Bioinformatics Tools Training Program

Designing oligonucleotide primers is a crucial step for successful molecular biology experiments
that require the use of PCR. PCR involves cycles of three steps: denaturation, annealing, and
extension. During denaturation, double-stranded DNA (dsDNA) molecules (templates) are
separated into single strands. During annealing, a pair of primers is annealed to the complementary
regions of the single-stranded molecules. In the extension step, DNA polymerase extends the
primers to produce DNA molecules that correspond to the region bracketed by the primers (the
amplicons). All of these steps are temperature sensitive, and the common choice of temperatures is
94C, 60C, and 70C, respectively. Poorly designed primers may lead to no amplification product or
additional undesired amplified fragments. The goals of primer design include good primer
specificity, high annealing efficiency, appropriate melting temperature, proper GC content, and the
prevention of primer hairpins or primer dimers.

Procedure
Detection
The Detection task can be used for designing standard PCR primers or hybridisation oligos to
DETECT a given sequence. The user can indicate: excluded regions primers are not allowed to
bind in this region targets – primers must amplify one of the targets included region – primers must
bind within this region.

Cloning
The Cloning task can be used to design primers ending or starting exactly at the boundary of the
included region. To clone for example open reading frames the 5’End of the primers should be
fixed. Primer3plus picks primers of various length, all forward primers starting at the same
nucleotide (A from ATG) and all reverse primers starting at the same nucleotide (G from TAG). To
have this functionality the parameter “Fix the x prime end of the primer” in general setting should
be set to 5 (default).
To distinguish between different alleles primers must bind with their 3’End fixed to the varying
nucleotides. If the parameter “Fix the x prime end of the primer” in general setting is set to 3,
primer3plus picks primers of various length, all primers ending at the same nucleotide.
Primer3Plus picks out of all primers the best pair and orders them by quality.

Sequencing
The Sequencing task is developed to design a series of primers on both the forward and reverse
strands that can be used for custom primer-based (re-)sequencing of clones. Targets can be defined
in the sequence which will be sequenced optimally. The pattern how Primer3Plus picks the primers
can be modified on the Advanced settings tab:

Lead
Defines the space from the start of the primer to the point were the trace signals are readable
(default 50 bp).

Spacing
Defines the space from the start of the primer to the start of the next primer on the same strand
(default 500 bp).

Interval
Defines the space from the start of the primer to the start of the next primer on the opposite strand
(default 250 bp).

Accuracy
Defines the size of the area in which primer3plus searches for the best primer (default 20 bp).

Pick Reverse Primers
Select “Pick Reverse Primers” to pick also primers on the reverse strand (selected by default).

Primer List
With the Primer List task all possible primers that can be designed on the target sequence and meet
the current settings will be returned with their corresponding characteristics. This task basically
allows manual selection of primers. The run time of the Primer List task can be relatively long,
especially when lengthy target sequences are submitted.

Primer Check
The Primer Check task can be used to obtain information on a specified primer, like its melting
temperature or self complementarity. For this task no template sequence is required and only the
primer sequence has to be provided.

Naming of Primers
Primer3Plus will create automatically a name for each primer based on the Sequence ID, the primer
number and the primer acronym.

Source Sequence
The sequence from which to select primers or hybridization oligos.

Sequence Id
An identifier that is reproduced in the output to enable you to identify the chosen primers.

Targets
If one or more Targets is specified then a legal primer pair must flank at least one of them. A Target
might be a simple sequence repeat site (for example a CA repeat) or a single-base-pair
polymorphism.

Excluded Regions
Primer oligos may not overlap any region specified in this tag. The associated value must be a
space-separated list of pairs where start is the index of the first base of the excluded region, and
length is its length. This tag is useful for tasks such as excluding regions of low sequence quality or
for excluding regions containing repetitive elements such as ALUs or LINEs.

Primer Size
Minimum, Optimum, and Maximum lengths (in bases) of a primer oligo. Primer3 will not pick
primers shorter than Min or longer than Max, and with default arguments will attempt to pick
primers close with size close to Opt. Min cannot be smaller than 1. Max cannot be larger than 36.
(This limit is governed by maximum oligo size for which melting-temperature calculations are
valid.) Min cannot be greater than Max.

Primer Tm
Minimum, Optimum, and Maximum melting temperatures (Celsius) for a primer oligo. Primer3 will
not pick oligos with temperatures smaller than Min or larger than Max, and with default conditions
will try to pick primers with melting temperatures closetoOpt.