A new algorithim, that enables a more accurate deduction of oligonucleotide (oligo) melting temperatures under laboratory conditions, could bring improvements in primers and probe design for use in polymerase chain reaction (PCR) techniques, writes Wai Lang Chu.
The new IDT Tm Algorithm enables more accurate temperature (Tm) prediction under the experimental conditions used by researchers. As well as PCR applications, the algorithm is set to have uses in DNA sequencing, and in functional genomics applications such as siRNA mediated gene knockdown.
"An oligonucleotide's melting temperature is its most critical property," noted Dr Mark Behlke, vice president for molecular genetics at IDT.
"It's the point at which half the oligo strands are hybridised to complementary sequences and half are free in solution as single strands. The problem is that existing algorithms can be off in their predictions by as much as 5-10 degrees Celsius when estimating the temperature at which melting takes place," he added.
The algorithm is set to eliminate the 'trial and error' approach that researchers are often forced to undertake when amplifying the amount of genetic material through PCR, using hybridisation, sequencing or antisense/RNAi applications.
Most extant algorithms estimate the Tm for oligos under standard conditions of 1M salt, and then scale from that to predict melting temperatures for the actual condition to be used in the experiment.
The key issue is that predictions are mostly based on a salt-correction equation, developed in the 1960s during studies of bacterial genomic DNA that is known by researchers to have significant shortcomings when used with oligonucleotides.
These inexact algorithms can have a serious impact on the work of biotech researchers. "Imprecise Tm prediction can adversely affect the design of experiments," said Dr Richard Owczarzy, who led the team of IDT scientists that developed the new algorithm.
"Indeed, the resultant errors can actually be large enough to ruin experiments, or, even worse, to give misleading or inaccurate results. Accurate estimation of Tm is too important to be left to 40-year old equations, so we built our own more accurate algorithm from the ground up," he added.
Dr Owczarzy's team of IDT scientists achieved this by studying the Tm of oligos for a wide variety of sequences and conditions. After performing over 4,000 melting curves, they succeeded in developing a new algorithm that accurately predicts the Tm of oligos as salt concentration is varied.
Hybridisation between complementary nucleic acids is an implicit feature in the DNA structure that is exploited for many applications of the biological and biomedical arts. For example, virtually all methods for replicating and/or amplifying nucleic acid molecules are initiated by a step in which a complementary oligonucleotide (typically referred to as a "primer") hybridises to some portion of a "target" nucleic acid molecule. A polymerase then synthesises a complementary nucleic acid from the primer, using the target nucleic acid as a "template."
In PCR, two or more primers are used that hybridise to separate regions of a target nucleic acid and its complementary sequence. The sample is then subjected to multiple cycles of heating and cooling, repeatedly hybridising and dissociating the complementary strands so that multiple replications of the target nucleic acid and its complement are performed.
As a result, even very small initial quantities of a target nucleic acid may be enormously increased or "amplified" for subsequent uses (e.g., for detection, sequencing, etc.).
Multiplex PCR is a particular version of PCR in which several different primers are used to amplify and detect a plurality of different nucleic acids in a sample-usually ten to a hundred different target nucleic acids. Thus, the technique allows a user to simultaneously amplify and evaluate large numbers of different nucleic acids simultaneously in a single sample.
The enormous benefits of high throughput, speed and efficiency offered by this technique have made multiplex PCR increasingly popular. However, achievement of successful multiplex PCR usually involves empirical testing as existing computer programs that pick and/or design PCR primers have errors. In multiplex PCR, the errors become additive and therefore good results are seldom achieved without some amount of trial and error.
IDT, which has been granted a patent on the Tm Algorithm (US06889143), has incorporated this new tool into its web-based suite of oligonucleotide design tools, which are freely available to researchers through the SciTools portal