Molecular Biology: A Practical Guide - Velikov V.A. 2013

PCR
Primer Design

Primers are synthetic oligonucleotides consisting of 16-30 bases. They are complementary to DNA regions flanking the target sequence. A primer acts as an essential starting component required for DNA polymerase activity: the enzyme attaches NUCLEOTIDES complementary to the template to its 3’-OH end.

The primer binding to the 5’ end of the Gene is called the forward primer (For), while the one binding to the 3’ end is called the reverse primer (Rev). Nucleotide sequence Databases typically provide only a single DNA strand—the sense strand, which is transcribed into mRNA. The forward primer is designed based on this strand, meaning it initiates the synthesis of this exact strand. The reverse primer is designed for the complementary strand, yet it is also written in the 5’→3’ direction.

In the practical exercise below, you are required to manually design primers for the Amplification of the NADH dehydrogenase gene in Nikolsky's viper (Vipera nikolskii) without the aid of specialized software, and to determine the PCR cycling conditions.

Procedure

1. Retrieve information on the Introduction/19.html">Primary Structure of the NADH dehydrogenase gene in the common viper, which will serve as a reference for designing primers for PCR amplification of this gene in other viper species. To do this, access the GenBank database via the US National Library of Medicine website (http://www.pubmed.com).

2. Enter the Organism's Latin name and the gene of interest (Vipera berus NADH dehydrogenase).

3. Select the “Nucleotide” tab and press “Enter”.

4. Copy the information into a file. An example of one of the sequences is shown in Fig. 1.

5. Design the forward and reverse PCR primers. For the forward primer, simply select a short segment of the gene near its 5’ end with an optimal length of approximately 20 nucleotides. Designing the reverse primer requires reconstructing the complementary DNA strand. All nucleotide sequences must be written in the 5’→3’ direction ^a.

6. Calculate the annealing Temperature for your primers and set up the PCR thermal cycling protocol.

Class="center">Fig. 1. NADH dehydrogenase gene of the common viper V. berus

Primer Design Guidelines

- Primer length should be 16-25(30) nucleotides.

- GC content should be within the range of 50-60%.

- The difference in annealing temperature between the two primers should not exceed 6°C.

- Primers must not be self-complementary or cross-complementary.

- Nucleotides at the 3’ end of the primer must be strictly complementary to the template (mismatches are permissible at the 5’ end of long primers ^b).

Calculating the Primer Annealing Temperature

Numerous software programs and algorithms exist for precise calculation of the optimal temperature. A simplified calculation can be performed using the following formulas:

Notes

^a Do not forget about the antiparallel Nature of DNA strands when designing the reverse primer. If it has the correct nucleotide composition but the wrong polarity, no PCR product will be generated. Such a primer (parallel) will fail to anneal to the template, making The formation of an oligo-/polynucleotide hybrid complex impossible.

Oligonucleotides of any composition can be custom-synthesized by specialized biotechnology companies. Upon request, a fluorescent label can be incorporated into the sequence. Additional nucleotides can also be added to the 5’ end during ordering, such as a recognition site for a specific restriction enzyme. The company can likewise sequence the resulting PCR product, as the sequencing primer is already available.