SPECTROPHOTOMETRY (QUANTIFICATION OF NUCLEIC ACIDS)

By

Pauline Asami

BecA-ILRI Hub

Introduction to Bioinformatics and Molecular Biology (IMBB)

May, 2015

Spectrophotometer • An instrument employed to measure the amount of light that a sample absorbs. • The instrument operates by passing a beam of light through a sample and measuring the intensity of light reaching a detector. A spectrophotometer consists of two instruments: • A spectrometer for producing light of any selected color (wavelength) • A photometer for measuring the intensity of light.

It operates on Beer’s law: • When monochromatic light (light of a specific wavelength) passes through a solution there is usually a quantitative relationship (Beer's law) between the solute concentration and the intensity of the transmitted light, that is, the more concentrated the specimen is, the less light is transmitted through it.

Therefore: The concentration of a substance in directly proportional to the amount of light absorbed.

Light source visible: incandescent bulb Light source UV: deuterium-discharge lamps Monochromator : wave selecting device Cuvette: round or square, come in sets, quartz must be used for UV Photoresistor: converts radiant energy to electrical energy

Source: NFS BIOTEKS

After extraction? •  Spectrophotometric analysis is the most commonly used method of

quantifying DNA. •  Agarose gel electrophoresis can also be used to analyse the DNA sample for

purity. •  The NanoDrop is a simple, easy-to-use spectrophotometer for measuring

DNA, RNA, and protein concentrations in small volume samples. Why Quantify? •  To check concentration and purity of DNA/RNA present in the solution. •  Knowing concentration and purity is useful for downstream applications like

PCR, Restriction digests.

Source: Google images

Spectrophotometric measures

•  Measures DNA, RNA (A260) and Proteins (A280) concentrations and sample purity (260:280).

•  Absorbance at 260 nm Nucleic acids absorb UV light at 260 nm due to the aromatic base moieties within their structure. Purines (thymine, cytosine and uracil) and pyrimidines (adenine and guanine) both have peak absorbances at 260 nm, thus making it the standard for quantitating nucleic acid samples. •  Absorbance at 280 nm The 280 nm absorbance is measured where proteins and phenolic compounds have a strong absorbance. Similarly, the aromaticity of phenol groups of organic compounds absorbs strongly near 280 nm. •  Absorbance at 230 nm Many organic compounds have strong absorbances at around 225 nm. In addition to phenol, TRIzol, and chaotropic salts, the peptide bonds in proteins absorb light between 200 and 230 nm.

Spectrophometric measurements • The A260/280 ratio shows the purity of the sample analysed. • Pure DNA sample gives a 260:280 ratio of ˷1.8 and for pure RNA the ratio is ˷ 2. • A260/230 ratio indicates the presence of organic contaminants, such as (but not limited to): phenol, TRIzol, chaotropic salts and other aromatic compounds. Samples with 260/230 ratios below 1.8 are considered to have a significant amount of these contaminants that will interfere with downstream applications • If the sample is contaminated with proteins or other organic compounds the ratios will vary from the above mentioned values. • Various nanodrop models are available e.g Nanodrop 8000, Nanodrop lite and Nanodrop 2000/2000c which is currently available at BecA labs.

Examples of Nano drop readings and interpretation Example 1:

RNA sample Interpretation: this sample is suitable for downstream applications

RNA Sample Interpretation: Due to the low purity , this sample should be re‐isolated. This reading is not at all reliable due to the contamination.

Example 2:

Exercise: Determine if the below are good quality or not

DNA samples

Note that: •  Because of the low concentrations, sometimes it is difficult to assess the

purity of the samples (esp. RNA) by analyzing the A260/280 and A260/230 ratios.

•  Quantification cannot be assessed by the NanoDrop because they are

outside the lowest concentrations the NanoDrop is designed to measure. •  A more sensitive method such as an Agilent Biolanalyzer or Qubit analysis

is recommended.

http://phagesdb.org/media/workflow/protocols/pdfs/PHProtocol_NanoDrop.pdf http://www.mlz-garching.de/files/nanodrop_2000_user_manual.pdf https://www.youtube.com/watch?v=FiGZnNs2xXY

How the Nano drop works:

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