| PCR Primer Design | 点击： 作者： 来源： 时间： 2007-03-07 本站论坛
|
|  |
Most of the reviews on PCR optimization (Erlich et al., 1991; Dieffenbach 1993; Roux 1995) consider different parameters of PCR but generally do not discuss basic concepts of PCR primer design. Because of the requirements for different strategies of PCR, more effective PCR studies would be attainable by considering the basic concepts of PCR primer design.
Primer Length: a Hard Core Factor
Length of a primer is a critical parameter (Wu et al., 1991). The rule-of-thumb is to use a primer with a minimal length that ensures a denaturation temperature of 55-56°C. This greatly enhances specificity and efficiency. For general studies, primers of typically 17-34 nucleotides in length are the best. Primer >16 nucleotides in length are not generally annealed specifically to non-target DNA sequence (e.g. human DNA in an assay for bacterial infection). For example, a short primer sequence, such as a 12 bp oligonucleotide, binds to 200 specific annealing sites in the human genome. (The genome consists of 3x109 nucleotides: 3 x 109/412=200). In contrast, a 20 mer sequence is expected to randomly exist only once every 420 nucleotides and as such, has only a 1 in 400 probability of existing by chance in the human genome. Primers, 18-24 mer are accepted as best in being sequence specific if the annealing temperature of the PCR reactions is set within 5°C of the primer Td (dissociation temperature of the primer/template duplex) (Dieffenbach, 1993). Primers work exceptionally well for the sequence with least intra-strand secondary structure. This is because secondary structure impedes primer annealing and extension. Longer primers (28-35 mer) are required only to discriminate homologous genes within different species or when a perfect complementary sequence to all the template is not expected. They could also be used when extra sequence information e.g. a motif binding site, restriction endonuclease site or GC clamp is attached to 5' end. Such extensions do not generally alter annealing to the sequence specific portion of the primer (Sheffield et al., 1989).
Although the following formula is generally used for determining melting temperature (Tm):
Tm = 4 (G C) 2(A T)
Frier et al. (1986) showed that the nearest-neighbor calculation is better for calculating the melting temperature of longer primers because this also takes account of thermodynamic parameters. Using improved nearest-neighbor thermodynamic values given by John SantaLucia (1995), a good estimate of melting temperature can be obtained for oligonucleotide analysis.
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).
上一篇：建立PCR实验室的基本条件 下一篇：试管婴儿植入前遗传学诊断单细胞PCR的方法 共7页: 上一页 [1] 2 [3] [4] [5] [6] [7] 下一页 | | |
|
|
|
