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

PCR
Primer design and PCR parameter calculation using specialized software

With some experience in Setting up PCR, you can use commercial software and online resources to save time when designing primers. Similarly, other PCR-related calculations can be performed. In particular, the MOLBIOL.RU website is a professional online community for Russian-speaking molecular biologists, geneticists, biochemists, biophysicists, and bioengineers (http://www.molbiol.ru). A specialized form available on the site helps calculate PCR parameters, although the site does not feature a Primer Design program.

Primers for PCR Amplification of a specific DNA sequence can be calculated (designed) using other online resources. Specifically, you can use the Primer3 program (http://frodo.wi.mit.edu) or alternatives.

The following practical exercise introduces web portals designed for PCR (Polymerase Chain Reaction) calculations, providing hands-on experience with these software tools.

Procedure

1. Open the Primer3 program website (http://frodo.wi.mit.edu). Enter The nucleotide sequence of the NADH dehydrogenase Gene from the common viper into the designated text box (source: http://www.pubmed.com).

2. Click "Pick Primers", and the program will generate several pairs of primers. The resulting PCR product, or amplicon, will be approximately 200 bp in length. This is convenient for DNA detection, but unsuitable for projects requiring a full-length gene or a specific gene segment.

3. Analyze the generated primer pairs and select the one you consider most appropriate from the default suggestions.

4. Specify the length of the DNA region to be amplified, for example, 851–1000 bp, and design the primers. Determine how many NUCLEOTIDES the amplicon will lose.

5. Specify one of the primers, for example, the ND2 For primer from Practical Work 6.2, and calculate the second primer. To do this, paste the primer sequence into the appropriate text box and click "Pick Left Primer".

6. Repeat these steps with the ND2 Rev primer, and then with your own primers previously designed "manually" (Practical Work 6.1).

7. Set additional requirements for the primers and run the calculation.

8. Open the homepage of MOLBIOL.RU (http://www.molbiol.ru). On the main page, find the "Calculations" section and select "PCR Parameter Calculation".

9. By entering varying parameters into the designated boxes—such as PCR product length, molar amounts of primers, Taq polymerase, and others—calculate the number of nucleotides consumed in the reaction and the maximum potential yield of the PCR product. Determine the number of cycles required to complete the reaction assuming "ideal" doubling.

10. Read the accompanying notes on the calculations. Please note that the calculations are based on ideal conditions using 0.5 µg of human genomic DNA AS A template. These data are for estimation purposes only.