Primers are short (normally 18 to 22 base pairs) DNA or RNA oligonucleotides which are used as starting points for the replication of DNA or RNA sequences. They are required for various techniques like:
• Quantification of mRNA Expression by Real-Time Polymerase chain reaction (qPCR)
• Replication of specific DNA sequences by End-Point PCR
• cDNA synthesis by reverse transcription of mRNA
• DNA or RNA sequencing including Next-Generation sequencing
• Generation of DNA probes for in-situ hybridization, Southern blotting or Northern blotting
In Vivo and In Vitro Techniques
In vivo, primers are essential for the replication of DNA during the S phase. The primase builds short RNA primers along the lagging strand which are then used as starting points for the DNA polymerase.
For in vitro techniques primers are often designed with a known sequence suitable for the amplification of the desired DNA sequence by PCR. The synthesis of oligonucleotides is a chemical reaction where nucleotides are coupled step by step in the sequence you want. Oligonucleotides are often isolated by high-performance liquid chromatography (HPLC) to enhance their purity.
When designing primers, one should keep secondary structures and the GC content in mind. Secondary structures might prevent primers from binding to its target sequence. The GC content is responsible for the temperature that is needed for the annealing of the primers. For techniques that use two different primers like all kinds of PCR, you should use primers with equal or similar melting temperatures. To keep track of those issues, you can use one of the freely available tools like Primer3Plus that help you to design your primers.
For problems with your PCR check our troubleshooting guides for Real-Time PCR or [End-Point PCR]
For cDNA synthesis, you have the choice between oligo-dT and random hexamer primers. The former ones have the advantage to solely synthesize mRNA with polyA tails, whereas the latter ones usually result in a higher amount of synthesized cDNA. In the case you want to perform a reverse transcription with RNA from prokaryotes, where no polyA tails are present, you should use random hexamer primers.
If you have problems regarding your cDNA synthesis, you should have a look at our troubleshooting section for cDNA synthesis.