bands are DNA size markers. Image supplied courtesy of PE
Biosystems, Warrington, UK, and reproduced with permission.
A second important feature of PCR is its ability to work with minuscule
amounts of starting DNA. This means that PCR can be used to obtain sequences from
the trace amounts of DNA that are present in hairs, bloodstains and other forensic
specimens, and from bones and other remains preserved at archaeological sites. In
clinical diagnosis, PCR is able to detect the presence of viral DNA well before the
virus has reached the levels needed to initiate a disease response. This is particularly
important in the early identification of viral-induced cancers because it means that
treatment programs can be initiated before the cancer becomes established.
These are just a few of the applications of PCR. The technique is now a major
component of the molecular biologist's toolkit and we will discover many more
examples of its use in the study of genomes.
Although PCR was first developed only a decade and a half ago, the simplicity and
the versatility of the technique have ensured that it is among the most ubiquitous of
molecular genetic methodologies, with a wide range of general applications
PCR enables rapid amplification of template DNA for screening of
uncharacterized mutations
Because of its rapidity and simplicity, PCR is ideally suited to providing
numerous DNA templates for mutation screening. Partial DNA sequences, at the
genomic or the cDNA level, from a gene associated with disease, or some other
interesting phenotype, immediately enable gene-specific PCR reactions to be
designed. Amplification of the appropriate gene segment then enables rapid testing for
the presence of associated mutations in large numbers of individuals. By contrast,
cell-based DNA cloning of the gene from numerous different individuals is far too
slow and labor-intensive to be considered as a serious alternative.
Typically, the identification of exon-intron boundaries and sequencing of the
ends of introns of a gene of interest offers the possibility of genomic mutation
screening. Individual exon-specific amplification reactions are developed by
designing primers which recognize intronic sequences located close to the exon-intron
boundary. The resulting PCR products are then analyzed by rapid mutation-screening
methods, in which the optimal size for mutation screening is usually about 200 bp.
Conveniently, the average size of a human exon is about 180 bp but, in the case of
very large exons, it is usual to design a series of primers to generate overlapping
exonic products. PCR can also quickly provide amplified cDNA sequences for
mutation screening. Such cDNA mutation screening may be the only way in which
mutations can be screened if the exon-intron organization of a gene has not been
established. To do this, mRNA is isolated from a convenient source of tissue, such as
blood cells, converted into cDNA using reverse transcriptase and the cDNA is used as
a template for a PCR reaction. This version of the standard genomic PCR reaction is
consequently often referred to as RT-PCR (reverse transcriptase-PCR). Clearly, the
24
method is ideally suited to genes expressed at high levels in easily accessible cells,
such as blood cells. However, as a result of low level ectopic transcription of genes in
all tissues, it has also been applied to transcript analysis of genes which are not
significantly expressed in blood cells, such as the dystrophin (DMD) gene (Chelly et
al., 1989).
PCR products for gene mutation screening are obtained from genomic DNA using intronspecific primers flanking exons or by RT-PCR. (A) Genomic DNA. Exons 1–4 can be
amplified separately from genomic DNA using pairs of intron-specific primers 1F + 1R, 2F +
2R, etc. (B) RT-PCR. This relies on at least some mRNA being present in easily accessible
cells such as blood cells, permitting conversion to cDNA. The cDNA can then be used as a
template for pairs of exon-specific primers (1F+1R, 2F+2R, etc.) to generate overlapping
DNA fragments.
PCR permits rapid genotyping for polymorphic markers
Restriction site polymorphisms (RSPs) result in alleles possessing or lacking a
specific restriction site. Such polymorphisms can be typed using Southern blot
hybridization. A DNA probe representing the locus is hybridized against genomic
DNA samples that have been digested with the appropriate restriction enzyme and
size-fractionated by agarose gel electrophoresis. The resulting RFLPs have two alleles
corresponding to the presence or absence of the restriction site. As a convenient
alternative to RFLPs, PCR can type RSPs by simply designing primers using
sequences which flank the polymorphic restriction site, amplifying from genomic
DNA, then cutting the PCR product with the appropriate restriction enzyme and
separating the fragments by agarose gel electrophoresis.
25
8- PCR Glossary
AFLP
AFLP Amplified Fragment Length Polymorphism
Genomic DNA is digested with MseI-EcoRI and the MseI-EcoRI fragments are PCR
amplified to reveal length polymorphism.
Asymmetric PCR
Preferential PCR amplification of one strand of DNA by lowering the concentration
of one primer.
Bisulfate sequencing
Used to detect CpG methylation in genomic DNA. Sodium bisulfate converts
unmethylated cytosine to Uracil. Comparison of sodium bisulfate treated and
untreated DNA reveal methylated cytosines.
DD-PCR
Differential Display PCR
A technique of comparing gene expression between related cells. Arbitrary primers
are used to amplify 3'-ends of subpopulations of polyA mRNAs.
DOR PCR
Degenerate Oligonucleotide-primed PCR (DOR PCR)
Degenerate PCR
Degenerate PCR primers used to amplify unknown DNA sequences based on similar
known sequences.
Forensic PCR
VNTR locus are PCR amplified to compare DNA samples from different sources.
Hairpin PCR
A method of error-free DNA amplification for mutation detection. It first converts a
DNA sequence to a hairpin. True mutations will maintain the hairpin structure during
amplification while PCR errors will disrupt the hairpin structure.
In situ PCR
PCR on fixed cells. DNA or RNA is immobilized in their subcellular locations.
Long PCR
Amplification of long target DNA sequences.
MSP PCR
Methylation Specific PCR Used to detect CpG methylation in genomic DNA.
Sodium bisulfate converts unmethylated cytosine to Uracil. Comparison of sodium
bisulfate treated and untreated DNA reveal methylated cytosines.
PCR-RFLP
See Restriction Fragment Length Polymorphism (PCR-RFLP)
26
By designing primers that will introduce or destroy a restriction site for one of the
alleles, the PCR products for SNP alleles can be distinguished by restriction fragment
lengths.
Competitive PCR (cPCR)
A method for quantify DNA using real-time PCR. A competitor internal standard is
co-amplified with the target DNA. Target is quantified from the melting curves of the
target and the competitor.
PCR-Single Strand Conformational Polymorphism (PCR-SSCP)
PCR followed by SSCP detection of point mutations.
Single Strand Conformational Polymorphism (SSCP)
Electrophoresis separation of single-stranded nucleic acids based on differences in
sequence. Single base mutations may be detected because single base mutations may
disrupt secondary structure of the SS DNA and leads to changes in mobility through
the gel.
Rapid Amplification of cDNA Ends (RACE)
Used for cloning full-length 5' or 3' ends of a cDNA. An adapter sequence is added to
either 5' or 3' ends of cDNA. The two PCR primers are either specific to the adapter
or specific to known sequences of cDNA.
RAPD
Random Amplified Polymorphic DNA (RAPD)
Random primers are used to
amplify genomic DNA. Patterns of bands may be different for individuals in a
population or closely related species.
Serial Analysis of Gene Expression (SAGE)
A technique for profiling gene expression in cells. SAGE characterizes a short
segment of DNA, called a SAGE tag, in each expressed gene. cDNA is digested with
the restriction enzyme Nla III, ligated to a linker, then digested with BsmF1, an
enzyme that cleaves 10-14 bases from the Nla III cut. SAGE tags are then
concatenated, cloned, and sequenced.
Touchdown
Decreasing the annealing temperature by 1 degree every one or second cycle.
27