Primers

A primer must be synthesized by an enzyme called primase, which is a type of RNA polymerase, before DNA replication can occur. The synthesis of a primer is necessary because the enzymes that synthesize DNA, which are called DNA polymerases, can only attach new DNA nucleotides to an existing strand of nucleotides.

A primer is a short strand of RNA or DNA (generally about 18-22 bases) that serves as a starting point for DNA synthesis. It is required for DNA replication because the enzymes that catalyze this process, DNA polymerases, can only add new nucleotides to an existing strand of DNA. The polymerase starts replication at the 3'-end of the primer, and copies the opposite strand.

PCR primer design  ---some are forward and some rearward. i.e. 3' to 5' or 5' to 3'.  usually 3' to 5' and design proceeds by creating or designing the reverse nucleotides. Best to end with a C-G bond as they have triple H bonds. The forward primer which is antisense to the bottom of the twp strands and will end up identical to the top strand. Length of oligo 18-22 but should end on a C-G. We then have to take the reverse complement, see video "Primer Design..."   You do need forward and reverse primers. Sticky ends may add up to 36-44 bp but keep it under 60 for cost of making.

Pairs of primers should have similar melting temperatures since annealing in a PCR occurs for both simultaneously. A primer with a Tm (melting temperature) significantly higher than the reaction's annealing temperature may mishybridize and extend at an incorrect location along the DNA sequence, while Tm significantly lower than the annealing temperature may fail to anneal and extend at all.

Primer sequences need to be chosen to uniquely select for a region of DNA, avoiding the possibility of mishybridization to a similar sequence nearby. A commonly used method is BLAST search whereby all the possible regions to which a primer may bind can be seen. Both the nucleotide sequence as well as the primer itself can be BLAST searched. The free NCBI tool Primer-BLAST integrates primer design and BLAST search into one application, as do commercial software products such as ePrime and Beacon Designer. Computer simulations of theoretical PCR results (Electronic PCR) may be performed to assist in primer design.

Many online tools are freely available for primer design, some of which focus on specific applications of PCR. The popular tools Primer3Plus and PrimerQuest can be used to find primers matching a wide variety of specifications. Highly degenerate primers for targeting a wide variety of DNA templates can be interactively designed using GeneFISHER. Primers with high specificity for a subset of DNA templates in the presence of many similar variants can be designed using DECIPHER.

Oligonucleotide synthesis is the chemical synthesis of relatively short fragments of nucleic acids with defined chemical structure (sequence). The technique is extremely useful in current laboratory practice because it provides a rapid and inexpensive access to custom-made oligonucleotides of the desired sequence. Whereas enzymes synthesize DNA and RNA only in a 5' to 3' direction, chemical oligonucleotide synthesis does not have this limitation, although it is, most often, carried out in the opposite, 3' to 5' direction. Currently, the process is implemented as solid-phase synthesis using phosphoramidite method and phosphoramidite building blocks derived from protected 2'-deoxynucleosides (dA, dC, dG, and T), ribonucleosides (A, C, G, and U), or chemically modified nucleosides, e.g. LNA or BNA.

To obtain the desired oligonucleotide, the building blocks are sequentially coupled to the growing oligonucleotide chain in the order required by the sequence of the product (see Synthetic cycle below). The process has been fully automated since the late 1970s. Upon the completion of the chain assembly, the product is released from the solid phase to solution, deprotected, and collected. The occurrence of side reactions sets practical limits for the length of synthetic oligonucleotides (up to about 200 nucleotide residues) because the number of errors accumulates with the length of the oligonucleotide being synthesized. Products are often isolated by high-performance liquid chromatography (HPLC) to obtain the desired oligonucleotides in high purity. Typically, synthetic oligonucleotides are single-stranded DNA or RNA molecules around 15–25 bases in length

Oligonucleotides find a variety of applications in molecular biology and medicine. They are most commonly used as antisense oligonucleotides, small interfering RNA, primers for DNA sequencing and amplification, probes for detecting complementary DNA or RNA via molecular hybridization, tools for the targeted introduction of mutations and restriction sites, and for the synthesis of artificial genes.

Oligonucleotides are short DNA or RNA molecules, oligomers, that have a wide range of applications in genetic testing, research, and forensics. Commonly made in the laboratory by solid-phase chemical synthesis, these small bits of nucleic acids can be manufactured as single-stranded molecules with any user-specified sequence, and so are vital for artificial gene synthesis, polymerase chain reaction (PCR), DNA sequencing, library construction and as molecular probes. In nature, oligonucleotides are usually found as small RNA molecules that function in the regulation of gene expression (e.g. microRNA), or are degradation intermediates derived from the breakdown of larger nucleic acid molecules.

