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Designing PCR Primers to Amplify Target Genes

Madison Pascual Munar has a Doctorate degree in Molecular Biotechnology.

Primers were designed based on the ORF sequence (A1) from a metagenome fragment to identify its possible origin (H1, H2, H3).

Primers were designed based on the ORF sequence (A1) from a metagenome fragment to identify its possible origin (H1, H2, H3).

Oligonucleotide PCR Primers

Primers are used to amplify a gene of interest into millions of copies using Polymerase Chain Reaction (PCR) for downstream analyses such as DNA fingerprinting, paternity testing, genotyping, disease detection, pathogen identification, genetic engineering, and DNA sequencing.

PCR Primers are short single-strand synthetic nucleotide about 18-24 bases that is complementary to the ends of the target DNA fragment to be amplified using PCR. Primers for gene expression may extend to more than 24 bases due to the addition of other sequences such as restriction enzyme sites and peptide tags (His tag, HA tag, FLAG tag, Myc tag). The sequence of PCR primers is responsible for the high fidelity and specificity of PCR analysis.

The synthesis of complementary DNA strand during PCR begins when the DNA sample is denatured at a certain temperature (94°C-98°C). This will be followed immediately by the annealing temperature (48°C-72°C), where primers starts to bind to the complementary bases on the target DNA sequence. During the annealing stage, forward and reverse primers will attach to the ends of the target gene to initiate the addition of new nucleotide bases facilitated by the DNA polymerase.

designing-pcr-primers-to-amplify-target-genes

Priming the polymerase chain reaction

As its name suggests, primers "prime" or initiate the synthesis of the complementary DNA strand by guiding the DNA polymerase on the target DNA sequence. DNA polymerase can only add new nucleotide bases on the free hydroxyl group (OH) found at the 3' end of the primer. Thus, primers are always designed with special consideration on the DNA sequence direction. 5' end and 3' end refers to the position of the carbon molecule in the pentose phosphate backbone in a DNA strand.

The 5' end of a nucleotide base has a free phosphate group which will form a phosphodiester bond with the free hydroxyl group at the 3' end of the existing nucleotide base with the action of the DNA polymerase. The phosphodiester bond joins two nucleotide bases during complementary DNA synthesis.

Nucleotide sequence showing the 5' end where the phosphate group (PO4) is attached and the 3' end with a free hydroxyl group (OH).

Nucleotide sequence showing the 5' end where the phosphate group (PO4) is attached and the 3' end with a free hydroxyl group (OH).

Targeting Protein-Coding Genes

If protein-coding genes are involved, one will look for the start codon (ATG) as a reference to the DNA direction. This can be done using an open source bioinformatics tool like ORFfinder in NCBI which can easily detect open reading frames (ORF) or DNA sequences starting with the bases ATG (start codon) and ends with any of the following sequences TAG, TAA, TGA (stop codon).

DNA sequence showing an ORF sequence.

DNA sequence showing an ORF sequence.

Two Simple Steps in Designing PCR Primers

There are two simple steps in designing primers, (1) identify the region to be amplified, (2) check for the properties of the generated primer sequence such as primer length, melting temperature (Tm), primer-dimerization, and stem-loop formation. OligoAnalyzer Tool is an open source bioinformatics software which can be used to analyze the properties of the generated primer sequences.

After identifying the region of the DNA to be amplified, the upstream bases will serve as the binding site for the forward primer while the end of the sequence will be the binding site for the reverse primer.

The sequence of the forward primer is just the same as the sequence of the coding strand. The forward primer will initiate the synthesis of the coding strand from the 5' to 3' direction.

Synthesis of the complementary bases occurs only from the existing hydroxyl group at the 3' end of the primer sequence, thus, the reverse primer sequence should also be designed on the 5' to 3' orientation. This only means that we need to get the reverse complement of the coding strand at the end of the target gene to get the primer sequence which will run from the 5' to 3' direction. The reverse primer initiates the synthesis of the template strand.

designing-pcr-primers-to-amplify-target-genes

Generating Reverse Complement Sequence

On-line tools are readily available to generate reverse complement sequence for the reverse primer sequence. The Sequence Manipulation Suite can be used to generate the reverse complement of the coding sequence for the reverse primer sequence.

designing-pcr-primers-to-amplify-target-genes

Checking Primer Consensus Sequence with the Target Gene Using CodonCode Aligner

CodonCode Aligner can be used to visualize the primer binding sites on the target gene using the sequence of the designed primers. CodonCode Aligner is also useful when assembling the quality trimmed forward and reverse sequences of your DNA samples.

Consensus sequence showing the primer binding sites on the target gene.

Consensus sequence showing the primer binding sites on the target gene.

Amplification of Target Gene Using the Designed Primers

The forward and reverse primers will bind with the complementary strand and guide the DNA polymerase to synthesize or add complementary bases on the free OH group at the 3' end of the primer sequence. After one cycle of PCR, the copy of the target DNA sequence will be doubled and will serve as the template for the succeeding cycles.

designing-pcr-primers-to-amplify-target-genes
designing-pcr-primers-to-amplify-target-genes

Important Points to Consider in Designing Primers

Forward and Reverse Primer Should Have Close Melting Temperature (Tm)

Make sure the forward and reverse primer melting temperature is similar or close to each other so they can work at the same annealing temperature during PCR. At least five degrees difference between the forward and reverse primer sequence melting temperature is ideal. Melting temperature should be within the range 65°C-75°C. GC content influences the melting temperature, the higher the GC content the higher the melting temperature. Find primer sequence with 40%-60% GC content.

Properties of the Forward Primer Sequence.

Properties of the Forward Primer Sequence.

Properties of the Reverse Primer Sequence.

Properties of the Reverse Primer Sequence.

Check for Primer Dimerization

Primer dimerization occurs when the forward or reverse primers self-anneal because of complementary bases rather than binding to the target DNA sequence. This may be prevented by increasing the annealing temperature on the PCR thermal cycling conditions and using less volume of primers on the PCR mix. Avoid choosing regions rich with repeating GC and AT bases because this may cause primer dimerization. Also check possible complementary bases on the 3' ends of the forward and reverse primer sequence to avoid primer-primer dimerization.

Formation of Primer Dimers.

Formation of Primer Dimers.

Check for Stem-Loop Formation

Stem-loop formation occurs when the nucleotide sequence in the primer can self-anneal due to the presence of complementary bases which can form secondary structures resembling a loop. Choose primer sequence with little to moderate chance of forming secondary structures.

Stem-loop formation as secondary structures.

Stem-loop formation as secondary structures.

Check for Homologous Sequences using BLAST

Once you have the forward and reverse primer sequences, you can do a simple BLAST search to determine homologous sequences in NCBI. This may determine whether the primer is highly specific to a specific gene or organism or may capture other homologous genes from different organisms.

Optimize Annealing Temperature

One of the most important factors in dealing with PCR primers is the optimization of the annealing temperature on the PCR thermal cycling condition. The annealing temperature induces the attachment of the primer on the target sequence. Too high annealing temperature may lessen the chances of primer attachment on the target DNA sequence. Also, avoid very low annealing temperature because this may cause mis-priming to non-target DNA sequences. Often this is evident as several non-specific bands appearing after gel electrophoresis. The highest temperature where primers are observed to anneal and provide the expected amplicon size should be used to ensure specificity to the target gene.

There you go, now try and design your own primers!

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References