Quality and quantity determination of DNA samples. Brief summary.
Abstract
DNA yield can be assessed using various methods including absorbance (optical density), agarose gel electrophoresis, or use of fluorescent DNA-binding dyes. Nucleic acids and proteins have absorbance maxima at 260 and 280 nm, respectively. The ratio of absorbance at 260 nm and 280 nm is used to assess the purity of DNA and RNA. A ratio of about 1.8 is generally accepted as pure for DNA while a ratio of about 2.0 is generally accepted as “pure” for RNA. In this lab, the samples were analysed only at 260 nm and 280 nm only. The negative absorbance values obtained in the results indicated that the DNA could have been degraded or it there were still traces of previously used reagents like phenol as well as the buffer could have had impurities. The negative results for absorbance recorded affected the absorbance ration which were way out of range. Ethidium bromide fluoresces orange in UV light, making it possible to visualize the DNA. If the DNA was intact, it would appear as a distinct band on the gel. If it was degraded, it would appear as a smear of thousands of small fragments. If the DNA was contaminated with protein, there will be a bright band of DNA at the bottom of the well and along the migration path from the wells where the slower moving protein trapped DNA. If the well was over loaded with DNA that is too concentrated, the band will have a jagged smear above it. In the gel electrophoresis conducted there was poor separation as only smear band of proteins could be seen on the gel. The combination of the two analysis methods of quality and quantity determination of DNA lead to the conclusion that the DNA obtained in Practical 5 (bacterial and fungal DNA) was of very poor quality and very little in amount. (Tiwari and Ghimire 2010).
Introduction
DNA yield can be assessed using various methods including absorbance (optical density), agarose gel electrophoresis, or use of fluorescent DNA-binding dyes. All three methods are convenient, but have varying requirements in terms of equipment needed, ease of use, and calculations to consider.
After isolation of DNA, quantification and analysis of quality are necessary to ascertain the approximate quantity of DNA obtained and the suitability of DNA sample for further analysis. This is important for many applications including gel electrophoresis, digestion of DNA by restriction enzymes or PCR amplification of target DNA, it is more important to have good quality DNA samples that is unsheared or undegraded DNA, than high quantities of DNA. Both the quality and quantity of nucleic acid starting template affect PCR, in particular the sensitivity and efficiency of amplification. PCR sensitivity and efficiency can be reduced by the presence of impurities in nucleic acid preparations or in biological samples. One of the most commonly used methodologies for quantifying the amount of nucleic acid in a preparation is spectrophotometric analysis. (Lalwan, et al,. 2014)
Spectrophotometer, will include the absorbance of all molecules in the sample that absorb at the wavelength set. Nucleotides, RNA, ssDNA, and dsDNA absorb at 260 nm; hence they will contribute to the total absorbance of the sample. Therefore, to ensure accurate results, nucleic acid samples require purification prior to measurement. Nucleic acids and proteins have absorbance maxima at 260 and 280 nm, respectively. The ratio of absorbance at 260 nm and 280 nm is used to assess the purity of DNA and RNA. A ratio of about 1.8 is generally accepted as pure for DNA while a ratio of about 2.0 is generally accepted as “pure” for RNA. If the ratio is appreciably lower in either case, it may indicate the presence of protein, phenol or other contaminants that absorb strongly at or near 280 nm. Similarly, absorbance at 230 nm is accepted as being the result of other contamination; therefore, the ratio of A260/A230 is frequently also calculated. The 260/230 values for “pure” nucleic acid are often higher than the respective 260/280 values. Expected 260/230 values are commonly in the range of 2.0-2.2. However, in this lab absorbance at 230 nm was not conducted. (William,et al,.1997) .
Residual chemical contamination from nucleic acids extraction procedures may result an overestimation of the nucleic acid concentration and negatively influence downstream analysis. Abnormal 260/280 ratios usually indicate that the sample is either contaminated by protein or a reagent such as phenol. A low A260/A280 ratio may be caused by residual phenol or other reagent associated with the extraction protocol, a very low concentration (> 10 ng/ul) of nucleic acid. High 260/280 purity ratios are not necessarily indicative of a problem. However, a very high ratio can suggest a poor-quality blank eliminating too much signal near the 280-nm wavelength. (Barbas, et al,. 2007)
DNA was run on an agarose gel. By running a molecular weight marker of known concentration, the extracted DNA concentration could then be determined. The appearance of the DNA on the gel also revealed if it is clean and intact. Agarose is a derivative of agar, a polysaccharide derived from algae. In solution, ionization of the phosphate groups along the backbone of the DNA results in many negative charges on the molecule. Once loaded into the gel, an electric current is applied and the negatively charged molecules of DNA move through the gel toward the positive electrode. The gel is exposed to ethidium bromide, a flat molecule that intercalates, or slides between, the stacked base pairs of the DNA. Ethidium bromide fluoresces orange in UV light, making it possible to visualize the DNA. If the DNA was intact, it would appear as a distinct band on the gel. If it was degraded, it would appear as a smear of thousands of small fragments. If the DNA was contaminated with protein, there will be a bright band of DNA at the bottom of the well and along the migration path from the wells where the slower moving protein trapped DNA. If the well was over loaded with DNA that is too concentrated, the band will have a jagged smear above it (Gill, et al,. 1987).
