Primers are an essential component in the amplification of DNA both in vivo and in vitro. In vivo, the enzyme, DNA polymerase requires a primer for the initiation of DNA replication. In vitro, primers are mostly used for the initiation of polymerase chain reaction (PCR). Some other techniques including sequencing, cloning, site-directed mutagenesis, etc. require primers. Hence, the designing of primers for in vitro techniques becomes quite simple but, a challenging process for molecular biologists. Therefore, the basic rules for the primer designing for both PCR and sequencing are discussed in this article.
Key Areas Covered
1. What is a Primer
– Definition, Types, Role
2. How Do Primers Work in a PCR
– Features of DNA, Process of PCR
3. How to Make Primers for PCR
– Basic Rules to Design PCR Primers
4. How to Design a Sequencing Primer
– Features of Sequencing Primers
Key Terms: DNA Synthesis, Forward Primers, Length, Melting Temperature, PCR, Reverse Primers, Sequencing Primers
What is a Primer
A primer is a short strand of DNA or RNA that serves as the starting point for DNA synthesis. The enzymes that catalyze DNA replication are capable of adding nucleotides to an existing 3′ end. Hence, the primer lays the foundation for DNA synthesis by serving as a prime. RNA primers are used inside the cell for the initiation of DNA replication by DNA polymerase. However, synthetic DNA primers can be used for the amplification of DNA, mainly by PCR and other techniques. Two types of primers are used in PCR, and they are known as forward and reverse primers. During PCR, millions of copies of the desired DNA fragment can be produced by flanking that particular DNA sequence in the genomic DNA by forward and reverse primers. Forward and reverse primer that flank a particular DNA sequence are shown in figure 1.
How Do Primers Work in PCR
DNA is a molecule having two strands that are held together. The base pair pattern is complementary to each in both strands. The two strands are held together by the hydrogen bonds between complementary nitrogen bases. In addition, each strand has its own directionality. One strand has 5’to 3′ directionality while the other has 3′ to 5′ directionality. Hence, the two strands are antiparallel. The strand with 5′ to 3′ direction is known as the sense strand while the strand with 3′ to 5′ direction is known as the antisense strand. Each two strands should be synthesized individually during PCR.
The three steps of PCR are denaturation, annealing, and elongation. In denaturation, the two strands of DNA are separated by breaking the hydrogen bonds by heating to 95 °C. Forward primer binds to the sense strand while the reverse primer binds to the antisense strand. The annealing of primers occurs when the temperature drops from 95 °C to 50-60 °C. Hence, both strands can be synthesized at the same time with the help of Taq polymerase. The amplification of both sense and the antisense strands occur in the 5′ to 3′ direction. As PCR is an exponential reaction, the three steps are repeated in 25-35 cycles. Both forward and reverse primers are used in each cycle to produce around 235 copies of desired DNA fragment. The role of primers in PCR is shown in figure 2.
How to Make Primers for PCR
In order to amplify a particular DNA fragment in the genome, that particular DNA fragment should be flanked by both forward and reverse primers. Hence, both primers should be complementary to the sequences that flank the DNA fragment. The basic guidelines for the successful design of PCR primers are described below.
- The direction of both forward and reverse primer should be 5′ to 3′.
- The length of each primer should be between 18 to 25 nucleotides in length.
- The GC content of primers be between 40 and 60% and the presence of a C or G in the 3’end of the primer may promote binding.
- The melting temperature and Tm (the temperature at which half of the primer has annealed to the template) of the primer pair should be similar and above 60 °C. The maximum difference should be 5 °C.
- The 3′ end of the primer should exactly match the template DNA.
- At least 2G or C bases (GC clamp) should be present in the last 5 bases at the 3′ end of the primer. The GC clamp promotes a strong binding to the target sequence.
- Restriction sites with 5-6 nucleotides can be added to the 5’end of the primer.
- The dinucleotide repeats (ATATATAT) or repeats of the same nucleotide more than 4 times (ACCCC) should be avoided in the primer sequences. This causes mispriming.
- Intra-primer homology or the secondary structures of primers should be avoided. Inter-primer homology or complementary sequences in the forward and reverse primers should be avoided. Both conditions may form self-dimers or primer-dimers.
- The ΔG value for dimer analysis should be between 0 to −9 kcal/mole.
Many online tools are available for the easiness of the primer design such as Primer 3, Primer X, NetPrimer, DNAstrar, etc. The specificity of the designed primers can be determined by the tools such as NCBI Primer-BLAST or UCSC in-silico PCR.
How to Design a Sequencing Primer
Sequencing primers are short, DNA strands, just like PCR primers. However, PCR primers are designed for the amplification of a particular DNA fragment while sequencing primers are used to reveal the nucleotide sequence of the amplified DNA fragment by PCR. Unlike PCR primers, a single primer can be used in the sequencing, if only the target sequence is less than 500 bp in length. As an example, the forward primer of the PCR can be used in sequencing, to amplify only the sense strand. Moreover, the degree of mismatches tolerated during the sequencing reaction is higher than the PCR. Generally, PCR primers are complementary to the target sequence. However, some sequencing primers are not related to the target sequence. They are known as universal primers. Universal primers such as T7 or SP6 anneal to the vector that carries the target sequence. They can be used for both a variety of vectors and various types of DNA fragments.
Primers are used in PCR and sequencing for the initiation of the DNA synthesis. Two types of PCR primers can be identified as forward and reverse primer. Forward primers anneal to the sense strand while reverse primers anneal to the antisense strand. In sequencing, either forward or reverse primer can be used to amplify the target. During the designing of primers, many factors should be considered such as primer length, Tm, and GC content. Many online tools are available that can be used for the designing of primers for a particular sequence.
1. “Primer Design: Tips for an Efficient Process.” Genome Compiler Corporation, 3 Nov. 2015, Available here.
2. “Sequencing primers and primer design.” Sequencing primers and primer design, University of Calgary, Available here.