Unknown DNA — Rational Primer Design and Analysis — the ...

PCR is a modified primer extension reaction using a thermostable DNA polymerase that allows for the heat
dissociation of newly formed complimentary DNA and subsequent hybridization of oligonucleotide probes to
the target regions for subsequent rounds of amplification. The scope and methods of PCR are huge and
many varied and beyond the aim of this tutorial, and I will not attempt to teach the procedure. Refer to any
good text in molecular biology techniques for details (for good, early, primary reviews of PCR methodology
see Mullis [1990], White et al. [1989], and Cherfas [1990]). What I will attempt to teach is a method for
inferring appropriate oligonucleotide probes, commonly known as primers, for PCR or hybridization screening
analysis. These oligonucleotides are usually about 20 or more bases in length and target the beginning and
ending locations of the PCR amplification process.
Coupled with PCR techniques and/or ultra sensitive hybridization screenings, oligonucleotide primers have
allowed the ‘fishing out’ of thousands of genes from complex genomes that would have previously been
extremely difficult to ever even find, yet alone sequence. Present-day economic, automated synthesis and
the ready availability of nucleotides, have made primers commonplace. (This has also facilitated the
development of reliable methods for the introduction of site-specific mutations into known sequences.)
Because of the high specificity and adjustable stringency of oligonucleotide hybridization, the sequence
knowledge of a relatively short stretch of unique DNA is sufficient to rapidly isolate and/or amplify, clone if
desired, and sequence the corresponding gene. However, whatever technique one may use, primers are
essential ingredients.
PCR and hybridization screening both require the design of appropriate primers. This can be a ‘hit-or-miss’
affair or you can use computational methods to greatly assist the efficiency of the process. Several strategies
can be imagined for the design of oligonucleotide primers. If an exact nucleotide sequence is known, then a
single oligonucleotide probe for hybridization or a pair of primers for PCR of a defined sequence can simply
be selected, tested, and synthesized. In the absence of a defined DNA sequence, sometimes a group of
similar DNA sequences can be aligned and a consensus sequence created from which primers can be
designed. However, this is often not possible because DNA can be very, very difficult to align. In some cases
one may even be forced to work off of either a small portion of a protein sequence from an Edman
degradation reaction or, as will be illustrated in this tutorial, a consensus pattern from a group of related
proteins — the luxury of using DNA directly will not always be available.
The Guessmer — from Proteins to Primers.
When nucleotide data is lacking or problematic, amino acid sequences can be back translated to provide the
necessary primers. In the absence of exact protein sequence data, a consensus pattern from a group of
related proteins can often be used. Using amino acid sequence information requires one to back translate the
sequence. This is not a trivial chore though, because of the degeneracy of the genetic code. There are 64
possible codons for the 20 amino acids. Because of this, many different back translation probe techniques
have been employed. Two are, either utilizing large pools of short oligonucleotides whose sequences are
highly degenerate, or using small pools, or even just one pair, of longer oligonucleotides of lesser or no
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