A primer is a short synthetic oligonucleotide which is used in many molecular techniques such as PCR. These primers are designed to have a sequence which is the reverse complement of a region of template or target DNA to which we wish the primer to anneal, where the polymerase starts replication at the 3'-end of the primer, and copies the opposite strand. Often, PCR is used to amplify a known sequence of DNA. Thus, the scientist chooses the sequence he or she wants to amplify, then designs and orders primers which will anneal to sequences flanking the sequence of interest. Thus PCR leads to the amplification of a particular segment of DNA.

Oligonucleotide synthesis is the method by which primers are manufactured. There are things to consider when designing a primer to ensure correct annealing to the required gene that is desired to be amplified.The optimal primer length should be between 17 and 28 bases, with base G+C composition of 50 to 60%. 3'-ends of primers should not be complementary, as otherwise primer dimers will be synthesized, also more than 3 G's or C's should be avoided in the last 5 bases at the 3' end of the primer. Pairs of primers should have similar melting temperatures since annealing in a PCR occurs for both simultaneously where Primer Melting Temperatures (Tm) between 55-65oC are preferred, otherwise self-dimerization and secondary annealing might occur. Also primer self-complementarities (ability to form 2o structures such as hairpins) should be avoided.

A number of primer design tools such as Primer Premier are available that can assist in PCR primer design, and those tools reduce the cost of experimentation. They are quite easy to use for the inexperienced user as well. Standard primers are HPLC purified and checked by CE to insure the high quality...

To begin the PCR process, the DNA polymerases (the enzymes which synthesise DNA) first need a base upon which to attach new DNA nucleotides. Primers are the strands of DNA (or RNA) that serve as this initial foundation for the DNA replication process, and they are used to demarcate the segment of the DNA template to be amplified. In the PCR process, two primers are matched to the segment of DNA. During the annealing process, one primer attaches to the top strand and the other attaches to the bottom strand at each end of the sample. Although primers composed of RNA can be used, DNA primers are more commonly seen in PCR as they are more temperature-stable, which is important during the annealing process.

Primer pairs consist of the upper and lower primers. Although two different primers may make up the primer pairs, it is important that the primers both have similar melting temperatures in order to facilitate a successful PCR reaction.

Universal Primers

Universal primers, as the name suggests, are primers that can anneal with many different types of DNA templates. Universal primers are used in many PCR reactions and relate to nucleotide sequences that are commonly found in cloning vectors and DNA molecules. For many PCR applications, universal primers are sufficient to promote a successful DNA amplification process.  See "Types of PCR Primers for more details re Specific Primers, Single Primers, Special Primers, Degenerate Primers

Definition of degenerate primers

A degenerate primer is defined as:  “A mix of oligonucleotide sequences in which some positions contain a number of possible bases, giving a population of primers with similar sequences that cover all possible nucleotide combinations for a given protein sequence” (Iserte 2013). For example:

ATCGTT[GC]AAGT[AGC]ATC

refers to a series of primers in which the seventh and twelfth nucleotides are degenerate. The amount of degeneracy is defined by the number of different primer combinations in the mix. The above example has a degeneracy value of six.

Using the International Union of Pure and Applied Chemistry (IUPAC) system for degenerate bases  we can insert a single-letter code to represent our unknown region. In the above example we would use ‘S’ in place of GC, and ‘V’ in place of AGC.

Designing degenerate primers

To design your own set of degenerate primers, follow some basic guidelines:

1)  Align multiple amino acid sequences using free online software such as EBIClustalO.

2)  Target an area approximately 200-500 base pairs in length for optimal PCR amplification.

3)  Position forward and reverse primers in more conserved regions – the less degenerate, the further apart these can be.

4)  Include between 6 and 7 amino acids in the primers, equating to ~15-20 base pairs.

5)  Try to include amino acids methionine and tryptophan, which are coded by a single codon (three-letter nucleotide code), and avoid amino acids leucine, serine and arginine, which can each be coded by six codon combinations (Table 2).

6)  For subsequent cloning procedures and to increase primer length (and therefore annealing temperature) add a 5’ tail (6-9 base pairs) containing a restriction enzyme site.

7)  If there is complete degeneracy (no matches among any given species), consider using the base inosine (structurally similar to guanine) as it can pair with any of the four bases, although it will bind to cytosine preferentially. Alternatively, insert N for aNy base to ensure equimolar concentrations of each base at that position in your primer mix.

8)  Avoid degeneracy at the 3’ terminus (this would not be a good place to insert inosine).

Depending on the goal, the play-off between primer specificity and efficiency can be modified by altering the degeneracy of the primer. For example, the more degenerate the primers, the less specific annealing will be; however, decreased degeneracy will allow more potential to identify unknown variants.

Primers

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