PCR Primer Design（二）
Terminal Nucleotides Make a Difference
Both
the terminals of the primer are of vital importance for a successful
amplification. The 3'-end position in the primer affects mispriming.
However, for certain reactions, such as amplification refractory
mutation system (ARMS), this mispriming is required (Newton et al.,
1989; Old, 1991; Tan et al., 1994). Runs (3 or more) of C's or G's at
the 3' end of the primer should be avoided as G + C rich sequence leads
to mispriming. Complementarity at the 3' end of the primer elevates
mispriming as this promotes the formation of a primer dimer artifact and
reduces the yield of the desired product (Huang et al., 1992). The
stability of the primer is determined by its false priming efficiency;
ideally it should have a stable 5' end and an unstable 3' end. If the
primer has a stable 3' end, it will bind to a site which is
complementary to the sequence rather than the target site and may lead
to secondary bands. It is adequate to have G or C in last 3 bases at 5'
termini for the efficient binding of the primer to the target site. This
GC clamp reduces spurious secondary bands (Sheffield et al., 1989).
GC Content, Tm and Ta are Interrelated
GC
content, melting temperature and annealing temperature are strictly
dependent on one another (Rychlik et al., 1990). GC% is an important
characteristic of DNA and provides information about the strength of
annealing. A GC of 50-60% is recommended. The value recommended by
Dieffenbach (1993) is 45-55%.
Secondary Structure
An
important factor to consider when designing a primer is the presence of
secondary structures. This greatly reduces the number of primer
molecules available for bonding in the reaction. The presence of hairpin
loops reduces the efficiency by limiting the ability to bind to the
target site (Singh et al., 2000). It is well established that under a
given set of conditions, the relative stability of a DNA duplex
structure depends on its nucleotide sequence (Cantor and Schimmel,
1980). More specifically, the stability of a DNA duplex appears to
depend primarily on the identity of the nearest-neighbor nucleotides.
The overall stability and the melting behavior of any DNA duplex
structure can be predicted from its primary sequence if the relative
stability (DG0) and the temperature dependent behavior (DH0, DCp0) of
each DNA's nearest-neighbor interaction is known (Marky and Breslauer,
1982). Tinoco et al., (1971, 1973) and Uhlenbeck et al., (1973) have
predicted stability and melting behavior of RNA molecules for which they
and others have determined the appropriate thermodynamic data. But, to
the best of our knowledge, no experimental data is available to support
the prediction of the thermodynamic properties of hairpin structures, an
important factor to consider when designing a primer. Single stranded
nucleic acid sequences may have secondary structures due to the presence
of complementary sequences within the primer length e.g. hairpin loops
and primer-dimer structures. We have recently shown experimentally that
hairpin loops, if present, can greatly reduce the efficiency of the
reaction by limiting primer availability and the ability to bind to the
target site (Singh et al., 2000). The effect of primer-template
mismatches on the PCR has been studied earlier in a Human
Immunodeficiency Virus (HIV) model (Kwok et al., 1990). Studies have
also been performed for the characterization of hairpins (Marky et al.,
1983, 1985), cruciforms (Marky et al., 1985), bulge and interior loops
(Patel et al., 1982 , 1983).
Dimers and False Priming Cause Misleading Results
Annealing
between the 3' end of one primer molecule and the 5' end of another
primer molecule and subsequent extension results in a sharp background
product known as primer dimer. Its subsequent amplification product can
compete with the amplification of the larger target. If the primer binds
anywhere else than the target site, the amplification specificity is
reduced significantly (Breslauer et al., 1986). This leads to a weak
output or a smear. This occurs again when some bases at 3' end of the
primer bind to target sequence and achieve favorable chances of
extension (Chou et al., 1992).
To minimize the possibility
of dimers and false priming, PCR is generally performed at high
temperature (>50°C), but primers may be extended non-specifically
prior to thermal cycling if the sample is completely mixed at room
temperature (RT) (Hung et al., 1990). To prevent this occurring the Hot
Start? protocol is recommended (Erlich et al., 1991). All reagents
except one (usually the Taq DNA Polymerase) are mixed at RT. The sample
is denatured completely for 3 to 7 min, kept on ice for 2 min and then
Taq DNA polymerase is added to start the reaction.
Know Your Product Before Amplification
PCR
product length is directly proportional to inefficiency of
amplification (Wu et al., 1991). Primers should be designed so that only
small regions of DNA (150-1000 bp) can be amplified from fixed tissue
samples or purified plasmid or genomic DNA. The product is ideal for
probe hybridization studies (Schowalter and Sommer, 1989). For reverse
transcriptase polymerase chain reaction (RT-PCR) as described by
Kawasaki (1990b), primers should only be designed in exons taking care
that both primers should be on different exons of mRNA to avoid spurious
product amplified from contaminating DNA in the mRNA preparation, if
any. If the desired restriciton enzyme site is not available within the
amplified product, it may be incorporated within the primer (Ponce and
Michal, 1989; Jung et al., 1990).
Mismatch to Improve Sensitivity and Specificity
There
is a good and a bad aspect to mismatches in primers. Single mismatches
at or near the terminal 3' nucleotide of a primer are known to affect
both oligonucleotide stability and efficiency of polymerase reaction;
mismatches in the primer at or near the 3' terminal end affect PCR more
dramatically than mismatches at other positions (Petruska et al., 1988).
Generally, mismatches at the 3' end terminal nucleotide reduce or
inhibit efficiency of amplification (Kwok et al., 1990; Liu et al.,
1994) but studies have shown that a mismatch 3-4 bases upstream of the
3' end of a primer used for the ARMS study actually increases
specificity. A mismatch may therefore be deliberately created while
designing a primer for ARMS PCR (Old, 1991).
