寡核苷酸(Oligo)引物酶标仪定量计算

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Absorbance (A), also known as optical density (OD), is the quantity of light absorbed by a solution. Transmittance is the quantity of light that passes through a solution^[1]. Absorbance and % transmittance are often used in spectrophotometry and can be expressed by the following:

Absorbance equation

• A = Log₁₀ (I₀/I)

where I₀ is the intensity of the incident light, and I is intensity of that light after it passed through the sample

• T = I/I₀ and %T = 100 (T)

The equation that allows one to calculate absorbance from % transmittance is

• A = 2 - Log₁₀ (%T)

Determine concentration using the Beer-Lambert Law

The concentration of a sample can be calculated from its absorbance using the Beer–Lambert law, which is expressed as follows:

• A = ε * c * p

Where ε is the molar absorptivity, or molar extinction coefficient, in L* mol^-1 *cm^-1;
c is the concentration of the solute in solution, in mol/L;
p is the path length of the sample, in cm, for example 1 cm for a cuvette.

ε₂₆₀^[2]

Would you believe that the extinction coefficient for a sequence as short as 6 bases can vary just by arranging the bases in a different order? Additionally, base composition can lead to significant differences in the extinction coefficient.

最邻近消光系数 单碱基的消光系数 image.png

OD₂₆₀

The amount of oligonucleotide in nanomoles that, when dissolved in 1 mL volume, results in 1 unit of absorbance at 260 nm with a standard 1 cm path-length cuvette. nmole/OD₂₆₀ is calculated from oligonucleotide's molar extinction coefficient. OD₂₆₀ is calculated from the following equation,

• OD₂₆₀ = (A₂₆₀ * V) / p

where A₂₆₀ is the absorbance at 260 nm, V is the solution volume in mL, and p is the length of the light path through the sample (cm). Thus, OD₂₆₀ has the units mL/cm.

Starting from the oligo molar extinction coefficient, ε₂₆₀ :

• ε₂₆₀ = L/(mol · cm) = 10³ mL/(mol · cm) = mL/(mmol · cm)

Since OD₂₆₀ has the units of mL/cm, the equation can be written as :

• 10³ OD₂₆₀/mol = 10³ OD₂₆₀ / 10⁹ nmol = OD₂₆₀ / 10⁶ nmol

Combination of both equations yields :

• ε₂₆₀ = OD₂₆₀ / 10⁶ nmol = OD₂₆₀ / 10³ umol = OD₂₆₀ / mmol

Molecular Weight^[4]

Molecular weight of an oligomer is a sum of the weights of individual bases and chemical modifications. Oligos are typically synthesized without 5’-phosphate group, which must be subtracted.

• MW = Σ_{Individual Base MW} + Σ_{Individual Mod MW} - PO₂H + H₂

where PO₂H = 63.980 and H₂ = 2.016

碱基	分子量
MWdA	313.209
MWdC	289.184
MWdG	329.208
MWdT	304.196
MWdU	290.169

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引物合成报告的生成

以“5'-ACGT-3'的计算方法”为例^[3]

第一步：计算消光系数

ε₂₆₀ =(εAC+εCG+εGT)-(εC+εG) L ⋅ mol^-1 ⋅ cm^-1

ε₂₆₀ = (21,200+18,000+20,000)-(7,400+11,500) L ⋅ mol^-1 ⋅ cm^-1

ε₂₆₀ = 40,300 L ⋅ mol^-1 ⋅ cm^-1 = 40.3 L ⋅ mmol^-1 ⋅ cm^-1 = 40.3 OD₂₆₀/umol

在所有网上技术信息和印刷的文件中，上述值40,300显示为40.3。这是因为单位是L ⋅ mmol^-1 ⋅ cm^-1（是毫摩尔而不是摩尔）。

第二步：计算浓度

A₂₆₀设定为1.0光密度（OD）单位;

c = A/(ε * p)= 1/( 40,300 L ⋅ mol^-1 ⋅ cm^-1 * 1 cm) = 2.48*10^-5 mol·L^-1

第三步：计算摩尔质量（MW）

MW = [A x n+T x n+C x n+G x n]-PO₂H + H₂

MW = [313.209 x 1+289.184 x 1+329.208 x 1+304.196 x 1]- 63.980+2.016 = 1173.83 g·mol^-1

第四步：将浓度换算为常用单位

c = 2.48 x 10^-5 mol·L^-1 x 1173.83 g·mol^-1 = 29.1 ug·ml^-1 = 29.1 ng·ul^-1

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引物酶标仪定量计算实例

引物信息如图：

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将引物按理论值稀释至 100 uM，然后取1 uL加入至199 uL水中进行稀释，充分混匀，全部吸入透明96孔板中，用酶标仪进行测量，测得A ₂₆₀为 0.4162 ，背景值为 0.0876 ;

引物实际摩尔浓度 c=(0.4162-0.0876)/(605.4 x 10^-3) x 200 = 108.56 uM

接近理论值100 uM。

计算工具

在线Oligo分析器

OligoAnalyzer^® Tool

OligoEvaluator™

参考资料

1. Absorbance

2. 寡核苷酸定量

3. Oligo quantification—getting it right

4. Definitions and Physical Models