Research Oligo

A common laboratory method is PAGE. Since primers are single-stranded DNA and can form complex secondary structures, denaturing PAGE is essential.

Gel Recommendations:

* Primers <12 bases: 20% polyacrylamide gel (7M urea)

* Primers 12 - 60 bases: 16% polyacrylamide gel (7M urea)

* Primers >60 bases: 12% polyacrylamide gel (7M urea)

Notes:

(1) Dissolve 0.2-0.5 OD primer with saturated urea solution (or add dry urea powder into primer solution until saturated) and heat denature (95°C, 2 min) before loading. The urea denatures the DNA and increases sample specific gravity for easier loading.

(2) Detect the bands with a fluorescent TLC plate under a UV lamp. Good purity is indicated by no impurities below the main band. (Sometimes due to insufficient denaturation, there may be bands above the main band, which are primer secondary structure bands.)

The OD value of a synthetic primer is determined by using a UV spectrophotometer at a wavelength of 260 nm and a 1 cm path length quartz cuvette.

* The solution must be diluted so that the optical density is between 0.2-0.8.

* The OD value of the stock solution must be calculated by multiplying the reading by the dilution ratio.

For example, the DNA was dissolved in 1 mL of water to create a stock solution. A 1:10 dilution of this stock (100 μL stock + 900 μL water) yielded an A260 of 0.2 when measured in a 1-cm path length quartz cuvette. This measurement corresponds to an original stock concentration of 2 OD.

This situation is more likely to occur with shorter oligonucleotide, The two main reasons are:

(1) Base composition: Different components of A, G, C, and T lead to different electrophoresis speeds.

(2) Steric structure: Differences in the DNA's three-dimensional structure result in varied electrophoresis speeds.

EB staining is primarily used to judge the amount of double-stranded DNA (dsDNA) because it stains by embedding itself between the double helix. Synthetic single-stranded DNA (ssDNA) can only be stained if it folds back on itself to form a local hairpin loop or partially forms a double-helix structure with other strands.

Since the possibility of forming a secondary structure differs greatly among primers, the degree of EB staining will also vary. For example, oligonucleotide(dT) does not form a secondary structure, and the EB staining effect is very poor. Therefore, EB staining cannot be used for quantification, and UV spectrophotometry should be applied for detection.

(1) Length: Generally between 15 and 30 bases.

(2) GC content: Between 40% and 60%.

(3) Base distribution: Bases should be randomly distributed.

(4) Complementarity: There should be no complementary sequences within the primer itself (hairpins) or between the two primers (primer dimers).

(5) Binding energy (∆G): Primers should have relatively high ∆G values at the 5' end, middle ∆G values, and low ∆G values at the 3' end.

(6) Product structure: The single strand of the amplification product should not form a secondary structure.

(7) Specificity: The primers must have high specificity for the target.

As oligonucleotide length increases, synthesis complexity and the risk of incomplete sequences also increase. Unless specifically required, we recommend limiting primer length to 80 bases or less. At this length, the proportion of full-length product in a crude synthesis typically does not exceed 40%, and additional losses may occur during purification, resulting in reduced final yield.

With optimized synthesis conditions, primers of up to 145 bases can be produced; however, yields and purity decrease significantly at extended lengths, and such designs should be considered only when shorter alternatives are not feasible.

Agarose gel electrophoresis is unsuitable for analyzing synthetic primers. Since primers are single-stranded DNA, they easily form complex steric structures, leading to multiple or no bands on an agarose gel. Therefore, denaturing PAGE electrophoresis is the recommended method.