Nested PCR
Nested
PCR is often successful in reducing unwanted products while
dramatically increasing sensitivity (Albert and Fenyo, 1990). It is used
when the actual quantity of target DNA is very low or when the target
DNA is impure. Nested PCR reduces background amplification thereby
enhancing target detection. The technique is especially helpful for
amplification of low copy number targets (<100 molecules) and while
doing quantitative PCR (Haff, 1994). The process involves one PCR
reaction followed by the next PCR extension which amplifies the first
PCR product. Two sets of primers are designed. PCR is first carried out
using outer primers and subsequently with inner primers positioned
within the product obtained in the first extension. It is also possible
to perform a nested PCR reaction in a single sample without dilution
between the two PCR reactions (Erlich et al., 1991). When designing
primers for nested PCR, care must be taken to eliminate potential primer
dimers and cross dimers within and between inner and outer primer sets.
Multiplex PCR
This technique
involves co-amplification of two or more target sequences within a
single sample (Chamberlain et al., 1991; Edwards and Gibbs, 1994). A
unique pair of primers for each target is preferred but primers can be
designed so that a single primer can amplify different regions with two
or more counterparts (Varawalla et al., 1991a; 1991b). When designing
primers for multiplex PCR systems, the basic rule is to have similar
annealing temperatures and similar GC% of the primers (Nicodeme and
Steyaret, 1997). Product length should also be taken into consideration
when designing primers so that they can be effectively separated and
studied by electrophoresis. Multiplex PCR may be used for detection of
genetic disorders (Old et al., 1990; Shuber et al., 1991). Zhu and Clark
(1996) demonstrated that addition of competitive primers may
dramatically increase PCR amplification efficiency.
Universal Primers
Molecular
biologists are well aware of the exponential increase in the DNA
sequence databanks with several thousands bases added every day. Many
genes of varied importance have been sequenced in several species.
However, the scientific community may require information on such genes
in other species, which are used as experimental models. Researchers are
often forced to re-sequence genes for new species in order to conduct
expression level or other PCR related studies of the gene (Kain et al.,
1991) Bulat et al. (1992) demonstrated the application of universal
primers. Universal primers facilitated the rapid study of novel genes in
new models. Rose et al., (1998) demonstrated a new primer design
strategy for PCR amplification of unknown targets that are related to
multiple-aligned protein sequences. Universal primers are designed in
the conserved region of the sequences (Singh et al., 2000). Universal
primers should be designed from amino-acid sequences in the regions of
lowest degeneracy using a multiple sequence alignment (Nomenclature
Committee of the International Union of Biochemistry, 1985). Universal
primers were used for differential display of eukaryotic mRNAs by PCR
(Liang and Pardee, 1992). A universal primer set for detection of
parasitic genomes was also designed using Dirofilaria immitis as a test
sample (Nagano et al., 1996), whereas Venta et al. (1996) designed
gene-specific universal primers for the canine genome. These were used
for developing a genetic map of dog-based markers. Universal primers may
be used for amplification as well as sequencing in one reaction (Berg
and Olaisen, 1994)
Degenerate Primers
Degeneracy
in primer sequence should also be taken into consideration. In fact
researchers pursuing the cloning of novel genes often face the problem
that only a partial protein sequence is known (Bindon et al., 1998). In
these circumstances several procedures can be used, some involve
universal primers or reverse translation of the protein sequence into a
DNA sequence and the design of primers from this sequence. However, due
to redundancy in the genetic code, primer design must account for the
ambiguous DNA bases and has to be designed in the region of lowest
degeneracy (Kwok et al., 1994). Le Guyader et al. (1996) evaluated the
effect of degenerate primers in the detection of caliciviruses. Mack and
Sninsky (1988) demonstrated the selection of conserved regions encoded
by amino acids with minimal codon degeneracy in order to reduce
mismatch. Degenerate primers based on the amino acid sequence of
conserved regions were also used to search for members of a gene family
(Wilks et al., 1989), homologous genes from different species (Kopin et
al., 1990) and related viruses (Mack and Sninsky, 1988; Manos et al.,
1989; Shih et al., 1989). A computer program was also developed
specifically for degenerate primer design (Chen and Zhu, 1997).
Software in Primer Design
Most
molecular biological applications are aided by software. The use of
software in biological applications has given a new dimension to the
field of bioinformatics. Many different programs for the design of
primers are now available. Freeware software is available on the
internet and many universities have established servers where a user can
log on and perform free analyses of proteins and nucleic acid
sequences. There are number of simple stand-alone programs as well as
complex integrated networked versions of the commercial software
available. These software packages may be for complete DNA and protein
analysis, secondary structure predictions, primer design, molecular
modeling, development of cloning strategies, plasmid drawing or
restriction enzyme analyses etc. Companies engaged in biosoftware
development include: Alkami Biosystems, Molecular Biology Insights,
PREMIER Biosoft International, IntelliGenetics Inc., Hitachi Inc., DNA
Star, Advanced American Biotechnology and Imaging.
Some
scientists have also developed algorithms and computer programs for
various purposes of primer design (Rychlik and Rhoades, 1989; Lowe et
al., 1990; Lucas et al., 1991; O'Hara and Venezia, 1991; Tamura et al.,
1991; Makarova et al., 1992; Osborne, 1992; Plasterer, 1997; Sze et al.,
1998).
Conclusion
Biological science,
and in particular biotechnology, is rapidly changing and cannot achieve
its objectives without the help of computer technology and information
technology tools. PCR primer design concepts are not new. However
constant upgrading and updating of the strategies and methods are
essential to maintain rapid and efficient progress. Computational
strategies in biotechnology are of particular importance. The algorithms
relevant to the efficient design of primers should be modified taking
into account experimental data.
-
产品技术
