Biotechnology - Nucleic acid synthesis - Part 1: Requirements for the production and quality control of synthesized oligonucleotides
1 Scope
This document specifies minimum requirements for the production and quality control of synthesized oligonucleotides (nominally up to 250 bases).
This document also describes general quality attributes for synthesized oligonucleotides as well as common methods for evaluating quality attributes.
2 Normative references
There are no normative references in this document.
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
certified reference material; CRM
reference material (3.4) characterized by a metrologically valid procedure for one or more specified properties, accompanied by an RM certificate that provides the value of the specified property, its associated uncertainty, and a statement of metrological traceability
[SOURCE: ISO Guide 30:2015, 2.1.2, modified]
3.2
performance
ability of oligonucleotides, which are synthesized for the specific intended use including biological assays, to fulfil the requirements for the specific use
Note 1: In the case of oligonucleotides synthesized as primers for a PCR, it is the ability of such synthetic oligonucleotides to function as primers.
Note 2: In the case of oligonucleotides synthesized as probes for use in DNA microarrays, real time PCR or NGS, it is the ability of such synthetic oligonucleotides to hybridize as probes with target oligonucleotide sequence.
Note 3: Performance is confirmed by testing that evaluates full functioning of oligonucleotides in their respective uses.
3.3
purity
ratio between the amount of expected-sequence and/or length synthetic oligonucleotides and the total amount of oligonucleotides
Note: Purity of synthetic oligonucleotides is the ratio of absorbance peak area corresponding to synthetic oligonucleotides of expected sequence and/or lengths in comparison with total peak area of oligonucleotides. The measurement of absorbance at 260 nm (OD260) is calculated by high performance liquid chromatography (HPLC), or capillary electrophoresis (CE). The purity is calculated with area normalization method from the result of HPLC or CE or mass spectrometer.
3.4
reference material; RM
material, sufficiently homogeneous and stable with respect to one or more specified properties, which has been established to be fit for its intended use in a measurement process
[SOURCE: ISO Guide 30:2015, 2.1.1, modified - Notes have been deleted.]
3.5
synthetic oligonucleotides
chemically synthesized DNA (deoxyribonucleic acid) or RNA (ribonucleic acid)
Note 1: Various compounds such as modified bases, base analogues, end-labelling reagents, fluorescent compounds, etc., can be introduced into the synthetic oligonucleotides.
Note 2: In this document, a synthetic oligonucleotide is considered single stranded, linear, and in length from approximately 10 nucleotide-bases to 250 nucleotide-bases.
4 Design and selection of suitable oligonucleotides that are fit for purpose
The quality and consistency requirements of synthetic oligonucleotides depend on their intended uses. For example, the quality requirements of primer and probe in a polymerase chain reaction (PCR), or in a microarray or NGS are significantly different.
In general, users are responsible for specifying quality requirements of the oligonucleotides for their specific application.
A quality grade list can be provided by the producer from which the user can select the grade that fits the intended use, for example “genomic grade” or “antisense grade” (see 5.2).
Robust quality management and testing, together with a common understanding of quality attributes can provide consistent information to allow:
——users to appropriately select oligonucleotides for the intended use;
——users and producers to better communicate and agree on specification for custom oligonucleotides.
