A primer is a short single-stranded nucleic acid used by all living organisms in the initiation of DNA synthesis. A synthetic primer may also be referred to as an oligo, short for oligonucleotide. DNA polymerase (responsible for DNA replication) enzymes are only capable of adding nucleotides to the 3’-end of an existing nucleic acid, requiring a primer be bound to the template before DNA polymerase can begin a complementary strand.[1] DNA polymerase adds nucleotides after binding to the RNA primer and synthesizes the whole strand. Later, the RNA strands must be removed accurately and replace them with DNA nucleotides forming a gap region known as a nick that is filled in using an enzyme called ligase.[2] The removal process of the RNA primer requires several enzymes, such as Fen1, Lig1, and others that work in coordination with DNA polymerase, to ensure the removal of the RNA nucleotides and the addition of DNA nucleotides. Living organisms use solely RNA primers, while laboratory techniques in biochemistry and molecular biology that require in vitro DNA synthesis (such as DNA sequencing and polymerase chain reaction) usually use DNA primers, since they are more temperature stable. Primers can be designed in laboratory for specific reactions such as polymerase chain reaction (PCR). When designing PCR primers, there are specific measures that must be taken into consideration, like the melting temperature of the primers and the annealing temperature of the reaction itself. Moreover, the DNA binding sequence of the primer in vitro has to be specifically chosen, which is done using a method called basic local alignment search tool (BLAST) that scans the DNA and finds specific and unique regions for the primer to bind.
RNA primers in vivo
Further information:
replication fork, requiring only an initial RNA primer to begin synthesis. In the lagging strand, the template DNA runs in the 5′→3′ direction. Since DNA polymerase cannot add bases in the 3′→5′ direction complementary to the template strand, DNA is synthesized ‘backward’ in short fragments moving away from the replication fork, known as Okazaki fragments. Unlike in the leading strand, this method results in the repeated starting and stopping of DNA synthesis, requiring multiple RNA primers. Along the DNA template, primase intersperses RNA primers that DNA polymerase uses to synthesize DNA from in the 5′→3′ direction.[1]
Another example of primers being used to enable DNA synthesis is reverse transcription. Reverse transcriptase is an enzyme that uses a template strand of RNA to synthesize a complementary strand of DNA. The DNA polymerase component of reverse transcriptase requires an existing 3' end to begin synthesis.[1]
Primer removal
After the insertion of
deoxyribonucleotides that fill the gaps where the RNA primer was present. DNA ligase then joins the fragmented strands together, completing the synthesis of the lagging strand.[1]
In prokaryotes, DNA polymerase I synthesizes the Okazaki fragment until it reaches the previous RNA primer. Then the enzyme simultaneously acts as a
deoxyribonucleotides. Later, a gap between the strands is formed called a nick, which is sealed using a DNA ligase
.
In eukaryotes the removal of RNA primers in the
deoxyribonucleotides using an enzyme known as ligase1, through a process called ligation
anneal to a specific site on the template DNA. In solution, the primer spontaneously hybridizes with the template through Watson-Crick base pairing before being extended by DNA polymerase. The ability to create and customize synthetic primers has proven an invaluable tool necessary to a variety of molecular biological approaches involving the analysis of DNA. Both the Sanger chain termination method and the “Next-Gen” method of DNA sequencing require primers to initiate the reaction.[1]
PCR primer design
The polymerase chain reaction (PCR) uses a pair of custom primers to direct DNA elongation toward each other at opposite ends of the sequence being amplified. These primers are typically between 18 and 24 bases in length and must code for only the specific upstream and downstream sites of the sequence being amplified. A primer that can bind to multiple regions along the DNA will amplify them all, eliminating the purpose of PCR.[1]
A few criteria must be brought into consideration when designing a pair of PCR primers. Pairs of primers should have similar melting temperatures since annealing during PCR occurs for both strands simultaneously, and this shared melting temperature must not be either too much higher or lower than the reaction's
annealing temperature
. A primer with a Tm (melting temperature) too much higher than the reaction's annealing temperature may mishybridize and extend at an incorrect location along the DNA sequence. A Tm significantly lower than the annealing temperature may fail to anneal and extend at all.
Additionally, primer sequences need to be chosen to uniquely select for a region of DNA, avoiding the possibility of hybridization to a similar sequence nearby. A commonly used method for selecting a primer site is
Beacon Designer. Computer simulations of theoretical PCR results (Electronic PCR) may be performed to assist in primer design by giving melting and annealing temperatures, etc.[7]
As of 2014, many online tools are freely available for primer design, some of which focus on specific applications of PCR. Primers with high specificity for a subset of DNA templates in the presence of many similar variants can be designed using by some software (e.g. DECIPHER[8]) or be developed independently for a specific group of animals.[9]
Selecting a specific region of DNA for primer binding requires some additional considerations. Regions high in mononucleotide and dinucleotide repeats should be avoided, as loop formation can occur and contribute to mishybridization. Primers should not easily anneal with other primers in the mixture; this phenomenon can lead to the production of 'primer dimer' products contaminating the end solution. Primers should also not anneal strongly to themselves, as internal hairpins and loops could hinder the annealing with the template DNA.
When designing primers, additional nucleotide bases can be added to the back ends of each primer, resulting in a customized cap sequence on each end of the amplified region. One application for this practice is for use in TA cloning, a special subcloning technique similar to PCR, where efficiency can be increased by adding AG tails to the 5′ and the 3′ ends.[10]
Degenerate primers
Main article:
Degenerate bases
Some situations may call for the use of degenerate primers. These are mixtures of primers that are similar, but not identical. These may be convenient when amplifying the same
degenerate bases
. Degenerate primers may not perfectly hybridize with a target sequence, which can greatly reduce the specificity of the PCR amplification.
Degenerate primers are widely used and extremely useful in the field of microbial ecology. They allow for the amplification of genes from thus far uncultivated microorganisms or allow the recovery of genes from organisms where genomic information is not available. Usually, degenerate primers are designed by aligning gene sequencing found in GenBank. Differences among sequences are accounted for by using IUPAC degeneracies for individual bases. PCR primers are then synthesized as a mixture of primers corresponding to all permutations of the codon sequence.
^Adenosine added on the primer 50 end improved TA cloning efficiency of polymerase chain reaction products, Ri-He Peng, Ai-Sheng Xiong, Jin-ge Liu, Fang Xu, Cai Bin, Hong Zhu, Quan-Hong Yao