What are modified oligonucleotides?
What are modified oligonucleotides?
Numerous reports have covered oligonucleotides for which modifications have been made of the phosphodiester backbone, of the purine and pyrimidine heterocyclic bases, and of the sugar moiety; these modifications serve as structural and mechanistic probes.
What are oligos used for?
Oligonucleotides, or oligos, are short single strands of synthetic DNA or RNA that serve as the starting point for many molecular biology and synthetic biology applications! From genetic testing to forensic research and next-generation sequencing, an oligo may very well be the starting point.
How are oligos used in research?
Why use oligos in your research? Oligos are commonly used in the polymerase chain reaction (PCR). PCR is the technique of taking a single strand of DNA and generating thousands or millions of copies of that DNA for use in other downstream applications (i.e. cloning, sequencing, etc.).
Are primers and oligos the same thing?
Oligonucleotides made up of 2′-deoxyribonucleotides are the molecules used in polymerase chain reaction (PCR). These are referred to as primers and are used to massively amplify a small amount of DNA.
How are oligonucleotides synthesized?
In solid-phase synthesis, an oligonucleotide being assembled is covalently bound, via its 3′-terminal hydroxy group, to a solid support material and remains attached to it over the entire course of the chain assembly.
What are oligos made of?
Oligos are short, synthetic strands of DNA or RNA. The word oligonucleotide is derived from the Greek word olígoi, meaning “few” or “small”, and nucleotide, which are the building blocks of nucleic acids, such as those in DNA.
What exactly is PCR used for and why is it an effective and important technique?
PCR is used in molecular biology to make many copies of (amplify) small sections of DNA? or a gene?. Using PCR it is possible to generate thousands to millions of copies of a particular section of DNA from a very small amount of DNA. PCR is a common tool used in medical and biological research labs.
How do you store oligos?
The best practice is to store oligos in a freezer (–20°C) in either TE buffer, nuclease-free water, or dried for up to 24 months. It is also best practice to minimize oligo exposure to UV light.
How do you reconstitute oligos?
Oligo resuspension
- During the dry-down process, oligos form a white flakey pellet at the bottom of the tube.
- If resuspension is difficult, try heating the oligo at 55°C for 1–5 minutes, then vortex thoroughly.
- IDT oligonucleotides (both DNA and RNA) are typically shipped dry.
What is oligonucleotide structure?
The oligonucleotide structure is characterized by the specific association with seven nickel ions, involving the N7 atom of every guanine. One of the Ni2+ions is shared between two guanines of symmetry related molecules. Until now no oligonucleotide has been crystallized in the presence of this metal ion.
What is the purpose of capping used at the time of oligonucleotide synthesis?
On some DNA synthesizers there is a second capping step after iodine oxidation. The purpose of this is to dry the resin, as residual water from the oxidation mixture can persist and inhibit the next coupling reaction.
What is oligo mixture?
Standard “wobbles” or “mixed bases” are mixtures of two or more different bases at a given position within the sequence. Wobbles are often incorporated into oligonucleotide probes designed to hybridize to an unknown gene that encodes a known amino acid sequence.
What is oligo manufacturing?
The oligonucleotide manufacturing process consists of five major steps, (1) synthesis, (2) cleavage and deprotection (C&D), (3) purification, (4) desalting and concentration and (5) lyophilization, as shown in Fig. 1. Additional steps can be added depending on the type of oligonucleotide being manufactured.
What are the four important applications of PCR?
We present a survey of the following applications of PCR: 1) The amplification of gene fragments as fast alternative of cloning. 2) The modification of DNA fragments. 3) The sensitive detection of pathogenic microorganisms, if desired followed by an accurate genotyping. 4) DNA analysis of arachaeological specimens.
What are the advantages and disadvantages of PCR?
Table 1
Advantages of PCR | Disadvantages of PCR |
---|---|
Shown to be more cost-effective with selective use than culture and staining | Becomes less cost-effective when performed with a multi-organism PCR approach |
Increased ability to detect less common organisms such as viruses | Supply costs, machinery fees, training expenses |
Are oligos stable at room temperature?
For room temperature or 4°C storage, oligos that are resuspended in TE buffer (10 mM Tris pH 8.0, 0.1 mM EDTA; such as IDTE), are more stable than dry oligos. Oligos that are stored in water are the least stable. However, at 4°C, oligos stored under all of these conditions are stable for at least 60 weeks.
What temperature should oligos be stored at?
We ship most of our oligos dry, and the short shipping time will not jeopardize oligo stability for any standard oligo. As mentioned earlier, dry oligos remain stable for up to 25 weeks when stored at 37°C (98°F).
How do you dissolve oligo?
Always briefly centrifuge oligos before opening for the first time. We dissolve the stock oligo in sterile dH2O which must be freshly autoclaved. Alternatively, TE buffer (10 mM Tris pH 8.0, 1 mM EDTA) can be used. For convenience, make a freezer stock at 100 µM concentration (which should be thawed infrequently).
How do you synthesize an oligo?
Synthesis cycle
- Step 1: De-blocking (detritylation) The DMT group is removed with a solution of an acid, such as 2% trichloroacetic acid (TCA) or 3% dichloroacetic acid (DCA), in an inert solvent (dichloromethane or toluene).
- Step 2: Coupling.
- Step 3: Capping.
- Step 4: Oxidation.