Electronic Thesis and Dissertation Repository

Thesis Format

Monograph

Degree

Master of Science

Program

Computer Science

Supervisor

Ilie, Lucian

Abstract

The Polymerase Chain Reaction (PCR) technology is widely used to create DNA copies. It has impacted many diverse fields including genetics, forensics, molecular paleontology, medical applications and environmental microbiology.

The main object in PCR is a primer, a short single strand of DNA, about 18-25 bases long, that serves as the starting point of DNA synthesis. Primers are essential for DNA replication because the enzymes that catalyze this process, DNA polymerases, can only add new nucleotides to an existing strand of DNA. The PCR starts at the 3’-end of the primer and copies the opposite strand.Designing good primers is essential for a successful PCR process. Primer3 is one of the main available programs that produce primers for a variety of applications. Other programs, such as PrimerBLAST, QuantPrime and PRIMEGENS, depend on Primer3 for generating candidate primers and then subject them to specificity checking. The primers produced must not have high similarity with sequences other than their targets, as this would enable unwanted cross-hybridization. We will discuss these techniques, focusing on the parameters required for designing good primers and the pros and cons of some of the above mentioned programs. We introduce a new program, bestPrimer, that produces better primers as shown by extensive comparison with Primer3.

Summary for Lay Audience

DNA samples needs to be amplified into multiple copies for research purposes. This can be done using a technology called Polymerase Chain Reaction (PCR), which has impacted many diverse fields including genetics, forensics, molecular paleontology, medical applications and environmental microbiology. The process requires a small fragment of DNA called a primer, which acts as the starting point of the DNA replication.Primers are essential for the PCR reaction to be successful, because the replication can only take place from one of the specific ends of the primer.

There are several programs available for designing primers. Primer3 is one popular program for primer design that considers several factors for designing primers. The primers produced must not have high similarity with sequences other than their targets, as this would enable unwanted cross-hybridization. We will discuss these techniques, focusing on the parameters required for designing good primers and introduce a new program, bestPrimer, that produces better primers and runs faster as shown by extensive comparison with Primer3.

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