PCR Primer Design

Bioinformatics Sub-centre, School of Biotechnology, Devi Ahilya University, Khandwa Road, Indore 452 017 MP, India

Abstract

To make PCR a specific, efficient and cost effective tool for researchers and clinicians the most important aspect is oligonucleotide primer design. This review discusses various aspects of primer design. Advice is provided for optimal design and the role of bioinformatic tools is highlighted. The authors discuss theoretical considerations and compare computational and experimental studies.

Introduction

Bioinformatics is a newly-emerged inter-disciplinary research area spanning a range of specialties that include molecular biology, biophysics, computer science, mathematics and statistics. It makes use of scientific and technological advances in the areas of computer science, information technology and communication technology to solve complex problems in life sciences, particularly problems in biotechnology. Bioinformatics comprises of the development and application of algorithms for the analysis and interpretation of data, for the design and construction of vital databases, and for the design of experiments.
Bioinformatics is used interchangeably with the terms biocomputing and computational biology. However, biocomputing is more correctly defined as the systematic development and application of computing systems and computational solution techniques to model biological phenomena. Polymerase chain reaction (PCR) is one such phenomenon. PCR is used for the in vitro amplification of DNA at the logarithmic scale. Various components of the PCR reaction such as Taq DNA polymerase, assay buffer, deoxynucleoside triphosphates, stabilizing agents, and primers make it possible for the DNA template to be amplified sufficiently in vitro to attain detectable quantities. PCR can be used for various purposes such as the amplification of human specific DNA sequences, differentiation of species, sub-species and strains, DNA sequencing, detection of mutations, monitoring cancer therapy, detection of bacterial and viral infections, pre-determination of sex, linkage analysis using single sperm cells, ascertaining recombinant clones and studying molecular evolution. PCR is a sensitive technique and therefore highly susceptible to contamination which may result in false positivity. To make PCR a specific, efficient and cost effective tool for researchers and clinicians the most important component of the PCR is the oligonucleotide primers. Literature searches indicate that insufficient experimental work has been done in the field of bioinformatics especially in the field of nucleic acid sequence analyses. Inadequate experimental data is available (at least in the public domain) for the establishment of primer design strategies. In this review the authors aim to establish various aspects and types of PCR and primer design theory, supported by computational and experimental data.

PCR Primer Design

Selective amplification of nucleic acid molecules, that are initially present in minute quantities, provides a powerful tool for analyzing nucleic acids (Saiki et al., 1985; Mullis et al., 1987). The polymerase chain reaction is an enzymatic reaction, which follows relatively simple, predictable and well understood mathematical principles. However the scientist often relies on intuition to optimise the reaction. To make PCR an efficient and cost effective tool, some components of PCR such as Taq DNA polymerase, assay buffer, deoxynucleoside triphosphates (dNTPs), stabilizing agents (Sarkar et al., 1990), DNA Template and oligonucleotide primers must be considered in greater detail (Linz et al., 1990). Efficacy and sensitivity of PCR largely depend on the efficiency of primers (He et al., 1994). The ability for an oligonucleotide to serve as a primer for PCR is dependent on several factors including: a) the kinetics of association and dissociation of primer-template duplexes at the annealing and extension temperatures; b) duplex stability of mismatched nucleotides and their location; and c) the efficiency with which the polymerase can recognize and extend a mismatched duplex. The primers which are unique for the target sequence to be amplified should fulfill certain criteria such as primer length, GC%, annealing and melting temperature, 5" end stability, 3" end specificity etc (Dieffenbach et al., 1993).
DNA template quality or purity is not particularly significant for amplification. Provided DNA does not contain any inhibitor of Taq DNA polymerase, it can be isolated by almost any method (Murray and Thompson, 1980; Sambrook et al., 1989; Kaneko et al., 1989; Mercier et al., 1990; Kawasaki 1990a; Green et al., 1991; Keller et al., 1993; Klebe et al., 1996; Singh and Naik, 2000).