CHAPTER 6 Primer Design

CHAPTER 6: Primer Design 47 Introduction Primers are designed to amplify a specific DNA sequence from either genomic or plasmid template DNA. For successful amplification of DNA sequences in PCR, primers need to fulfill several basic requirements: 1. The 3��� portion of each primer (about 15-20 bases) must be complementary to the DNA template. 2. The 3��� portions of primers used in PCR should not be complementary to each other, because this facilitates the formation of primer-dimers. 3. An additional sequence of bases that is noncomplementary to the template DNA may be added at the 5��� end of a primer. Extra bases are often added to primers to incorporate restriction sites into the PCR product. Restriction sites at each end of the PCR product enable future manipulations of the PCR product, such as its introduction into plasmids for gene expression. Primer Design Each PCR requires unique primers. The nucleotide sequence of primers depends on the nucleotide sequence of the template DNA. The objective of this lab project is to amplify the ArsR gene from K12 E. coli genomic DNA using primers complementary to this gene, thus you must first obtain the DNA sequence of this gene. DNA sequences may be found in the NCBI (National Center for Biotechnology) nucleotide database at www.ncbi.nlm.nih.gov. Each sequence that has been deposited in this database has its own ���ID��� referred to as the accession number. If you know the accession number for a DNA sequence, you may use it to search for the desired gene, or alternatively, you may type in the name of the gene and species. The database displays the requested DNA sequence and relevant data (see website or pages 58-59). In the display you will find information on the authors and journal where the DNA